US008178536B2
(12) UnitedO States Patent (10) Patent No.: US 8,178,536 B2 Nunes et al. (45) Date of Patent: May 15, 2012
(54) SIRTUIN MODULATING COMPOUNDS 2004/0010033 A1 1/2004 Anderson et al. 2004.0034037 A1 2/2004 Harbeson et al. 2004/0044203 A1 3/2004 Wittman et al. (75) Inventors: Joseph J. Nunes, Andover, MA (US); 2004/0048843 A1 3/2004 Ting et al. Jill Milne, Brookline, MA (US); Jean 2004/0072760 A1 4/2004 Carboni et al. Bemis, Arlington, MA (US); Roger Xie, 2004/O142997 A1 7, 2004 Chen et al. Stably M. S. St. Vu, 2004/O1578452004/O152743 A1 8/2004 DohertySchoenafinger et al. et al. rlington, MA (US) Pui Yee Ng. 2004/0171073 Al 9, 2004 Neiland et al. Boston, MA (US); Jeremy S. Disch, 2004/O180905 A1 9, 2004 Munchhof Natick, MA (US); Thomas Salzmann, 2004/022O189 A1 11/2004 Sun et al. 2005.000984.0 A1 1/2005 Cui et al. Warren, NJ (US); David Armistead, 2005, OO65151 A1 3, 2005 Norcross Sudbury, MA (US) 2005, OO65,196 A1 3, 2005 Inaba et al. 2005/0085519 A1 4/2005 Rubinet al. (73) Assignee: Sirtris Pharmaceuticals, Inc., 2005. O197353 A1 9, 2005 Ritzeler et al. 2005/O197375 A1 9, 2005 Sircar et al. Cambridge, MA (US) 2005.0245513 A1 11/2005 Gallant et al. 2005/0266515 A1 12/2005 O'Brien et al. (*) Notice: Subject to any disclaimer, the term of this 2006, OO14756 A1 1/2006 Edwards et al. patent is extended or adjusted under 35 2006.0036098 A1 2/2006 Kim et al. U.S.C. 154(b) by 0 days. 2006, OO74O75 A1 4/2006 Hadida-Ruah et al. 2007/0O37809 A1 2/2007 Nunes et al. 2007/0O3781.0 A1 2/2007 Nunes et al. (21) Appl. No.: 12/955,663 2007, 0037827 A1 2/2007 Nunes et al. 2007/0O37865 A1 2/2007 Nunes et al. (22) Filed: Nov. 29, 2010 2009.0099.170 A1 4, 2009 Nunes et al. 2009, O163476 A1 6/2009 Milburn et al. (65) Prior Publication Data 2010/0168084 A1 7, 2010 Huber et al. US 2011 FO130387 A1 Jun. 2, 2011 FOREIGN PATENT DOCUMENTS O O AU 30932.89 9, 1989 Related U.S. Application Data DE 1 108 698 6, 1961 (62) Division of application No. 1 1/499,876, filed on Aug. P 28 92 3.28. 4, 2006, now Pat. No. 7,855,289. (Continued) (60) Provisional application No. 60/705,612, filed on Aug. 4, 2005, provisional application No. 60/741,783, filed OTHER PUBLICATIONS on Dec. 2, 2005, provisional application No. 60/779.370, filed on Mar. 3, 2006, provisional Bundgaard (Design and application of prodrugs. In A Textbook of application No. 60/792.276, filed on Apr. 14, 2006. DrugKubinyi Design (3D QSARand Development, in Drug Design: (1991), Ligand-Protein p. 113-191). Interactions and (51) Int. Cl Molecular Similarity, vol. 2-3, Springer, 1998, 800 pages) TOC and Aiki/50 2006.O1 pp. 243-244 provided.* ( .01) Wermuth, The Practice of Medicinal Chemsitry, 2d ed. (2003), 768 C07D 237/26 (2006.01) pages. CHS. 9-10 provided.* (52) U.S. Cl...... 514/249; 544/235 Pacholec et al. (J. Biol. Chem., 285(11), 8340-51).* (58) Field of Classification Search ...... 514/249; Bemis et al. (Bioorg. Med. Chem. Lett., 19 (2009), 2350-53).* 544/235 Parket al. (Toxicology Lett., 120 (2001), 281-91).* See application file for complete search history. (Continued) (56) References Cited Primary Examiner — Robert Havlin U.S. PATENT DOCUMENTS (74) Attorney, Agent, or Firm — Ropes & Gray LLP 3,164,603 A 1/1965 McCafferty 3,503,929 A 3, 1970 Loudas (57) ABSTRACT 3,517,007 A 6, 1970 Kim et al. 3,712,888 A 1/1973 Kaempfen Provided herein are novel sirtuin-modulating compounds and 3,928,228 A 12/1975 Crounse methods of use thereof. The sirtuin-modulating compounds it's A 38. SE al may be used for increasing the lifespan of a cell, and treating 4471,040 A 9, 1984 RAF et al. and/or preventing a wide variety of diseases and disorders 4,939,133 A 7, 1990 Connor et al. including, for example, diseases or disorders related to aging 5,808,087 A 9, 1998 Matsunaga et al. or stress, diabetes, obesity, neurodegenerative diseases, car s: A 3. RE et al diovascular disease, blood clotting disorders, inflammation, 5958,950 A 9, 1999 Miss et al. cancer, and/or flushing as well as diseases or disorders that 6.29I476 B1 9/2001 Kordket al. would benefit from increased mitochondrial activity. Also 6.479,508 B1 1 1/2002 Beaulieu et al. provided are compositions comprising a sirtuin-modulating 6,653,309 B1 1 1/2003 Saunders et al. compound in combination with another therapeutic agent. 7,345,178 B2 3/2008 Nunes et al. 2003,01995.16 A1 10, 2003 Moser et al. 2003/023281.6 A1 12/2003 Beaulieu et al. 6 Claims, 2 Drawing Sheets US 8,178,536 B2 Page 2
FOREIGN PATENT DOCUMENTS Borra et al., “Mechanism of Human SIRT1 Activation by FR 1439 129 5, 1966 Resveratrol”, J. Biol. Chem., 280(17): 17187-195 (2005). FR 1476,529 4f1967 Brandon et al., “Monoclonal Antibody-Based ELISA for GB 1382 861 2, 1975 Thiabendazole in Liver.” Journal of Agric. Food Chem., 40: 1722-26 GB 1421 619 1, 1976 (1992). GB 2405793 3, 2005 Briehn et al., “Alternative heterocycles for DNA recognition: The JP S41006584 4, 1966 benzimidazolefimidazole pair.” Chemistry-A European Journal, JP O4190232 7, 1992 9(9): 2110-22 (2003). JP 06247969 9, 1994 Bukowski "Some reactions of 2-cyanobenzimidazoles.” Acta JP 2002161084 6, 2002 JP 200330O875 10, 2003 Poloniae Pharmaceutica, 35(3):295-299 (1978) (abstract only). JP 200330O886 10, 2003 Birli et al., “DNA binding ligands targeting drug-resistant Gram JP 2003313176 11, 2003 positive bacteria. Part 1: Internal benzimidazole derivatives.” JP 200475614 11, 2004 Bioorganic and Medicinal Chemistry Letters, 14(5): 1253-57 (2004). JP 2005162855 6, 2005 Birli et al., “DNA binding ligands targeting drug-resistant Gram JP 2005330284 12/2005 positive bacteria. Part 2: C-terminal benzimidazole and derivatives.” PL 96241 12, 1977 Bioorganic and Medicinal Chemistry Letters, 14(5): 1259-63 (2004). WO WO 97,04776 2, 1997 Dahlbom et al., “N-Alkyl-3-piperidyl Phenothiazine-10 WO WO99/33824 7, 1999 carboxylates'. Acta Chemica Scandinavica, 15(10):2043-46 (1961). WO WOOOf 69849 11, 2000 Database Chemcats Chemical Abstracts Service, Columbus, OH, WO WOO1 OO610 1, 2001 US; Jan. 18, 2005, XP002384 121 ON's STK199474, STK199472, WO WOO1/21615 3, 2001 STK1994.73, STK180355, STK174405, STK196060, STK115373, WO WOO1,27119 4/2001 WO WOO1,96336 12/2001 STK164162, STK136073, STK164152, STK120473, STKO52285 WO WO O2/O66454 8, 2002 -& "Interchim Intermediates' Jan. 18, 2005, Interchim, Montlucon, WO WO O2/O79.192 10, 2002 France, XP002386059. WO WOO3,OO7959 1, 2003 Dubey et al., “A convenient one-pot synthesis of 1-alkyl WO WOO3,O11219 2, 2003 benzimidazole-2-substituted aminothiazoles.” Indian J. Hetero. WO WOO3,0666.29 8, 2003 Chem., 12(2): 95-98 (2002) (abstract only). WO WOO3,O74516 9, 2003 Dubey et al., “Studies on syntheses of 1-alkyl-2-(substituted WO WO 03/080545 10, 2003 thiazolyl) benzimidazoles.” Indian Journal of Chemistry, Section WO WO 2004/O16600 2, 2004 B:42B(4):931-34 (2003) (abstract only). WO WO 2004/030625 4/2004 Elgemeie et al., “Synthesis of Benzimidazole Ketene N.S-Acetals WO WO 2004/033666 4/2004 and Their Reactions with Nucleophiles.” Synthetic Communications, WO WO 2004/0393.18 5, 2004 WO WO 2004/041277 5, 2004 33(4):555-62 (2003). WO WO 2004/062663 T 2004 Fekner et al., “Synthesis and Metalation of a Chiral, Pyridine WO WO 2004/069 160 8, 2004 Strapped, Cyclic Bis(benzimidazole) Ligand.” Organic Letters, WO WO 2004/084.813 10, 2004 6(6): 989-92 (2004). WO WO 2005/OO2552 1, 2005 Halluska et al., “In vitro and in vivo Antitumor Effects of the Dual WO WO 2005/025574 3, 2005 Insulin-Like Growth Factor-I/Insulin Receptor Inhibitor, WO WO 2005/043630 5, 2005 BMS-554417.” Cancer Research, 66(1):362-71 (2006). WO WO 2005, O77939 8, 2005 Huang et al., “Synthesis and Anticancer Evaluation of WO WO 2005,10O342 10/2005 Bis(Benzimidazoles), Bis(Benzoxazoles), and Benzothiazoles.” WO WO 2005,105798 11, 2005 Bioorganic & Medicinal Chemistry, 14:6106-19 (2006). WO WO 2006/O1828O 2, 2006 Jules et al., “Derivatives of 3-, 4-, and 5-Phenylsalicylamides,” J. Am. WO WO 2006/02O767 2, 2006 WO WO 2006/034833 4/2006 Pharma. Assoc., 45(5):277-281 (1956). WO WO 2006/050506 5, 2006 Kaeberlein et al., “Substrate-specific Activation of Sirtuins by WO WO 2006/053227 5, 2006 Resveratrol.” J. Biol. Chem., 280(17): 17038-45 (2005). WO WO 2006/094236 9, 2006 Katagiri et al., “Studies on Ketene and Its Derivatives. Part 1191). WO WO 2006/113458 10, 2006 Reactions of Haloketenes with 2-Arylideneaminopyridines,” J. WO WO 2007/019344 2, 2007 Hetero. Chem. 21:407-12 (1984). WO WO 2007/019346 2, 2007 Kubinyi, "3D QSAR in Drug Design Ligand-Protein Interactions and WO WO 2008/073451 6, 2008 Molecular Similarity.” Springer, 800 pages, 2-3:243-44 provided WO WO 2008, 106692 9, 2008 (1998). WO WO 2008,156869 12/2008 Kuster et al., “Synthese von Substituierten Benzotriazolen Zur WO WO 2009/058348 5, 2009 Stabilisierung aromatischer Polyamide gegen UV-Licht.” Die WO WO 2009/061.453 5, 2009 Angewandte Makromolekulare Chemie, 54:55-70 (1976). OTHER PUBLICATIONS Ma et al., "Combinatorial Synthesis of Substituted Biaryls and Het erocyclic Arylamines,” J. Combinatorial Chem. 6:426-30 (2004). Attanasi et al., “Conjugated Azoalkenes. Part 14. Synthesis of New Marques et al., “Expanding the Repertoire of Heterocycle Ring Pairs 1-Amino-and 1,2-Diamino-pyrrole Derivatives by Reaction of some for Programmable Minor Groove DNA Recognition.” J. Amer. Chem. Soc., 126:10339-349 (2004). Conjugated AZoalkenes with Activated Methylene Compounds Nawwar et al., “Aroylisothiocyanates in Heterocyclic Synthesis: RCH2Ac and RCH2CN (R=Aryl, Heteroaryl).” J. Chem. Soc., Synthesis of New Benzimidazole Derivatives with Anticipated Fun Perkin Transactions 1, Organic and Bioorganic Chemistry, 3:315-320 gicidal Activity.” Phosphorus, Sulfur and Silicon and the Related (1993). Elements, 57:65-73 (1991). Bamford et al., “(1H-Imidazo[4,5-cpyridin-2-yl)-1,2,5-oxadiazol Newsome et al., “Enzyme-linked immunosorbent assay of benomyl 3-ylamine derivatives: A novel class of potent MSK-1-inhibitors.” and thiabendazole in some foods.” Association of Official Analytical Bioorganic and Medicinal Chemistry Letters, 15:3402-06 (2005). Chemists, 70(6):1025-27 (1987) (abstract only). Barraclough et al., “Inotropic activity of heterocyclic analogues of Pacholec et al., “SRT1720, SRT2183 and SRT1460 Do Not Activate isomazole.” European J. Med. Chem, 25:467-477 (1990). Sirt1 with Native Substrates’, FASEB Summer Research Confer Bauser et al., “Discovery and optimization of 2-aryl oxazolo ences; NAD Metabolism and Signaling, Jun. 21-26, 2009. pyrimidines as adenosine kinase inhibitors using liquid phaseparallel Pacholec et al., “SRT1720, SRT2183, SRT1460, and Resveratrol are synthesis.” Bioorganic & Medicinal Chemistry Letters, 14: 1997 not Direct Activators of SIRT1’, JBC Papers in Press, Manuscript 2000 (2004). M109.088.682, Jan. 8, 2010. US 8,178,536 B2 Page 3
Pacholec et al., “SRT1720, SRT2183, SRT1460, and Resveratrol are Bauer et al., “dSir2 and Dmp53 interact to mediate aspects of CR not Direct Activators of SIRT1, J. of Bio. Chem., 285(11): 8340 dependent lifespan extension in D. melanogaster;' AGING, 1(1):1- 8351, Mar. 12, 2010. 11, (2009). Beher et al., “Resveratrol is Nota Direct Activator of SIRT1 Enzyme Papers of the Week, “A Resveratrol Reversal.” DOI 10.1074/bc. Activity”, Chem. Biol. Drug Des. 74: 619-624 (2009). P109.088.682, Mar. 10, 2010 (abstract). Blander et al., “SIRT1 Shows No Substrate Specificity in Vitro.” J. Pessoa-Mahana, H. et al., “Solvent-Free Synthesis of Biol. Chem., 180(11):9780-9785 (2005). 6-Arylbenzimidazo 1.2-cquinazolines under Microwave Irradia Blum et al., “SIRT1 Modulation as a Novel Approach to the Treat tion.” Synthesis, 3:436-40 (2004). ment of Diseases of Aging.” J. Med. Chem. 54:417-432 (2011). Porcu et al., “The emerging therapeutic potential of sirtuin-interact Buchen, “Health Benefits of Red-Wine Chemical Unclear', Nature, ing drugs: from cell death to lifespan extension.” TRENDS in Phar http://www.nature.com/news/2010/1001 19/full/news.2010.18.html. macological Sciences, 26(2):94-103 (2005). Burnett et al., “Absence of Effects of Sir2 Overexpression on Prakash et al., “Synthesis of 1-methyl-2-(2-hydrazino-4-thiazolyl) Lifespan in C. Elegans and Drosophila,” Nature, 477:482-486 (2011). benzimidazole and its hydrazones,” J. Indian Chem. Soc., 55(9):919 Cantó et al., “Don’t write sirtuins off.” Nature, 477:41.1 (2011). 21 (1978) (abstract only). ClinicalTrials.gov Search of SRT2104—List Results http:// Prakash et al., “Thin-layer chromatographic separation of some clinicaltrials.gov/ct2/results?term=Srt2104 (retrieved on Jan. 27. thiosemicarbazones and 4-(1-methyl-2-benzimidazolyl)-2- 2011). thiazolylhydrazones.” Chemia Analityczna, 26(6):1065-67 (1981) ClinicalTrials.gov Search of SRT2379—List Results—http:// (abstract only). clinicaltrials.gov/ct2/results?term=Srt2379 (retrieved on Jan. 27. Raslan et al., “Studies with heterocyclic beta-enaminonitriles: A 2011). simple route for the synthesis of polyfunctionally substituted Csiszar et al., “Vasoprotective effects of resveratrol and SIRT1: thiophene, imidazo 1.2:1",6'pyrimido 5,4-bithiophene and thieno attenuation of cigarette Smoke-induced oxidative stress and proinflammatoryphenotypic alterations.” Am. J. Physiol. Heart Circ. 3.2-dpyrimidine derivatives.” J. Chinese Chem. Soc., 50(4):909-16 Physiol., 294:H2721-H2735 (2008). (2003) (abstract only). Dai et al., “SIRT1 Activation by Small Molecules Kinetic and Bio Reddy et al., “Synthesis of 6-arylpyrido2',3':4.5 pyrimido 1,6-a physical Evidence for Direct Interaction of Enzyme and Activator.”.J. benzimidazoles.” Indian J. Chem. Section B:23B(11): 1106-07 Bio. Chem..., 285(43):32695-32703 (2010). (1984) (abstract only). Davis et al., The Synthesis and Reactions of Certain 6-Substituted Renneberg et al., “Imidazopyridine/Pyrrole and Benz-imidazo 1, 2-c Quinazolines, J. Chem. Soc., 945-54 (1962) Hydroxybenzimidazole Pyrrole Pairs for DNA Minor Groove Rec Abstract. ognition.” J. Am. Chem. Soc., 125:5707-16 (2003). Dittenhafer-Reed et al., “Catalysis and Mechanistic Insights into Santra et al., “Excited-state intramolecular proton transfer in the Sirtuin Activation'. Chem. Bio. Chem., 12(2): 281-289 (2011). anionic species of 2-(2'-acetamidophenyl)benzimidazole in acqueous Feige et al., “Specific SIRT1 Activation Mimics Low Energy Levels medium.” Chemical Physics Letters, 327:230-37 (2000). and Protects against Diet-Induced Metabolic Disorders by Enhanc Sergievskii et al., “4-Aminofurazan-3-carboxylic Acid Iminoester in ing Fat Oxidation.” Cell Metabolism, 8:347-358 (2008). Reactions with N.O-Nucleophiles.” Russian J. Org. Chem. Huber et al., “SIRT1-independent mechanisms of the putative sirtuin 38(6):872-74 (2002). enzyme activators SRT1720 and SRT2183.” Future Med. Chem. Sergievskii et al., “Reactions of Methyl 4-Aminofurazan-3- 2(12): 1751-1759 (2010). carboximidate with Nitrogen-Containing Nucleophiles.” Russian J. Korshak et al., “General Method of the Synthesis of Step-Ladder Org. Chem. 37(5):717-20 (2001). Polymers', Makromolekulare Chemie, 176(5):1233-71 (1975) Takahashi et al., “Syntheses of Heterocyclic Compounds of Nitro Abstract. gen. CXXVI. Syntheses of Oxazolopyridines and Related Com Lombard et al., “Longevity Hits a Roadblock.” Nature, 477:410-411 pounds.” Chemical and Pharmaceutical Bulletin, 9:426-432 (1961). (2011). Thiel et al., “1,3,4-Thiadiazoles by reaction of dithiocarboxylic Milne et al., “Small molecule activators of SIRT1 as therapeutics for esters with carbonic hydrazides,” Journal fuer Praktische Chemie, the treatment of type 2 diabetes.” Nature, 450:712-717 (2007). 332(1):55-64 (1990) (abstract only). Minor et al., “SRT17290 Improves Survival and Healthspan of Obese Thompson et al., “Tyrosine Kinase Inhibitors. 7. 7-Amino-4- Mice.” Scientific Reports, 1-38 (2011). (phenylamino)- and 7-Amino-4-(phenylmethyl)aminolpyrido4.3- Pfister et al., “Opposing Effects of Sirtuins on Neuronal Survival: dpyrimidines: A New Class of Inhibitors of the Tyrosine Kinase SIRT1-Mediated Neuroprotection Is Independent of Its Deacetylase Activity of the Epidermal Growth Factor Receptor.” J. Med. Chem. Activity.” PLOS One, 3(12): 1-8 (2008). 38(19):3780-88 (1995). Silverman, “The Organic Chemistry of Drug Design and Drug Von Angerer “Product subclass 3: 1,3,5-triazines and phosphorus Action.” Elsevier, 29-32 (2004). analogues.” Science of Synthesis, 17:449-83 (2004) (abstract only). Viswanathan & Guarente “Regulation of Caenorhabditis elegans Walser et al., “Pentacyclic Triazolodiazepines as PAF-Antagonists.” Lifespan by Sir-2.1 Transgenes.” Nature, 477:E1-E2 (2011). J. Hetero. Chem., 28:1121-25 (1991). Yamazaki et al., “Treatment with SRT1720, a SIRT1 activator, ame Wittman et al., “Discovery of a 1H-Benzoimidazol-2-yl)-1H liorates fatty liver with reduced expression of lipogenic enzymes in pyridin-2-one (BMS-536924) Inhibitor of Insulin-like Growth Factor MSG mice.” Am J. Physiol. Endocrinol. Metab., 297:E1179-E 1186 I Receptor Kinase with in Vivo Antitumor Activity.” J. Med. Chem. (2009). 48:5639-43 (2005). Yoshizaki et al., “SIRT1 inhibits inflammatory pathways in macroph Yamaguchi et al., “Structure and Properties of Ethyl ages and modulates insulin sensitivity.” Am. J. Physiol. Endocrinol. (2-Benzimidazolyl)cyanoacetimidate.” J. Hetero. Chem. 36:841-47 Metab., 298:E419-E428 (2010). (1999). Yoshizaki et al., “SIRT1 Exerts Anti-Inflammatory Effects and Yamori “Information based on human cancer cell line-panel—its Improves Insulin Sensitivity in Adipocytes.” Molecular and Cellular application to the discovery of molecular target-based drugs and the Biology, 29(5): 1363-1374 (2009). diagnosis of chemosensitvity.” Drug Delivery System, 18(4):385-93 Zarse et al., “Differential Effects of Resveratrol and SRT1720 on (2003) (abstract only). Yogi et al., “Synthesis of Arylthio-Substituted 3.8-Diphenyl-1,2- Lifespan of Adult Caenorhabditis elegans," Horm. Metab. Res., diazacycloocta-2,4,6,8-tetraenes and Their Thermolysis.” Bull. 42(12): 837-839 (2010). Chem. Soc. Jpn. 60(1):335-342 (987). * cited by examiner U.S. Patent May 15, 2012 Sheet 1 of 2 US 8,178,536 B2
SUBCULTUREH-358 CELLS IN 150mm PLATES 48 HOURS 37°C 5% CO PLATE 100 uL CELLS/WELL (104 CELLS/WELL) INTO 96 WELL ASSAY PLATES 24 HOURS 37°C 5% CO ADD 0.5 L COMPOUNDS FROM TEST COMPOUND PLATE INTO ASSAY PLATE 48 HOURS 37°C 5% CO ADD 15uL/WELLALAMARBLUETM 2 HOURS 37°C 5% CO, READ FLUORESCENT ALAMARBLUETM
WASH ASSAY PLATES 1X 100 uL/WELL WITH PBS
ADD 200 uL/WELL ATP ASSAY REAGENT
READLUMINESCENT SIGNAL FIG. 1 U.S. Patent May 15, 2012 Sheet 2 of 2 US 8,178,536 B2
3.5
3.0
2.5
2.0
15
1.0
0.5 0.0 -9 -8 -7 -6 -5 -4 CONC. LogM) RESVERATROL
FIG. 2 US 8,178,536 B2 1. 2 SIRTUIN MODULATING COMPOUNDS SIRT3 is a homolog of SIRT1 that is conserved in prokary otes and eukaryotes (P. Onyango et al., Proc. Natl. Acad. Sci. RELATED APPLICATIONS USA99: 13653-13658 (2002)). The SIRT3 protein is targeted to the mitochondrial cristae by a unique domain located at the N-terminus. SIRT3 has NAD+-dependent protein deacety This application is a Divisional of U.S. application Ser. No. 5 lase activity and is upbiquitously expressed, particularly in 1 1/499,876, filed Aug. 4, 2006, which claims the benefit of metabolically active tissues. Upon transfer to the mitochon U.S. Provisional Application Nos. 60/705,612, filed Aug. 4. dria, SIRT3 is believed to be cleaved into a smaller, active 2005, 60/741,783, filed Dec. 2, 2005, 60/779,370, filed Mar. form by a mitochondrial matrix processing peptidase (MPP) 3, 2006 and 60/792.276, filed Apr. 14, 2006, the contents of (B. Schwer et al., J. Cell Biol. 158: 647-657 (2002)). which are incorporated by reference in their entirety. 10 Caloric restriction has been known for over 70 years to improve the health and extend the lifespan of mammals (Ma SEQUENCE LISTING soro, 2000). Yeast life span, like that of metazoans, is also extended by interventions that resemble caloric restriction, The instant application contains a Sequence Listing which such as low glucose. The discovery that both yeast and flies has been submitted via EFS-Web and is hereby incorporated 15 lacking the SIR2 gene do not live longer when calorically by reference in its entirety. Said ASCII copy, created on Feb. restricted provides evidence that SIR2 genes mediate the 14, 2011, is named SIRTP12006 and is 4,096 bytes in size. beneficial health effects of this diet (Anderson et al., 2003: Helfand and Rogina, 2004). Moreover, mutations that reduce BACKGROUND the activity of the yeast glucose-responsive cAMP (adenosine 3',5'-monophosphate)-dependent (PKA) pathway extend life The Silent Information Regulator (SIR) family of genes span in wild type cells but not in mutant sir2 Strains, demon represents a highly conserved group of genes present in the strating that SIR2 is likely to be a key downstream component genomes of organisms ranging from archaebacteria to a vari of the caloric restriction pathway (Lin et al., 2001). ety of eukaryotes (Frye, 2000). The encoded SIR proteins are SUMMARY involved in diverse processes from regulation of gene silenc 25 ing to DNA repair. The proteins encoded by members of the Provided herein are novel sirtuin-modulating compounds SIR gene family show high sequence conservation in a 250 and methods of use thereof. amino acid core domain. A well-characterized gene in this In one aspect, the invention provides sirtuin-modulating family is S. cerevisiae SIR2, which is involved in silencing compounds of Formula (I): HM loci that contain information specifying yeast mating 30 type, telomere position effects and cell aging (Guarente, (I) 1999: Kaeberlein et al., 1999: Shore, 2000). The yeast Sir2 protein belongs to a family of histone deacetylases (reviewed in Guarente, 2000; Shore, 2000). The Sir2 homolog, CobB, in Salmonella typhimurium, functions as an NAD (nicotinamide adenine dinucleotide)-dependent ADP-ribosyl transferase 35 N (Tsang and Escalante-Semerena, 1998). The Sir2 protein is a class III deacetylase which uses NAD as a cosubstrate (Imai et al., 2000; Moazed, 2001; Smith et al., or a salt thereof, where: 2000; Tanner et al., 2000; Tanny and Moazed, 2001). Unlike Ring A is optionally Substituted, fused to another ring or other deacetylases, many of which are involved in gene 40 both; and silencing, Sir2 is insensitive to class I and II histone deacety Ring B is substituted with at least one carboxy, substituted lase inhibitors like trichostatin A (TSA) (Imai et al., 2000; or unsubstituted arylcarboxamine, substituted or unsubsti Landry et al., 2000a: Smith et al., 2000). tuted aralkylcarboxamine, substituted or unsubstituted het Deacetylation of acetyl-lysine by Sir2 is tightly coupled to eroaryl group, Substituted or unsubstituted heterocyclylcar NAD hydrolysis, producing nicotinamide and a novel acetyl 45 bonylethenyl, or polycyclic aryl group or is fused to an aryl ADP ribose compound (Tanner et al., 2000; Landry et al., ring and is optionally Substituted by one or more additional 2000b: Tanny and Moazed, 2001). The NAD-dependent groups. deacetylase activity of Sir2 is essential for its functions which In another aspect, the invention provides sirtuin-modulat can connect its biological role with cellular metabolism in ing compounds of Formula (II): yeast (Guarente, 2000: Imai et al., 2000; Lin et al., 2000; 50 Smith et al., 2000). Mammalian Sir2 homologs have NAD (II) dependent histone deacetylase activity (Imai et al., 2000; Smith et al., 2000). Most information about Sir2 mediated functions comes from the studies in yeast (Gartenberg, 2000; Gottschling, 2000). Biochemical studies have shown that Sir2 can readily 55 deacetylate the amino-terminal tails of histones H3 and H4, resulting in the formation of 1-O-acetyl-ADP-ribose and nicotinamide. Strains with additional copies of SIR2 display O OH increased rNA silencing and a 30% longer life span. It has recently been shown that additional copies of the C. elegans 60 or a salt thereof, where: SIR2 homolog, Sir-2.1, and the D. melanogaster dSir2 gene Ring A is optionally Substituted; greatly extend life span in those organisms. This implies that R. R. R. and Ra are independently selected from the the SIR2-dependent regulatory pathway foraging arose early group consisting of —H, halogen, —ORs, —CN, —CORs. in evolution and has been well conserved. Today, Sir2 genes –OCORs –OCORs –C(O)NRR - OC(O)NRR, are believed to have evolved to enhance an organisms health 65 C(O)Rs, —CORs, —SRs—OSOH, -S(O),Rs—S(O), and stress resistance to increase its chance of surviving adver ORs, S(O)NRR —NRR —NRC(O)OR —NRC S1ty. (O)R and—NO;
US 8,178,536 B2 11 12 -continued In one aspect, the invention provides sirtuin-modulating O compounds of Structural Formula (X):
N (X) O -NR) {\ NH-C-R1, 21 O o and 10 2 M R" is selected from an optionally substituted monocyclic s!N ( \ ié, or bicyclic aryl, or an optionally Substituted monocyclic or bicyclic heteroaryl, with the provisos that: or a salt thereof, wherein: when R is methyl, and R is NH-C(O)—, R is not 15 R" is selected from H or optionally substituted C-C, straight or branched alkyl, and R" is selected from an ooptionally substituted monocyclic N C heteroaryl, an optionally Substituted bicyclic heteroaryl, oran optionally substituted naphthyl, wherein R is not chloro O benzo(b)thienyl, unsubstituted benzodioxolyl, unsubstituted benzofuranyl, methyl-benzofuranyl, unsubstituted furanyl, phenyl-, bromo-, or nitro-furyl, chlorophenyl-isoxazolyl, oXobenzopyranyl, unsubstituted naphthyl, methoxy-, 1-methoxynaphthyl: 2-methoxynaphthyl, or unsubstituted methyl-, or halo-naphthyl, unsubstituted thienyl, unsubsti 2-thienyl: 25 tuted pyridinyl, or chloropyridinyl. when R is methyl, and R is NH-C(O)— In another aspect, the invention provides sirtuin-modulat CH-CH , R is not ing compounds of Structural Formula (XI):
30 (XI) N C /*R31 R22 O 35 C 2 o A. when R is methyl, and R is NH-C(O). CH-O-, s!!N? \ ^ R" is not unsubstituted naphthyl: 2-methoxy, 4-nitrophenyl: 4-chloro, 2-methylphenyl; or 4-t-butylphenyl; and or a salt thereof, wherein: 40 when R is NH C(O)—, R is not optionally substi R" is selected from H or optionally substituted C-C, tuted phenyl. straight or branched alkyl, In a further embodiment, the invention provides sirtuin R’ is selected NR’ C(O)—, NR' S(O) , modulating compounds of Structural Formula (IX): - NR' C(O) NR' , - NR' C(S)- NR' , 45 NR' C(S)- NR' CR'R' , NR, C(O) CR'R' NR' , - NR' C(=NR") NR' , (IX) C(O) NR' , C(O) NR' S(O) , NR' , O —CR'R' , – NR' C(O) CR'—CR' , —NR' S | NH-C-R30, (O) NR' , – NR' C(O) NR' S(O) = NR, 50 CR'R' C(O) NR' , CR'R' C(O) NR' , 21 O o - NR' C(O)—CR'—CR-CR'R' , - NR' C (—N CN) NR' , NR' C(O) CR'R' O , 2 M NR' C(O) CR'R' CR'R' O-, - NR' S(O), su? \ 5¢ CR'R' , NR' S(O), CR'R' CR'R' , 55 NR' C(O) CR'R' CR'R' , NR' C(S) NR, CR, 'R' CR'R', , NR, C(O) O O or a salt thereof, wherein: —NR, C(O)—CR'R' , wherein R is an optionally R" is selected from H or optionally substituted C-C, Substituted C-C straight or branched alkyl, and straight or branched alkyl, and R" is selected from an optionally substituted monocyclic R" is selected from 2,3-dimethoxyphenyl, phenoxyphe 60 or bicyclic aryl, or an optionally Substituted monocyclic or nyl, 2-methyl-3-methoxyphenyl, 2-methoxy-4-methylphe bicyclic heteroaryl, with the provisos that: nyl, or phenyl substituted with 1 to 3 substituents, wherein when R’ is NH-C(O)-CH=CH-, R is not unsub one of said Substituents is a solubilizing group; with the stituted furyl, 5-(2-methyl-3-chlorophenyl)-furanyl. 2,4- provisos that R' is not substituted simultaneously with a dichlorophenyl, 3,5-dichloro-2-methoxyphenyl, 3-nitrophe solubilizing group and a nitro group, and R' is not singly 65 nyl, 4-chlorophenyl, 4-chloro-3-nitrophenyl, Substituted at the 4-position with cyclic Solubilizing group or 4-isopropylphenyl, 4-methoxyphenyl, 2-methoxy-5-bro at the 2-position with a morpholino group. mophenyl, or unsubstituted phenyl: US 8,178,536 B2 13 when R’ is NH C(O) CH , R is not 3,4- dimethoxyphenyl, 4-chlorophenyl, or unsubstituted phenyl: when R’ is NH C(O) CH-O-, R is not 2,4- dimethyl-6-nitrophenyl, 2- or 4-nitrophenyl, 4-cyclohexy lphenyl, 4-methoxyphenyl, unsubstituted naphthyl, or unsub stituted phenyl, or phenyl monosubstituted, disubstituted or trisubstituted solely with substituents selected from straight or branched-chain alkyl or halo: when R’ is NH C(O)-CH(CH)-O-, R is not 2,4-dichlorophenyl, 4-chlorophenyl, or unsubstituted phenyl: 10 and when R’ is NH S(O) , R is not unsubstituted phe nyl. In yet another aspect, the invention provides sirtuin-modu 15 lating compounds of Structural Formula (XII):
(XII) Y R31 s XaX7 S 8 X-ke Kisu?X10 25 or a salt thereof, wherein: each of X7, Xs, X and Xois independently selected from N. CR, or CR", wherein: and R" is selected from an optionally substituted monocyclic each R' is independently selected from Hora solubilizing 30 group; or bicyclic aryl, or an optionally Substituted monocyclic or each R" is independently selected from H or optionally bicyclic heteroaryl, with the proviso that when R' is substituted C-C straight or branched alkyl; one of X7, Xs, X and X is N and the others are selected from CR' or CR.'; and Zero to one R is a solubilizing group: 35 R" is selected from:
40 A. Ally Zo. Z, Z, and Z are each CH, and R is —NHC(O)—, Z13, 27.11 Z132 Z11 R" is not an optionally substituted phenyl. Z2 s Z2 s In certain embodiments, the compounds of Structural For 45 mula (XI) have the following values: each of X, Xs, X and Xo is independently selected from N, CR'', or CR.", wherein: ^asZ A.ZN-1/ 16 NZ O 16 NZ s each R" is independently selected from Horasolubilizing O) (C). 50 group; wherein: each R" is independently selected from H or optionally each Zo. Z, Z and Z is independently selected from substituted C-C straight or branched alkyl; N, CR, or CR.'; and one of X, Xs, X and X is N and the others are selected each Z, Zs and Z is independently selected from N. 55 from CR' or CR.'; and NR', S, O, CR, or CR", wherein: zero to one R is a solubilizing group; Zero to two of Zo. Z, Z or Z are N: at least one of Z, Zs and Z is N, NR', O or S. R" is selected from: Zero to one of Z, Zs and Z is S or O. Zero to two of Z, Zs and Z are N or NR'; 60 Zero to one R is a solubilizing group: Zero to one R is an optionally Substituted C-C straight or branched alkyl; and Z10 R’ is selected from NR' C(O) = NR, S(O) , Sz S —NR' C(O) NR' , - NR' C(S)- NR' , 65 Z13, 27.11 Z132 Z11 NR' C(S)- NR' CR'R' , NR' C(O) Z2 s Z2 s CR'R' NR' , - NR' C(=NR) NR' ,
US 8,178,536 B2 19
s-s , or
\ 10 and and 15 each R" is independently selected from H or optionally each R" is independently selected from H or optionally Substituted C-C straight or branched alkyl; and Substituted C-C straight or branched alkyl, and R is an optionally substituted phenyl, wherein: R is selected from an optionally substituted bicyclic aryl, when R is NH C(O) , R is a substituted phenyl or an optionally Substituted monocyclic or bicyclic het other than phenyl singly substituted with halo, methyl, nitro eroaryl, wherein: or methoxy: 2-carboxyphenyl: 4-n-pentylphenyl: 4-ethox yphenyl: 2-carboxy-3-nitrophenyl: 2-chloro-4-nitrophenyl: when R is NH C(O) , R’ is not unsubstituted 2-fu 2-methoxy-5-ethylphenyl; 2,4-dimethoxyphenyl: 3,4,5-tri ryl, 2-(3-bromofuryl), unsubstituted 2-thienyl, unsubstituted methoxyphenyl; 2.4 dichlorophenyl; 2,6-difluorophenyl: 3.5- 3-pyridyl, unsubstituted 4-pyridyl, dinitrophenyl: or 3,4-dimethylphenyl: 25 when R’ is NR' C(O)—CR'R' or -NH C(O)— CH(CH)–O, R is a substituted phenyl: when R is NH-C(O)-CH, R is not unsubstituted phenyl, 4-methoxyphenyl: 3,4-dimethoxyphenyl or 4-chlo rophenyl: 30 when R is NH C(O)-CH, O, R is not 2,4-bis(1, 1-dimethylpropyl)phenyl: when R is NH C(O) NH-, R is not 4-methox yphenyl; and when R is NH-S(O) , R is a substituted phenyl and 35 other than 3-methylphenyl, 3-trifluoromethylphenyl, 2,4,5- when R is NR' S(O) , R is not unsubstituted or 2,4,6-trimethylphenyl, 2,4- or 3,4-dimethylphenyl, 2.5- or 2-thienyl or unsubstituted naphthyl. 3,4-dimethoxyphenyl, 2,5-dimethoxy-4-chlorophenyl, 3.6- In yet another aspect, the invention provides sirtuin-modu dimethoxy, 4-methylphenyl, 2.5- or 3,4-dichlorophenyl, 2.5- lating compounds of Structural Formula (XVI): diethoxyphenyl, 2-methyl -5-nitrophenyl, 2-ethoxy-5-bro 40 mophenyl, 2-methoxy-5-bromophenyl, 2-methoxy-3,4- dichlorophenyl, 2-methoxy-4-methyl-5-bromophenyl, 3.5- (XVI) dinitro-4-methylphenyl, 3-methyl-4-methoxyphenyl, R33, 3-nitro-4-methylphenyl, 3-methoxy-4-halophenyl, 3-meth oxy-5-chlorophenyl, 4-n-butoxyphenyl, 4-halophenyl, 4-eth k 45 ylphenyl, 4-methylphenyl, 4-nitrophenyl, 4-ethoxyphenyl, 21Ne.N. 4-acetylaminophenyl, 4-methoxyphenyl, 4-t-butylphenyl, or para-biphenyl. N-N / In a further ascept, the invention provides sirtuin-modulat 50 ing compounds of Structural Formula (XVII): or a salt thereof, wherein: R’ is selected from NR' C(O) = NR, S(O) , O (XVII) -NR' C(O) NR' , - NR' C(S)-NR' , 55 R24 5-( NR' C(S)- NR' CR'R' , NR' C(O) 23 CR'R' NR' , - NR' C(=NR) NR' , R 21 2N. R29, C(O) NR' , —C(O)—NR' S(O)— —NR' . —CR'R' , – NR' C(O) CR'—CR' , – NR' S (O) NR' , – NR' C(O) NR' S(O) = NR, N-N/ CR'R' C(O) NR' , CR'R' C(O) NR' , 60 -NR' C(O) CR'—CR' CR'R' , -NR' C or a salt thereof, wherein: each of R and R is independently selected from Hor —CH, wherein at least one of RandR is H; and R’ is phenyl substituted with: 65 a) two —O-CH groups; b) three —O-CH groups located at the 2.3 and 4 posi tions; or US 8,178,536 B2 21 c) one —N(CH) group; and; d) when R is CH, one —O-CH group at the 2 or 3 position, wherein R’ is optionally additionally substituted with a solubilizing group. In one aspect, the invention provides sirtuin-modulating s-> compounds of Structural Formula (XVIII):
(XVIII) R31 R31 10 )- S 2N -NR
wherein each R" is independently selected from H or option & X 15 ally substituted C-C straight or branched alkyl, and R" is selected from an optionally substituted monocyclic or a salt thereof, wherein or bicyclic aryl, oran optionally Substituted monocyclic R" is selected from: or bicyclic heteroaryl, with the proviso that when R' is A-X A. al-X. 25 Z132 Z11 Z132 Z11 Z132 Z11 Z12 s Z12 s Z12 s Zo. Z, Z and Z are each CH, R” is H, and R is NHC(O)—, R is not an optionally substituted phe ^asO A.O)4 30 nyl. Z16 ZN1/ In another aspect, the invention provides sirtuin-modulat Z15 O Z15 s ing compounds of Structural Formula (XX): wherein: each Zo. Z, Z and Z is independently selected from 35 (XX) N, CR, or CR.'; and each Z, Zs and Z is independently selected from N. NR', S, O, CR, or CR.", wherein: 40 Zero to two of Zo. Z, Z or Z are N: or a salt thereof, wherein at least one of Z, Zs and Z is N, NR', S or O. R" is selected from:
Zero to one of Za Zs and Z is S or O. 45 Zero to two of Z, Zs and Z are N or NR'; Zero to one R is a solubilizing group; and Zero to one R' is an optionally substituted C-C straight or branched alkyl: Z13, a Z11 each R' is independently selected from Hora solubilizing 50 Z12 O ZióSA. group; R’ is selected from NR' C(O) = NR, S(O) , wherein: -NR' C(O) NR' , - NR' C(S)-NR' , each Zo. Z, Z and Z is independently selected from NR' C(S)- NR' CR'R' , NR' C(O) 55 N, CR, or CR.'; and CR'R' NR' , - NR' C(=NR) NR' , each Z, Zs and Z is independently selected from N. C(O) NR' , C(O) NR' S(O) , NR' , NR', S, O, CR, or CR', —CR'R' , – NR' C(O) CR'—CR' , – NR' S wherein: (O) NR' , – NR' C(O) NR' S(O) = NR, Zero to two of Zo. Z, Z or Z are N: CR'R' C(O) NR' , CR'R' C(O) NR' , 60 at least one of Za Zs and Z is N, NR', O or S. -NR' C(O) CR'—CR' CR'R' , -NR' C Zero to one of Z, Zs and Z is S or O. (—N CN) NR' , NR' C(O) CR'R' O , Zero to two of Z, Zs and Z are N or NR'; NR' C(O) CR'R' CR'R' O-, - NR S(O), Zero to one R is a solubilizing group; and CR'R' , NR' S(O). CR'R' CR'R' , Zero to one R is an optionally Substituted C-C straight or NR' C(O) CR'R' ; NR' C(O) CR'R' 65 branched alkyl: CR'R' , NR' C(S) NR' CR'R' CR'R' , each R' is independently selected from Hora solubilizing group;
US 8,178,536 B2
10 -NR)- V R" is selected from an optionally substituted monocyclic wherein each R" is independently selected from H or option or bicyclic aryl, or an optionally Substituted monocyclic or ally substituted C-C straight or branched alkyl; and bicyclic heteroaryl, with the provisos that: R is an optionally substituted phenyl, wherein: 15 when R is NH C(O) , R is not is not 3,5-dinitro when R is NR' C(O) , R." is not H: phenyl, 4-butoxyphenyl, when R is NH C(O)-CH, or -NH C(O)— CH. O. , R is not unsubstituted phenyl or 4-halophenyl: F. F and e when R is NH S(O) , R is not unsubstituted phe N F, nyl, 2,4- or 3,4-dimethylphenyl, 2,4-dimethyl-5-methox N / yphenyl, 2-methoxy-3,4-dichlorophenyl, 2-methoxy, 5-bro N mophenyl-3,4-dioxyethylenephenyl, 3,4-dimethoxyphenyl, 3,4-dichlorophenyl, 3,4-dimethylphenyl, 3- or 4-methylphe nyl, 4-alkoxyphenyl, 4-phenoxyphenyl, 4-halophenyl, 4-bi 25 phenyl, or 4-acetylaminophenyl. In one aspect, the invention provides sirtuin-modulating , or compounds of Structural Formula (XXII): (XXII) 30 R33 V R21, S N 2N N 35 & when R is NH C(O) and each of R, R2, R', or a salt thereof wherein: R" and R." is hydrogen, R is not R’ is selected from NH C(O) , or - NH C(O) CH2—, and O R is phenyl substituted with 40 a) one —N(CH) group; b) one CN group at the 3 position; c) one —S(CH) group; or Y-,N-NH - d) 45 unsubstituted phenyl, 2- or 4-nitrophenyl, 2,4-dinitrophenyl, 2- or 4-chlorophenyl, 2-bromophenyl, 4-fluorophenyl, 2,4- dichlorophenyl, 2-carboxyphenyl, 2-azidophenyl, 2- or .C. O A-l.Cl 4-aminophenyl, 2-acetamidophenyl, 4-methylphenyl, or bridging the 3 and 4 positions. 50 4-methoxyphenyl: In another aspect, the invention provides sirtuin-modulat when R is NH CO) , R." is methyl; and each of ing compounds of Structural Formula (XXIII): R. R', Rand R." is hydrogen, R is not 2-methylami nophenyl, (XXIII) R31 55 M s O R" R. Os N OnNY N R'-- 7 60 Nes?R20a \/iii. R when R’ is NH C(O)-CH or NH C(S)- or a salt thereof, wherein: NH , and each of R', R', R., R." and R." is hydrogen, each R and R' is independently selected from H or a R" is not unsubstituted phenyl: solubilizing group; 65 when R is NH S(O) , R." is hydrogen or methyl, each R', R." and R." is independently selected from Hor and each of R. R. R." and R." is hydrogen, R is not optionally Substituted C-C straight or branched alkyl; 4-methylphenyl; and US 8,178,536 B2 27 28 when R is -NH S(O) , R' is hydrogen or or a salt thereof, wherein: —CH N(CHCH), and each of R. R. R." and R." is each R and R' is independently selected from Hora hydrogen, R is not solubilizing group; each R', R" and R." is independently selected from Hor optionally substituted C-C straight or branched alkyl: Y-O- O Y-O- 10 In a particular aspect, the invention provides sirtuin-modu 15 lating compounds of Structural Formula (XXIII): (XXIII) R" is selected from an optionally substituted monocyclic R31 or bicyclic aryl, or an optionally Substituted monocyclic or M bicyclic heteroaryl, with the provisos that: R" R21 when R is NH-C(O)-CH , R is not 2-meth ylphenyl, or 3,4-dimethoxyphenyl: when R is NH-C(O)-CH=CH-, R is not R'-- 7 2-chlorophenyl: 25 Nes?R20a \/iii. when R is NH C(O) NH-, R is not unsubsti R tuted benzimidazolyl: when R is NH S(O) , and each of R. R', R', or a salt thereof, wherein: R" and R." is hydrogen, R is not unsubstituted phenyl, each R' and R' is independently selected from H or a 4-chlorophenyl, 4-methylphenyl, or 4-acetoamidophenyl: solubilizing group; 30 when R is NH S(O) , each of R and R." is each R', R." and R" is independently selected from Hor methyl or hydrogen, and each of R. R', and R." is hydro optionally Substituted C-C straight or branched alkyl; gen, R is not 4-nitrophenyl: when R is NH C(O) CH-O-, R." is methyl or hydrogen, and each of R', R', R', and R." is hydrogen, 35 R" is not 2.3-, 2.5-, 2,6-, 3,4- or 3,5-dimethylphenyl, 2.4- dichloromethyl, 2,4-dimethyl-6-bromophenyl, 2- or 4-chlo rophenyl, 2-(1-methylpropyl)phenyl, 5-methyl-2-(1-methyl ethyl)phenyl, 2- or 4-methylphenyl, 2,4-dichloro-6- 40 methylphenyl, nitrophenyl, 2,4-dimethyl-6-nitrophenyl, 2- or 4-methoxyphenyl, 4-acetyl-2-methoxyphenyl, 4-chloro-3,5- dimethylphenyl, 3-ethylphenyl, 4-bromophenyl, 4-cyclohex yphenyl, 4-(1-methylpropyl)phenyl, 4-(1-methylethyl)phe nyl, 4-(1,1-dimethylethyl)phenyl, or unsubstituted phenyl: R" is selected from an optionally substituted monocyclic 45 when R is NH-C(O)—CH2—, R." is methyl or or bicyclic aryl, or an optionally Substituted monocyclic or hydrogen, and each of R. R', R', and R." is hydrogen, bicyclic heteroaryl, R" is not unsubstituted naphthyl, 4-chlorophenyl, 4-nitro wherein: phenyl, 4-methoxyphenyl, unsubstituted phenyl, unsubsti i) at least one R is a solubilizing group or at least one R." is tuted thienyl an optionally Substituted C-C straight or branched alkyl 50 or both; or ii) R' is a solubilizing group other than CH N(CH N O CH). In yet another aspect, the invention provides sirtuin-modu 55 A. sy" asy/ lating compounds of Structural Formula (XXIV): R31 (XXIV) V when R is NH CO)—CH2—, R is methyl, and R it R21 60 each of R', R', R", and R." is hydrogen, R is not A unsubstituted phenyl: when R is NH-C(O)-CH=CH, R." is methyl or R'-- hydrogen, and each of R. R', R', and R." is hydrogen, O)-(s" R" is not unsubstituted furyl, nitrophenyl-substituted furyl, R20a R iii. 65 2,4-dichlorophenyl, 3,5-dichloro-2-methoxyphenyl, 3- or 4-nitrophenyl, 4-methoxyphenyl, unsubstituted phenyl, or nitro-substituted thienyl: US 8,178,536 B2 29 30 when R is NH-C(O)-CH(CHCH)—, and each of In one aspect, the invention provides sirtuin-modulating R. R. R. R.", and R." is hydrogen, R is not unsub compounds of Structural Formula (XXVI): stituted phenyl: when R is NH C(O)-CH(CH) O-, R." is methyl or hydrogen, and each of R. R', R', and R." is O (XXVI) hydrogen, R is not 2,4-dichlorophenyl. In a particular aspect, the invention provides sirtuin-modu R" ins, lating compounds of Structural Formula (XXIV): W 10 R'--O) ( )." R20a
15 or a salt thereof, wherein: each R and R' is independently selected from Hora solubilizing group; each R', R" and R." is independently selected from Hor optionally Substituted C-C straight or branched alkyl, and R is selected from an optionally substituted heteroaryl or or a salt thereof, wherein: an optionally substituted bicyclic aryl, with the provisos that: each R and R' is independently selected from H or a when each of R and R." is hydrogen or methyl and each solubilizing group and at least one of R and R' is a solu 25 of R", Ro and Ro, is hydrogen, R is not 5,6,7,8-tetrahy bilizing group; dronaphthyl, unsubstituted benzofuryl, unsubstituted ben each R', R." and R" is independently selected from Hor Zothiazolyl, chloro- or nitro-substituted benzothienyl, unsub optionally substituted C-C straight or branched alkyl: stituted furyl, phenyl-, bromo- or nitro-substituted furyl, dimethyl-substituted isoxazolyl, unsubstituted naphthyl, 30 5-bromonaphthyl, 4-methylnaphthyl, 1- or 3-methoxynaph thyl, azo-substituted naphthyl, unsubstituted pyrazinyl, S-methyl-substituted pyridyl, unsubstituted pyridyl, thienyl or phenyl-substituted quinolinyl, chloro-, bromo- or nitro substituted thienyl, unsubstituted thienyl, or 35 4N1 optionally Substituted C-C straight or branched alkyl, and 40 R" is selected from an optionally substituted monocyclic ON or bicyclic aryl, or an optionally Substituted monocyclic or bicyclic heteroaryl. In a particular aspect, the invention provides sirtuin-modu In a further aspect, the invention provides sirtuin-modulat 45 ing compounds of Structural Formula (XXV): lating compounds of Structural Formula (XXVI):
(XXVI) (XXV) O 50 R" ins, M
R'-- R'-- NMN \ y 55 N-xO) / \{ 7 R20a
or a salt thereof, wherein: or a salt thereof, wherein: 60 each R and R' is independently selected from Hora each R' and R' is independently selected from H or a solubilizing group, wherein at least one of R' or R' is a solubilizing group, wherein at least one of R'' and R' is a solubilizing group; solubilizing group; each R', R" and R." is independently selected from Hor each R', R." and R" is independently selected from Hor 65 optionally substituted C-C straight or branched alkyl; and optionally Substituted C-C straight or branched alkyl, and R is selected from an optionally substituted heteroaryl or R is an optionally substituted phenyl. an optionally substituted bicyclic aryl. US 8,178,536 B2 31 32 In another aspect, the invention provides sirtuin-modulat provided that when R is -NH C(O)—and R' is ing compounds of Structural Formula (XXVII):
(XXVII) 5 J. R31 21 N AR21 R'-- X- R19 lsSás N R20a 10 R" is not unsubstituted pyridyl, 2,6-dimethoxyphenyl, 3.4. 5-trimethoxyphenyl or unsubstituted furyl. In a particular aspect, the invention provides sirtuin-modu or a salt thereof, wherein: lating compounds of Structural Formula (XXVII): each R and R' is independently selected from H or a Solubilizing group; 15 each R" and R." is independently selected from H or (XXVII) optionally Substituted C-C straight or branched alkyl, , R31 21 N AR21 R" is selected from: R'-- X- R19 lsSás N R20a Z10 Sz, saS or a salt thereof, wherein: 25 each R and R' is independently selected from Hora Z13, 2 Z11: Z13, 24.11: Solubilizing group; Z2 Z2 each R" and R." is independently selected from H or optionally Substituted C-C straight or branched alkyl, R" is selected from: ^esO) or Oz. 30 ZS Z15 ZN17Z15 Z10 Sz sa wherein: 35 each Zo. Z, Z and Z is independently selected from Z132 Z11: Z13, 24.11: N, CR, or CR.'; and Z2 Z2 each Z, Zs and Z is independently selected from N. NR', S, O, CR, or CR", wherein: 40 Zero to two of Zo. Z, Z or Z are N: ^rsO) or OZ. at least one of Z, Zs and Z is N, NR', S or O. Zis, 15 ZS215 Zero to one of Z, Zs and Z is S or O. 45 wherein: Zero to two of Z, Zs and Z are N or NR'; each Zo. Z, Z and Z is independently selected from zero to one R is a solubilizing group; N, CR, or CR.'; and Zero to one R is an optionally Substituted C-C straight or each Z, Zs and Z is independently selected from N. branched alkyl; and NR', S, O, CR, or CR", wherein: 50 Zero to two of Zo. Z, Z or Z are N: R’ is selected from NR' C(O) = NR, S(O) , at least one of Za Zs and Z is N, NR', S or O; NR' C(O) NR' , NR' C(S)- NR' , Zero to one of Z, Zs and Z is S or O; NR' C(S) NR' CR'R' , – NR' C(O) Zero to two of Z, Zs and Z are N or NR'; CR'R' NR' , - NR' C(=NR) NR' , zero to one R is a solubilizing group; —C(O) NR' , —C(O) NR S(O) , 55 Zero to one R is an optionally Substituted C-C straight or NR' , CR'R' , NR' C(O) branched alkyl; and CR'—CR, ' , NR' S(O), NR' , NR' C R’ is selected from NR' C(O) = NR, S(O) , (O) NR' S(O) , NR' CR'R' C(O) - NR' C(O) NR' , - NR' C(S)- NR' , NR' , —CR'R' C(O) NR' NR' C(O) NR' C(S) NR' CR'R' , NR' C(O) CR'—CR, CR 'R' , NR, C(=N CN) 60 CR'R' NR' , - NR' C(=NR") NR' , NR' , – NR' C(O) CR'R' O. , – NR' C C(O) NR' , C(O) NR' S(O) , NR' , (O) CR'R' CR'R' O , NR' S(O), —CR'R' , – NR' C(O) CR'—CR' NR' S CR'R' , NR' S(O). CR'R' CR'R' , or (O). NR = NR, C(O) NR' S(O), , – NR CR'R' C(O) NR' , CR'R' C(O) NR' , R" is selected from an optionally substituted monocyclic 65 - NR' C(O) CR'—CR' CR'R' , - NR' C or bicyclic aryl, or an optionally Substituted monocyclic or (—N CN) NR' , NR' C(O) CR'R'-O-, bicyclic heteroaryl, NR' C(O) CR'R' CR'R' O-, - NR' S(O), US 8,178,536 B2 33 34 CR'R' , NR' S(O), CR'R' CR'R' , or when R is NH C(O) and R' is thiazolyl or pyri —NR' C(O)—CR'R' ; and midinyl, R' is not unsubstituted phenyl: R" is selected from an optionally substituted monocyclic when R is NH C(O) and R' is or bicyclic aryl, or an optionally Substituted monocyclic or bicyclic heteroaryl, with the provisos that: when R is NH-C(O) , R' is not pyrazolyl: when R’ is NH , and R' is thiazolyl, R' is not optionally substituted phenyl or optionally substituted pyridyl; when R is NH CO)—CH2—, and R' is pyrazolyl, 10 R" is not unsubstituted indolyl or unsubstituted phenyl: when R is NH C(O)-CH , and R' is R" is not unsubstituted pyridyl, unsubstituted thienyl, unsub stituted phenyl, 2-methylphenyl, 4-fluorophenyl, 4-methox 15 yphenyl, 4-methylphenyl, 3,4-dioxyethylenephenyl, 3-acety lamino-4-methylphenyl, 3-(6-amino-1-oxohexyl)amino-4- methylphenyl, 3-amino-4-methylphenyl, 2,6- dimethoxyphenyl, 3,5-dimethoxyphenyl, 3-halo -4-methoxyphenyl, 3-nitro-4-methylphenyl, 4-propoxyphe nyl, 3,4,5-trimethoxyphenyl or unsubstituted furyl; R" is not 2-methylphenyl or 3,4-dimethoxyphenyl: when R is NH C(O) and R' is when R is NH C(O)-CH=CH-, and R' is 25
30
R" is not 3,5-dinitrophenyl, 4-butoxyphenyl, R" is not 2-chlorophenyl: when R is NH-C(O) NH-, and R' is pyrazolyl, 35 R" is not unsubstituted isoxazolyl, unsubstituted naphthyl, F F unsubstituted phenyl, 2,6-difluorophenyl, 2,5-dimethylphe nyl, 3,4-dichlorophenyl, or 4-chlorophenyl; F when R is NH C(O) NH , and R' is N -N s 40 CC
/ \ s O 45
R" is not unsubstituted benzimidazolyl: when R is NH-, and R' is pyrazolyl, R is not 50 unsubstituted pyridyl: when R' is a solubilizing group, R' is 1-methylpyrrolyl and R is NH C(O) , R is not unsubstituted phenyl, unsubstituted furyl, unsubstituted pyrrolyl, unsubstituted In a more particular embodiment, the invention provides pyrazolyl, unsubstituted isoquinolinyl, unsubstituted ben 55 sirtuin-modulating compounds of Structural Formula (XX Zothienyl, chloro-substituted benzothienyl, 2-fluoro-4-chlo rophenyl or phenyl singly Substituted with a solubilizing VII): group; when R' is a solubilizing group, R is thienyl and R is (XXVII) —NH C(O) , R is not unsubstituted phenyl: 60 when R' is a solubilizing group, R is methylimidazolyl ,, R31 and R is NH-C(O) , R is not 1-methyl-4-(1,1-dim 21 N R ethylethyloxycarbonylamino)pyrrol-2-yl or phenyl singly R M R19 Substituted with a solubilizing group; lyS. N when R is NH- and R' is pyridyl, oxadiazolyl or 65 R20a thiadiazolyl, R is not unsubstituted phenyl, 3-methoxyphe nyl or 4-methoxyphenyl: US 8,178,536 B2 35 36 or a salt thereof, wherein: when R is NH C(O) and R' is thiazolyl or pyri each R and R' is independently selected from H or a midinyl, R' is not unsubstituted phenyl. solubilizing group; In yet another aspect, the invention provides compounds of each R" and R." is independently selected from H or Structural Formula (XXVIII): optionally substituted C-C straight or branched alkyl: 5 R" is selected from: (XXVIII) R" M / 21 N R21 10 Z10 X-ré "'s N s A. X R20a Z13,2711:Z2 Z13,241.1;Z2 15 or a salt thereof, wherein: each R and R' is independently selected from Hora solubilizing group; ^rs( ) O ( )Z4. each R" and R." is independently selected from H or ZS Z15 ZS Z15/ optionally Substituted C-C straight or branched alkyl; R’ is selected from: wherein: each Zo. Z, Z and Z is independently selected from N, CR, or CR.'; and each Z, Zs and Z is independently selected from N. 25 NR', S, O, CR, or CR", wherein: All A-X one to two of Zo. Z11, Z12 or Z are N: Z132 Z11 Z1327.11 at least one of Z, Zs and Z is N, NR', S or O. Z12 O Z2 s Zero to one of Z, Zs and Z is S or O. Zero to two of Z, Zs and Z are N or NR'; 30 wherein: zero to one R is a solubilizing group; each Zo. Z, Z and Z is independently selected from Zero to one R" is an optionally substituted C-C straight N,CR, or CR.", wherein one of Zo. ZZ or Z is N; and or branched alkyl; and zero to one R is a solubilizing group; R’ is selected from NR' C(O) = NR, S(O) , Zero to one R" is an optionally Substituted C-C straight -NR' C(O) NR' , —NR' C(S)- NR' , 35 or branched alkyl; and NR' C(S)- NR' CR'R' , NR' C(O) R’ is selected from NR' C(O) = NR, S(O) , CR'R' NR' , - NR' C(=NR) NR' , NR' C(O) NR' , - NR' C(S)- NR' , —NR' C(O) CR'—CR, ' , NR' S(O), NR' , NR' C(S) NR' CR'R' , NR' C(O) NR' C(O) NR' S(O) , NR' CR'R' C CR'R' NR' , - NR' C(=NR") NR' , (O) NR' , -NR' C(O)—CR'—CR' CR'R' , 40 – NR' C(O) CR'—CR' , NR' S(O), NR' , NR' C(=N CN) NR' , NR' C(O) NR' C(O) NR' S(O) , NR' CR'R' C CR'R'-O-, -NR' C(O)—CR'R' CR'R' O-, (O) NR' , -NR' C(O)—CR'—CR' CR'R' , NR' S(O). CR'R' , NR' S(O). CR'R'' NR' C(=N CN) NR' , - NR' C(O)– CR'R' , or - NR' C(O) CR'R' ; and CR'R'-O-, -NR' C(O) CR'R' CR'R' O , R" is selected from an optionally substituted monocyclic 45 - NR' S(O). CR'R' , NR' S(O). CR'R'' or bicyclic aryl, or an optionally Substituted monocyclic or CR'R' , or - NR' C(O) CR'R' ; and bicyclic heteroaryl, with the provisos that: R" is selected from an optionally substituted monocyclic when R is NH-C(O) , R' is not pyrazolyl: or bicyclic aryl, or an optionally Substituted monocyclic or when R is NH CO)—CH2—, and R' is pyrazolyl, bicyclic heteroaryl. R" is not unsubstituted indolyl or unsubstituted phenyl: 50 Also provided are pharmaceutical compositions compris when R is NH-C(O) NH-, and R' is pyrazolyl, ing one or more compounds of Formulas (I)-(XXVIII) or a R" is not unsubstituted isoxazolyl, unsubstituted naphthyl, salt, prodrug or metabolite thereof. unsubstituted phenyl, 2,6-difluorophenyl; 2.5-dimethylphe- In another aspect, the invention provides methods for using nyl: 3,4-dichlorophenyl; or 4-chlorophenyl: sirtuin-modulating compounds, or compostions comprising when R" is a solubilizing group, R' is 1-methylpyrrolyl 55 sirtuin-modulating compounds. In certain embodiments, sir and R is NH CO) , R is not unsubstituted phenyl: tuin-modulating compounds that increase the level and/or unsubstituted furyl; unsubstituted pyrrolyl; unsubstituted activity of a sirtuin protein may be used for a variety of pyrazolyl; unsubstituted isoquinolinyl; unsubstituted ben- therapeutic applications including, for example, increasing Zothienyl: chloro-substituted benzothienyl: 2-fluoro-4-chlo- the lifespan of a cell, and treating and/or preventing a wide rophenyl or phenyl singly Substituted with a solubilizing 60 variety of diseases and disorders including, for example, dis group; eases or disorders related to aging or stress, diabetes, obesity, when R” is a solubilizing group, R' is thienyland R is neurodegenerative diseases, chemotherapeutic induced neu —NH CO) , R is not unsubstituted phenyl: ropathy, neuropathy associated with an ischemic event, ocu when R' is a solubilizing group, R' is methylimidazolyl lar diseases and/or disorders, cardiovascular disease, blood and R is NH-C(O) , R is not 1-methyl-4-(1,1-dim- 65 clotting disorders, inflammation, and/or flushing, etc. Sirtuin ethylethyloxycarbonylamino)pyrrol-2-yl or phenyl singly modulating compounds that increase the level and/or activity Substituted with a solubilizing group; and of a sirtuin protein may also be used for treating a disease or US 8,178,536 B2 37 38 disorder in a subject that would benefit from increased mito Accession No. NP 036370, which are encoded by nucle chondrial activity, for enhancing muscle performance, for otides 777 to 1532 of GenBank Accession No. NM 012238; increasing muscle ATP levels, or for treating or preventing or about amino acids 254 to 495 of GenBank Accession No. muscle tissue damage associated with hypoxiaorischemia. In NP 036370, which are encoded by nucleotides 813 to 1538 other embodiments, sirtuin-modulating compounds that of GenBank Accession No. NM 012238. decrease the level and/or activity of a sirtuin protein may be The term “companion animals' refers to cats and dogs. As used for a variety of therapeutic applications including, for used herein, the term “dog(s) denotes any member of the example, increasing cellular sensitivity to stress, increasing species Canis familiaris, of which there are a large number of apoptosis, treatment of cancer, stimulation of appetite, and/or different breeds. The term “cat(s) refers to a feline animal stimulation of weight gain, etc. As described further below, 10 including domestic cats and other members of the family the methods comprise administering to a Subject in need Felidae, genus Felis. thereof a pharmaceutically effective amount of a sirtuin The terms “comprise' and “comprising are used in the modulating compound. inclusive, open sense, meaning that additional elements may In certain aspects, the sirtuin-modulating compounds may be included. be administered alone or in combination with other com 15 pounds, including other sirtuin-modulating compounds, or The term “conserved residue' refers to an amino acid that other therapeutic agents. is a member of a group of amino acids having certain common properties. The term “conservative amino acid substitution BRIEF DESCRIPTION OF THE FIGURES refers to the substitution (conceptually or otherwise) of an amino acid from one such group with a different amino acid FIG. 1 shows a schematic of the Cellular ATP Assay from the same group. A functional way to define common described in Example 5. properties between individual amino acids is to analyze the FIG. 2 shows a dose-response curve for ATP levels in cells normalized frequencies of amino acid changes between cor following resveratrol treatment. responding proteins of homologous organisms (Schulz, G. E. 25 and R. H. Schirmer. Principles of Protein Structure, DETAILED DESCRIPTION Springer-Verlag). According to Such analyses, groups of amino acids may be defined where amino acids within a group 1. Definitions exchange preferentially with each other, and therefore As used herein, the following terms and phrases shall have resemble each other most in their impact on the overall pro the meanings set forth below. Unless defined otherwise, all 30 tein structure (Schulz, G. E. and R. H. Schirmer, Principles of technical and Scientific terms used herein have the same Protein Structure, Springer-Verlag). One example of a set of meaning as commonly understood to one of ordinary skill in amino acid groups defined in this manner include: (i) a the art. charged group, consisting of Glu and Asp, Lys, Arg and His, The singular forms “a,” “an and “the include plural (ii) a positively-charged group, consisting of Lys, Arg and reference unless the context clearly dictates otherwise. 35 The term "agent' is used herein to denote a chemical com His, (iii) a negatively-charged group, consisting of Glu and pound, a mixture of chemical compounds, a biological mac Asp, (iv) an aromatic group, consisting of Phe, Tyr and Trp, romolecule (such as a nucleic acid, an antibody, a protein or (v) a nitrogen ring group, consisting of His and Trp. (vi) a portion thereof, e.g., a peptide), or an extract made from large aliphatic nonpolar group, consisting of Val, Leu and Ile, biological materials such as bacteria, plants, fungi, or animal 40 (vii) a slightly-polar group, consisting of Met and Cys, (viii) (particularly mammalian) cells or tissues. The activity of Such a small-residue group, consisting of Ser, Thr, Asp, ASn, Gly, agents may render it Suitable as a “therapeutic agent' which is Ala, Glu, Gln and Pro, (ix) an aliphatic group consisting of a biologically, physiologically, or pharmacologically active Val, Leu, Ile, Met and Cys, and (X) a small hydroxyl group Substance (or Substances) that acts locally or systemically in consisting of Ser and Thr. a subject. 45 “Diabetes’ refers to high blood sugar or ketoacidosis, as The term “bioavailable' when referring to a compound is well as chronic, general metabolic abnormalities arising from art-recognized and refers to a form of a compound that allows a prolonged high blood Sugar status or a decrease in glucose for it, or a portion of the amount of compound administered, tolerance. “Diabetes’ encompasses both the type I and type II to be absorbed by, incorporated to, or otherwise physiologi (Non Insulin Dependent Diabetes Mellitus or NIDDM) forms cally available to a subject or patient to whom it is adminis 50 of the disease. The risk factors for diabetes include the fol tered. lowing factors: waistline of more than 40 inches for men or 35 “Biologically active portion of a sirtuin” refers to a portion inches for women, blood pressure of 130/85 mmHg or higher, of a sirtuin protein having a biological activity, such as the triglycerides above 150 mg/dl., fasting blood glucose greater ability to deacetylate. Biologically active portions of a sirtuin than 100 mg/dl or high-density lipoprotein of less than 40 may comprise the core domain of sirtuins. Biologically active 55 mg/dl in men or 50 mg/dl in women. portions of SIRT1 having GenBank Accession No. A "direct activator of a sirtuin is a molecule that activates NP 036370 that encompass the NAD+ binding domain and a sirtuin by binding to it. A “direct inhibitor of a sirtuin is a the Substrate binding domain, for example, may include with molecule inhibits a sirtuin by binding to it. out limitation, amino acids 62-293 of GenBank Accession The term “EDs is art-recognized. In certain embodi No. NP 036370, which are encoded by nucleotides 237 to 60 ments, EDso means the dose of a drug which produces 50% of 932 of GenBankAccession No. NM 012238. Therefore, this its maximum response or effect, or alternatively, the dose region is sometimes referred to as the core domain. Other which produces a pre-determined response in 50% of test biologically active portions of SIRT1, also sometimes Subjects or preparations. The term “LDso is art-recognized. referred to as core domains, include about amino acids 261 to In certain embodiments, LDso means the dose of a drug which 447 of GenBank Accession No. NP 036370, which are 65 is lethal in 50% of test subjects. The term “therapeutic index' encoded by nucleotides 834 to 1394 of GenBank Accession is an art-recognized term which refers to the therapeutic index No. NM 012238; about amino acids 242 to 493 of GenBank of a drug, defined as LDso/EDso. US 8,178,536 B2 39 40 The term “hyperinsulinemia' refers to a state in an indi “Obese individuals or individuals suffering from obesity vidual in which the level of insulin in the blood is higher than are generally individuals having a body mass index (BMI) of normal. at least 25 or greater. Obesity may or may not be associated The term “including is used to mean “including but not with insulin resistance. limited to”. “Including and “including but not limited to are The terms “parenteral administration” and “administered used interchangeably. parenterally are art-recognized and refer to modes of admin The term “insulin resistance” refers to a state in which a istration other than enteral and topical administration, usually normal amount of insulin produces a Subnormal biologic by injection, and includes, without limitation, intravenous, response relative to the biological response in a Subject that intramuscular, intraarterial, intrathecal, intracapsular, does not have insulin resistance. 10 intraorbital, intracardiac, intradermal, intraperitoneal, tran An “insulin resistance disorder, as discussed herein, refers stracheal, Subcutaneous, Subcuticular, intra-articulare, Sub to any disease or condition that is caused by or contributed to capsular, Subarachnoid, intraspinal, and intrasternal injection by insulin resistance. Examples include: diabetes, obesity, and infusion. metabolic syndrome, insulin-resistance syndromes, Syn A “patient”, “subject”, “individual” or “host” refers to drome X, insulin resistance, high blood pressure, hyperten 15 either a human or a non-human animal. Sion, high blood cholesterol, dyslipidemia, hyperlipidemia, The term “percent identical refers to sequence identity dyslipidemia, atherosclerotic disease including stroke, coro between two amino acid sequences or between two nucle nary artery disease or myocardial infarction, hyperglycemia, otide sequences. Identity can each be determined by compar hyperinsulinemia and/or hyperproinsulinemia, impaired glu ing a position in each sequence which may be aligned for cose tolerance, delayed insulin release, diabetic complica purposes of comparison. When an equivalent position in the tions, including coronary heart disease, angina pectoris, con compared sequences is occupied by the same base or amino gestive heart failure, stroke, cognitive functions in dementia, acid, then the molecules are identical at that position; when retinopathy, peripheral neuropathy, nephropathy, glomerulo the equivalent site occupied by the same or a similar amino nephritis, glomerulosclerosis, nephrotic syndrome, hyperten acid residue (e.g., similar in steric and/or electronic nature), sive nephrosclerosis some types of cancer (such as endome 25 then the molecules can be referred to as homologous (similar) trial, breast, prostate, and colon), complications of pregnancy, at that position. Expression as a percentage of homology, poor female reproductive health (such as menstrual irregu similarity, or identity refers to a function of the number of larities, infertility, irregular ovulation, polycystic ovarian identical or similar amino acids at positions shared by the syndrome (PCOS)), lipodystrophy, cholesterol related disor compared sequences. Expression as a percentage of homol ders, such as gallstones, cholescystitis and cholelithiasis, 30 ogy, similarity, or identity refers to a function of the number gout, obstructive sleep apnea and respiratory problems, of identical or similar amino acids at positions shared by the osteoarthritis, and prevention and treatment of bone loss, e.g. compared sequences. Various alignment algorithms and/or osteoporosis. programs may be used, including FASTA, BLAST, or The term “livestock animals' refers to domesticated quad ENTREZ. FASTA and BLAST are available as a part of the rupeds, which includes those being raised for meat and Vari 35 GCG sequence analysis package (University of Wisconsin, ous byproducts, e.g., a bovine animal including cattle and Madison, Wis.), and can be used with, e.g., default settings. other members of the genus Bos, a porcine animal including ENTREZ is available through the National Center for Bio domestic Swine and other members of the genus Sus, an ovine technology Information, National Library of Medicine, animal including sheep and other members of the genus Ovis, National Institutes of Health, Bethesda, Md. In one embodi domestic goats and other members of the genus Capra. 40 ment, the percent identity of two sequences can be deter domesticated quadrupeds being raised for specialized tasks mined by the GCG program with a gap weight of 1, e.g., each Such as use as a beast of burden, e.g., an equine animal amino acid gap is weighted as if it were a singleamino acid or including domestic horses and other members of the family nucleotide mismatch between the two sequences. Equidae, genus Equus. Other techniques for alignment are described in Methods The term “mammal’ is known in the art, and exemplary 45 in Enzymology, Vol. 266: Computer Methods for Macromo mammals include humans, primates, livestock animals (in lecular Sequence Analysis (1996), ed. Doolittle, Academic cluding bovines, porcines, etc.), companion animals (e.g., Press, Inc., a division of Harcourt Brace & Co., San Diego, canines, felines, etc.) and rodents (e.g., mice and rats). Calif., USA. Preferably, an alignment program that permits The term “naturally occurring form' when referring to a gaps in the sequence is utilized to align the sequences. The compound means a compound that is in a form, e.g., a com 50 Smith-Waterman is one type of algorithm that permits gaps in position, in which it can be found naturally. For example, sequence alignments. See Meth. Mol. Biol. 70: 173-187 since resveratrol can be found in red wine, it is present in red (1997). Also, the GAP program using the Needleman and wine in a form that is naturally occurring. A compound is not Wunsch alignment method can be utilized to align sequences. in a form that is naturally occurring if, e.g., the compound has An alternative search strategy uses MPSRCH software, been purified and separated from at least some of the other 55 which runs on a MASPAR computer. MPSRCH uses a Smith molecules that are found with the compound in nature. A Waterman algorithm to score sequences on a massively par “naturally occurring compound” refers to a compound that allel computer. This approach improves ability to pick up can be found in nature, i.e., a compound that has not been distantly related matches, and is especially tolerant of Small designed by man. A naturally occurring compound may have gaps and nucleotide sequence errors. Nucleic acid-encoded been made by man or by nature. 60 amino acid sequences can be used to search both protein and A "naturally occurring compound” refers to a compound DNA databases. that can be found in nature, i.e., a compound that has not been The term “pharmaceutically acceptable carrier is art-rec designed by man. A naturally occurring compound may have ognized and refers to a pharmaceutically-acceptable material, been made by man or by nature. For example, resveratrol is a composition or vehicle. Such as a liquid or solid filler, diluent, naturally-occurring compound. A "non-naturally occurring 65 excipient, solvent or encapsulating material, involved in car compound is a compound that is not known to existin nature rying or transporting any Subject composition or component or that does not occur in nature. thereof. Each carrier must be “acceptable' in the sense of US 8,178,536 B2 41 42 being compatible with the Subject composition and its com administered. For example, the term is meant to encompass ponents and not injurious to the patient. Some examples of compositions that are free of, or Substantially free of an materials which may serve as pharmaceutically acceptable endotoxin Such as, for example, a lipopolysaccharide (LPS). carriers include: (1) Sugars, such as lactose, glucose and “Replicative lifespan of a cell refers to the number of Sucrose; (2) starches, such as corn starch and potato starch; daughter cells produced by an individual “mother cell.” (3) cellulose, and its derivatives, such as Sodium carboxym "Chronological aging or "chronological lifespan on the ethyl cellulose, ethyl cellulose and cellulose acetate; (4) pow other hand, refers to the length of time a population of non dered tragacanth; (5) malt; (6) gelatin; (7) talc.; (8) excipients, dividing cells remains viable when deprived of nutrients. Such as cocoa butter and Suppository waxes; (9) oils, such as “Increasing the lifespan of a cell' or “extending the lifespan peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, 10 corn oil and Soybean oil, (10) glycols, such as propylene of a cell as applied to cells or organisms, refers to increasing glycol, (11) polyols, such as glycerin, Sorbitol, mannitol and the number of daughter cells produced by one cell; increasing polyethylene glycol; (12) esters, such as ethyl oleate and ethyl the ability of cells or organisms to cope with stresses and laurate; (13) agar, (14) buffering agents, such as magnesium combat damage, e.g., to DNA, proteins; and/or increasing the hydroxide and aluminum hydroxide; (15) alginic acid, (16) 15 ability of cells or organisms to Survive and exist in a living pyrogen-free water, (17) isotonic Saline; (18) Ringer's Solu state for longer under a particular condition, e.g., stress (for tion; (19) ethyl alcohol; (20) phosphate buffer solutions; and example, heatshock, osmotic stress, high energy radiation, (21) other non-toxic compatible Substances employed in chemically-induced stress, DNA damage, inadequate salt pharmaceutical formulations. level, inadequate nitrogen level, or inadequate nutrient level). The terms “polynucleotide', and “nucleic acid are used Lifespan can be increased by at least about 20%, 30%, 40%, interchangeably. They refer to a polymeric form of nucle 50%, 60% or between 20% and 70%, 30% and 60%, 40% and otides of any length, either deoxyribonucleotides or ribo 60% or more using methods described herein. nucleotides, or analogs thereof. Polynucleotides may have "Sirtuin-activating compound” refers to a compound that any three-dimensional structure, and may perform any func increases the level of a sirtuin protein and/or increases at least tion, known or unknown. The following are non-limiting 25 one activity of a sirtuin protein. In an exemplary embodiment, examples of polynucleotides: coding or non-coding regions a sirtuin-activating compound may increase at least one bio of a gene or gene fragment, loci (locus) defined from linkage logical activity of a sirtuin protein by at least about 10%, 25%, analysis, exons, introns, messenger RNA (mRNA), transfer 50%, 75%, 100%, or more. Exemplary biological activities of RNA, ribosomal RNA, ribozymes, cDNA, recombinant poly sirtuin proteins include deacetylation, e.g., of histones and nucleotides, branched polynucleotides, plasmids, vectors, 30 p53; extending lifespan; increasing genomic stability; silenc isolated DNA of any sequence, isolated RNA of any ing transcription; and controlling the segregation of oxidized sequence, nucleic acid probes, and primers. A polynucleotide proteins between mother and daughter cells. may comprise modified nucleotides, such as methylated “Sirtuin-inhibiting compound” refers to a compound that nucleotides and nucleotide analogs. If present, modifications decreases the level of a sirtuin protein and/or decreases at to the nucleotide structure may be imparted before or after 35 least one activity of a sirtuin protein. In an exemplary embodi assembly of the polymer. The sequence of nucleotides may be ment, a sirtuin-inhibiting compound may decrease at least interrupted by non-nucleotide components. A polynucleotide one biological activity of a sirtuin protein by at least about may be further modified, such as by conjugation with a label 10%, 25%, 50%, 75%, 100%, or more. Exemplary biological ing component. The term “recombinant polynucleotide activities of sirtuin proteins include deacetylation, e.g., of means a polynucleotide of genomic, cDNA, semisynthetic, or 40 histones and p53; extending lifespan; increasing genomic synthetic origin which either does not occur in nature or is stability; silencing transcription; and controlling the segrega linked to another polynucleotide in a nonnatural arrange tion of oxidized proteins between mother and daughter cells. ment. "Sirtuin-modulating compound” refers to a compound of The term “prophylactic' or “therapeutic treatment is art Formulas (I)-CXXVIII) as described herein. In exemplary recognized and refers to administration of a drug to a host. If 45 embodiments, a sirtuin-modulating compound may either up it is administered prior to clinical manifestation of the regulate (e.g., activate or stimulate), down regulate (e.g., unwanted condition (e.g., disease or other unwanted State of inhibit or Suppress) or otherwise change a functional property the host animal) then the treatment is prophylactic, i.e., it or biological activity of a sirtuin protein. Sirtuin-modulating protects the host against developing the unwanted condition, compounds may act to modulate a sirtuin protein either whereas if administered after manifestation of the unwanted 50 directly or indirectly. In certain embodiments, a sirtuin condition, the treatment is therapeutic (i.e., it is intended to modulating compound may be a sirtuin-activating compound diminish, ameliorate or maintain the existing unwanted con or a sirtuin-inhibiting compound. dition or side effects therefrom). “Sirtuin protein’ refers to a member of the sirtuin deacety The term “protecting group' is art-recognized and refers to lase protein family, or preferably to the sir2 family, which temporary Substituents that protect a potentially reactive 55 include yeast Sir2 (GenBank Accession No. P53685), C. functional group from undesired chemical transformations. elegans Sir-2.1 (GenBank Accession No. NP 501912), and Examples of Such protecting groups include esters of car human SIRT1 (GenBank Accession No. NM 012238 and boxylic acids, silyl ethers of alcohols, and acetals and ketals NP 036370 (or AF083106)) and SIRT2 (GenBank Acces of aldehydes and ketones, respectively. The field of protecting sion No. NM 012237, NM 030593, NP 036369, group chemistry has been reviewed by Greene and Wuts in 60 NP 085096, and AF083107) proteins. Other family mem Protective Groups in Organic Synthesis (2" ed., Wiley: New bers include the four additional yeast Sir2-like genes termed York, 1991). “HST genes” (homologues of Sir two) HST1, HST2, HST3 The term "pyrogen-free', with reference to a composition, and HST4, and the five other human homologues hSIRT3, refers to a composition that does not contain a pyrogen in an hSIRT4, hSIRT5, hSIRT6 and hSIRT7 (Brachmann et al. amount that would lead to an adverse effect (e.g., irritation, 65 (1995) Genes Dev. 9:2888 and Frye et al. (1999) BBRC fever, inflammation, diarrhea, respiratory distress, endotoxic 260:273). Preferred sirtuins are those that share more simi shock, etc.) in a Subject to which the composition has been larities with SIRT1, i.e., hSIRT1, and/or Sir2 than with US 8,178,536 B2 43 44 SIRT2, such as those members having at least part of the The term “synthetic' is art-recognized and refers to pro N-terminal sequence present in SIRT1 and absent in SIRT2 duction by in vitro chemical or enzymatic synthesis. such as SIRT3 has. The terms “systemic administration.” “administered sys “SIRT1 protein” refers to a member of the sir2 family of temically.” “peripheral administration” and “administered sirtuin deacetylases. In one embodiment, a SIRT1 protein 5 peripherally are art-recognized and refer to the administra includes yeast Sir2 (GenBank Accession No. P53685), C. tion of a Subject composition, therapeutic or other material elegans Sir-2.1 (GenBank Accession No. NP 501912), other than directly into the central nervous system, such that human SIRT1 (GenBank Accession No. NM 012238 or it enters the patient's system and, thus, is subject to metabo NP 036370 (or AF083106)), and human SIRT2 (GenBank lism and other like processes. Accession No. NM 012237, NM 030593, NP 036369, 10 The term “therapeutic agent' is art-recognized and refers NP 085096, or AF083107) proteins, and equivalents and to any chemical moiety that is a biologically, physiologically, fragments thereof. In another embodiment, a SIRT1 protein or pharmacologically active Substance that acts locally or includes a polypeptide comprising a sequence consisting of systemically in a Subject. The term also means any Substance or consisting essentially of the amino acid sequence set forth intended for use in the diagnosis, cure, mitigation, treatment in GenBank Accession Nos. NP 036370, NP 501912, 15 or prevention of disease or in the enhancement of desirable NP 085096, NP 036369, or P53685. SIRT1 proteins physical or mental development and/or conditions in an ani include polypeptides comprising all or a portion of the amino mal or human. acid sequence set forth in GenBank Accession Nos. The term “therapeutic effect” is art-recognized and refers NP 036370, NP 501912, NP 085096, NP 036369, or to a local or systemic effect in animals, particularly mammals, P53685; the amino acid sequence set forth in GenBank and more particularly humans caused by a pharmacologically Accession Nos. NP 036370, NP 501912, NP 085096, active substance. The phrase “therapeutically-effective NP 036369, or P53685 with 1 to about 2, 3, 5, 7, 10, 15, 20, amount’ means that amount of such a Substance that produces 30, 50, 75 or more conservative amino acid substitutions; an some desired local or systemic effect at a reasonable benefit/ amino acid sequence that is at least 60%, 70%, 80%, 90%, risk ratio applicable to any treatment. The therapeutically 95%, 96%, 97%, 98%, or 99% identical to GenBank Acces 25 effective amount of such substance will vary depending upon sion Nos. NP 036370, NP 501912, NP 085096, the Subject and disease condition being treated, the weight NP 036369, or P53685, and functional fragments thereof. and age of the Subject, the severity of the disease condition, Polypeptides of the invention also include homologs (e.g., the manner of administration and the like, which can readily orthologs and paralogs), variants, or fragments, of GenBank be determined by one of ordinary skill in the art. For example, Accession Nos. NP 036370, NP 501912, NP 085096, 30 certain compositions described herein may be administered NP 036369, or P53685. in a sufficient amount to produce a desired effect at a reason “SIRT3 protein” refers to a member of the sirtuin deacety able benefit/risk ratio applicable to such treatment. lase protein family and/or to a homolog of a SIRT1 protein. In “Transcriptional regulatory sequence' is a generic term one embodiment, a SIRT3 protein includes human SIRT3 used throughout the specification to refer to DNA sequences, (GenBank Accession No. AAHO1042, NP 036371, or 35 Such as initiation signals, enhancers, and promoters, which NP 001017524) and mouse SIRT3 (GenBank Accession induce or control transcription of protein coding sequences No. NP 071878) proteins, and equivalents and fragments with which they are operable linked. In preferred embodi thereof. In another embodiment, a SIRT3 protein includes a ments, transcription of one of the recombinant genes is under polypeptide comprising a sequence consisting of, or consist the control of a promoter sequence (or other transcriptional ing essentially of the amino acid sequence set forth in Gen 40 regulatory sequence) which controls the expression of the Bank Accession Nos. AAHO1042, NP 036371, recombinant gene in a cell-type which expression is intended. NP 001017524, or NP 071878. SIRT3 proteins include It will also be understood that the recombinant gene can be polypeptides comprising all or a portion of the amino acid under the control of transcriptional regulatory sequences sequence set forth in GenBank Accession AAHO1042, which are the same or which are different from those NP 036371, NP 001017524, or NP 071878; the amino 45 sequences which control transcription of the naturally-occur acid sequence set forth in GenBank Accession Nos. ring forms of genes as described herein. AAHO1042, NP 036371, NP 001017524, or NP 071878 “Treating a condition or disease refers to curing as well as with 1 to about 2, 3, 5, 7, 10, 15, 20, 30, 50, 75 or more ameliorating at least one symptom of the condition or disease. conservative amino acid Substitutions; an amino acid A “vector is a self-replicating nucleic acid molecule that sequence that is at least 60%, 70%, 80%, 90%. 95%, 96%, 50 transfers an inserted nucleic acid molecule into and/or 97%, 98%, or 99% identical to GenBank Accession Nos. between host cells. The term includes vectors that function AAHO1042, NP 036371, NP 001017524, or NP 071878, primarily for insertion of a nucleic acid molecule into a cell, and functional fragments thereof. Polypeptides of the inven replication of vectors that function primarily for the replica tion also include homologs (e.g., orthologs and paralogs), tion of nucleic acid, and expression vectors that function for variants, or fragments, of GenBank Accession Nos. 55 transcription and/or translation of the DNA or RNA. Also AAHO1042, NP 036371, NP 001017524, or NP 071878. included are vectors that provide more than one of the above In one embodiment, a SIRT3 protein includes a fragment of functions. As used herein, “expression vectors' are defined as SIRT3 protein that is produced by cleavage with a mitochon polynucleotides which, when introduced into an appropriate drial matrix processing peptidase (MPP) and/or a mitochon host cell, can be transcribed and translated into a drial intermediate peptidase (MIP). 60 polypeptide(s). An "expression system' usually connotes a The term “substantially homologous' when used in con Suitable host cell comprised of an expression vector that can nection with amino acid sequences, refers to sequences which function to yield a desired expression product. are substantially identical to or similar in sequence with each The term “vision impairment” refers to diminished vision, other, giving rise to a homology of conformation and thus to which is often only partially reversible or irreversible upon retention, to a useful degree, of one or more biological (in 65 treatment (e.g., Surgery). Particularly severe vision impair cluding immunological) activities. The term is not intended to ment is termed “blindness” or “vision loss’, which refers to a imply a common evolution of the sequences. complete loss of vision, vision worse than 20/200 that cannot US 8,178,536 B2 45 46 be improved with corrective lenses, or a visual field of less –OCORs –OCORs –C(O)NRR - OC(O)NRR, than 20 degrees diameter (10 degrees radius). C(O)Rs, —CORs, SRs—OSOH, -S(O).Rs—S(O), 2. Sirtuin Modulators ORs, S(O)NRR —NRR —NRC(O)OR —NRC In one aspect, the invention provides novel sirtuin-modu (O)R and—NO; lating compounds for treating and/or preventing a wide vari 5 Rs and R are independently —H, a Substituted or unsub ety of diseases and disorders including, for example, diseases stituted alkyl group, a Substituted or unsubstituted aryl group or disorders related to aging or stress, diabetes, obesity, neu or a substituted or unsubstituted heterocyclic group; and rodegenerative diseases, ocular diseases and disorders, car n is 1 or 2. diovascular disease, blood clotting disorders, inflammation, In a further embodiment, sirtuin-modulating compounds cancer, and/or flushing, etc. Sirtuin-modulating compounds O of the invention are represented by Structural Formula (IIa): that increase the level and/or activity of a sirtuin protein may also be used for treating a disease or disorder in a Subject that would benefit from increased mitochondrial activity, for (IIa) enhancing muscle performance, for increasing muscle ATP levels, or for treating or preventing muscle tissue damage 15 associated with hypoxia or ischemia. Other compounds dis closed herein may be Suitable for use in a pharmaceutical OH, composition and/or one or more methods disclosed herein. N In one embodiment, sirtuin-modulating compounds of the invention are represented by Structural Formula (I):
or a salt thereof, where: (I) Ring A is optionally Substituted; 25 R. R. R. and Ra are independently selected from the group consisting of —H, halogen, —ORs, —CN, —CORs, –OCORs –OCORs –C(O)NRR - OC(O)NRR, N C(O)Rs, —CORs, SRs—OSOH, -S(O).Rs—S(O), ORs, S(O)NRR —NRR —NRC(O)OR —NRC 30 (O)R and - NO; Rs and R are independently —H, a Substituted or unsub or a salt thereof, where: stituted alkyl group, a substituted or unsubstituted aryl group Ring A is optionally substituted; and or a substituted or unsubstituted heterocyclic group; and Ring B is substituted with at least one carboxy, substituted n is 1 or 2. or unsubstituted arylcarboxamine, substituted or unsubsti 35 In yet another embodiment, sirtuin-modulating com tuted aralkylcarboxamine, substituted or unsubstituted het pounds of the invention are represented by Structural For eroaryl group, Substituted or unsubstituted heterocyclylcar mula (II): bonylethenyl, or polycyclic aryl group or is fused to an aryl ring and is optionally Substituted by one or more additional groups. 40 (IIb) In certain embodiments, Ring B is substituted with at least a carboxy group. In certain embodiments, Ring B is substituted with at least a substituted or unsubstituted arylcarboxamine, a substituted or unsubstituted aralkylcarboxamine or a polycyclic aryl 45 group. In certain embodiments, Ring B is substituted with at least a Substituted or unsubstituted heteroaryl group or a Substi tuted or unsubstituted heterocyclylcarbonylethenyl group. In another embodiment, sirtuin-modulating compounds of 50 or a salt thereof, where: the invention are represented by Structural Formula (II): Ring A is optionally Substituted; R. R. R. and Ra are independently selected from the group consisting of —H, halogen, —ORs, —CN, —CORs, (II) –OCOR –OCORs –C(O)NRR —OC(O)NRR, 55 C(O)Rs, —CORs, —SRs—OSOH, -S(O),Rs—S(O), ORs, S(O)NRR - NRR - NRC(O)CR - NRC (O)R and—NO; Rs and R are independently —H, a Substituted or unsub R stituted alkyl group, a Substituted or unsubstituted aryl group N 60 or a substituted or unsubstituted heterocyclic group; and n is 1 or 2. In certain embodiments, R. R. R. and R in Structural Formulas (II)-(IIb) are independently selected from the group or a salt thereof, where: consisting of —H. —ORs and —SRs, particularly —H and Ring A is optionally substituted; 65 —ORs (e.g., -H, -OH, - OCH). R. R. R. and Ra are independently selected from the Ring A is preferably substituted. Suitable substituents group consisting of —H, halogen, —ORs, —CN, —CORs, include halogens (e.g., bromine), acyloxy groups (e.g., US 8,178,536 B2 47 48 acetoxy), aminocarbonyl groups (e.g., arylaminocarbonyl In certain Such embodiments, Rs is selected from oxazol Such as Substituted, particularly carboxy-Substituted, pheny opyridyl, benzothienyl, benzofuryl, indolyl, quinoxalinyl, laminocarbonyl groups) and alkoxy (e.g., methoxy, ethoxy) benzothiazolyl, benzoxazolyl, benzimidazolyl, quinolinyl, groups. isoquinolinyl or isolindolyl. In certain such embodiments. Rs In yet another aspect, the invention provides novel sirtuin is selected from thiazolopyridyl, imidazothiazolyl, benzox modulating compounds of Formula (III): aZinonyl, or imidazopyridyl. Particular examples of Rs, where indicates attachment to (III) the remainder of Structural Formula (III), include: 10
15 CC) CIC) or a salt thereof, where: Ring A is optionally substituted; C-C Rs and R are independently —H, a substituted or unsub stituted alkyl group, a Substituted or unsubstituted aryl group OC) C) or a Substituted or unsubstituted heterocyclic group; R7, Ro Ro and R are independently selected from the group consisting of —H, halogen, —Rs —ORs, —CN. 25 —CORs, —OCORs —OCORs, —C(O)NRR —OC(O) NRSR —C(O)Rs, —CORs, —SRs —OSOH, -S(O),Rs. Cy C. —S(O), ORs, S(O)NRR - NRR - NRC(O)CR, —NRC(O)R and —NO; N Rs is a polycyclic aryl group; and 30 n n is 1 or 2. In certain embodiments, one or more of R7, R. RandR O 21N2N. are —H. In particular embodiments, R. R. Ro and R are each —H. O 's-N-N/ . In certain embodiments. Rs is a heteroaryl group, such as 35 an oxazolo 4,5-b]pyridyl group. In particular embodiments, Rs is a heteroaryl group and one or more of R7, Ro, Ro and where up to 2 ring carbons not immediately adjacent to the Rare —H. indicated attachment point are independently Substituted Ring A is preferably substituted. Suitable substituents with O—C-C straight or branched alkyl, C-C straight or include halogens (e.g., bromine), acyloxy groups (e.g., 40 branched alkyl or halo, particularly C-C straight or acetoxy), aminocarbonyl groups (e.g., arylaminocarbonyl, branched alkyl or halo. In certain embodiments, Rs is Such as Substituted, particularly carboxy-Substituted, pheny laminocarbonyl groups) and alkoxy (e.g., methoxy, ethoxy) groups, particularly alkoxy groups. In certain embodiments, N Ring A is Substituted with at least one alkoxy or halo group, 45 N-y particularly methoxy. 21 No In certain embodiments, Ring A is optionally substituted with up to 3 substituents independently selected from (C-C, straight or branched alkyl). O—(C-C straight or branched In certain embodiments (e.g., when the modulator is a alkyl), N(C-C straight or branched alkyl), halo, or a 5 to 50 sirtuin activator), Rs is 6-membered heterocycle. In certain embodiments, Ring A is not substituted with a nitrile or pyrrolidyl group. N N In certain embodiments, Rs is a substituted or unsubsti tuted bicyclic heteroaryl group. Such as a bicyclic heteroaryl 55 group that includes a ring Natom and 1 to 2 additional ring O)2No. heteroatoms independently selected from N, O or S. Prefer ably, Rs is attached to the remainder of the compound by a and Ring A is optionally substituted with up to 3 substituents carbon-carbon bond. In certain Such embodiments, 2 addi independently selected from (C-C straight or branched tional ring heteroatoms are present, and typically at least one 60 alkyl). O—(C-C straight or branched alkyl), N(C-C, of said additional ring heteroatoms is O or S. In certain Such straight or branched alkyl), halo, or a 5 to 6-membered embodiments, 2 total ring nitrogen atoms are present (with heterocycle. In certain such embodiments, Ring A is not Zero or one O or S present), and the nitrogen atoms are simultaneously substituted at the 2- and 6-positions with typically each in a different ring. In certain Such embodi O—(C-C straight or branched alkyl). In certain Such ments, Rs is not substituted with a carbonyl-containing moi 65 embodiments, Ring A is not simultaneously substituted at the ety, particularly when Rs is thienopyrimidyl or thienopyridi 2-, 4- and 6-positions with O—(C-C straight or branched nyl. alkyl). In certain such embodiments, Ring A is not simulta US 8,178,536 B2 49 50 neously Substituted at the 2-, 3-, and 4-positions with -continued O—(C-C straight or branched alkyl). In certain such embodiments, Ring A is not substituted at the 4-position with Sl-N N a 5 to 6-membered heterocycle. In certain such embodiments, N Ring A is not singly substituted at the 3- or 4-position (typi 5 cally 4-position) with O—(C-C straight or branched alkyl). Na2Ns s 4. s In certain such embodiments, Ring A is not substituted at the 4-position with O—(C-C straight or branched alkyl) and at N the 2- or 3-position with C-C straight or branched alkyl. n 10 In certain embodiments, Rs is O 21Ne.N.
N N O 's-N-N/ . 15 where up to 2 ring carbons not immediately adjacent to the OC)2No. indicated attachment point are independently Substituted with C-C straight or branched alkyl or halo; each of R7, Ro, and Ring A is optionally substituted with up to 3 substituents and R is —H; and Rio is selected from —H, —CH2OH. independently selected from (C-C straight or branched —COH, -COCH, —CH-piperazinyl, CHN(CH), alkyl), (C-C straight or branched haloalkyl, where a —C(O) NH (CH), N(CH), or —C(O)-piperazinyl. haloalkyl group is an alkyl group Substituted with one or more In certain Such embodiments, when Rs is halogen atoms), O—(C-C straight or branched alkyl). N(C-C straight or branched alkyl), halo, or a 5 to 6-mem N bered heterocycle. In certain such embodiments, Ring A is not 25 n N singly substituted at the 3- or 4-position with O (C-C, N straight or branched alkyl). In certain such embodiments, 2No. Ring A is not substituted at the 4-position with O—(C-C, straight or branched alkyl) and at the 2- or 3-position with C-C straight or branched alkyl. 30 and Ring A is 3-dimethylaminophenyl, none of R7, Ro, Rio In certain embodiments, Rs is and R is —CH N(CH), or —C(O) NH-(CH), N (CH), and/or when Rs is
halo N N 35 N N O y 21 No halo 40 and Ring A is 3.4 dimethoxyphenyl, none of R7, RoRo and (e.g., where one or both halo is chlorine) and Ring A is R is C(O)OCH or C(O)OH. optionally substituted with up to 3 substituents independently In certain embodiments, such as when Rs has one of the selected from (C-C straight or branched alkyl). O—(C-C, values described above and/or Ring A is optionally substi straight or branched alkyl), N(C-C straight or branched 45 tuted as described above, at least one of R7, RoRo and R is alkyl), halo, or a 5 to 6-membered heterocycle, but not singly —H. In certain such embodiments, each of R. R. Ro and substituted at the 3-position with O (C-C straight or R is —H. branched alkyl). In certain embodiments, R-7, RoRo or R is selected from In certain embodiments, such as when Rs has one of the - C(O)OH, -N(CH), —CHOH, -CHOCH CH values described above, Ring A is substituted with up to 3 50 piperazinyl, —CH2-methylpiperazinyl, —CH-pyrrolidyl, substituents independently selected from chloro, methyl, —CH-piperidyl, -CH2-morpholino. —CH2—N(CH), O-methyl, N(CH) or morpholino. In certain such embodi —C(O)—NH-(CH), piperazinyl, - C(O) NH ments, Rs is selected from (CH2), methylpiperazinyl, —C(O) NH (CH), pyr rolidyl, —C(O) NH (CH), morpholino. —C(O)— 55 NH-(CH), piperidyl, or—C(O) NH (CH), N(CH), N wherein n is 1 or 2. In certain such embodiments, Ro is selected from —C(O)OH, - N(CH), —CH2OH. —CH2OCH-CH2-piperazinyl, -CH2-methylpiperazi CC)21 No CI)N4No s nyl, -CH2-pyrrolidyl, -CH2-piperidyl, -CH2-mor N N 60 pholino, —CH2—N(CH), —C(O)—NH-(CH2), piper azinyl, —C(O) NH-(CH), methylpiperazinyl, —C(O) NH (CH), pyrrolidyl, —C(O) NH (CH), O)O s ON s N N morpholino. —C(O) NH-(CH), piperidyl, or —C(O)— NH-(CH2), N(CH), wherein n is 1 or 2, and each of R7, 65 Ro, and R is H. O)S s (CC) s In certain embodiments, Ring A is substituted with a nitrile group or is substituted at the paraposition with a 5- or 6-mem
US 8,178,536 B2 53 54 In another embodiment, the compounds of the formula wherein above are those wherein J is NR", Kis absent, and Mis C(O). X, X2, Xs, X, and Xs are independently selected from In yet another embodiment, the compounds of the formula CR" and N; and above are those wherein J is absent, K is NR', and M is C(O). In a further embodiment, compounds of formula (IV) are X is selected from NR", O, and S. those where when J is absent and K is NR', M is not C(O) and In a preferred embodiment, Ring B is phenyl or pyridinyl. when J is NR' and K is absent, M is not C(O). In a further aspect, the invention provides novel sirtuin In a preferred embodiment, the compounds above are those modulating compounds of Formula (IVa): wherein L is an optionally substituted 5- to 7-membered 10 carbocyclic or heterocyclic aryl group. Het-L-Q-Ar" (IVa) In yet another preferred embodiment, the compounds are those wherein L is an optionally Substituted phenylene, or a salt thereof, wherein: pyridinylene, imidazolylene, oxazolylene, or thiazolylene. Het is an optionally Substituted heterocyclic aryl group; In a particularly preferred embodiment, L is an optionally substituted phenylene. 15 L is an optionally substituted carbocyclic or heterocyclic In another particularly preferred embodiment, L is an arylene group; optionally substituted pyridinylene. Ar' is an optionally substituted carbocyclic or heterocyclic In an even more preferred embodiment, L is phenylene. aryl group; and In another even more preferred embodiment, L is pyridi nylene. Q is selected from NR In either of these embodiments, Arand J may be attached to L at the ortho-, meta-, or para-positions. Particularly pre ferred are those embodiments where attachment is at the meta-position. In certain embodiments, L is not phenylene when Ar' is 25 phenyl. Examples of such embodiments include embodi ments where L is an optionally substituted heterocyclic aryl group and Aris an optionally Substituted carbocyclic or het erocyclic aryl group, or wherein L is an optionally Substituted carbocyclic or heterocyclic aryl group and Ar' is an optionally 30 Substituted heterocyclic aryl group. In yet another aspect, the invention provides novel sirtuin modulating compounds of Formula (I) or a salt thereof, wherein Ring A is Substituted with at least one R' group; 35 R. R. R. R. R. R. R. R. R. Ro', and Rare as s-> defined above; each haloalkyl is independently a C1-C10 alkyl substituted with one or more halogenatoms, selected from F, Cl, Br, or I, wherein the number of halogen atoms may not exceed that 40 number that results in a perhaloalkyl group; each aryl is independently a 5- to 7-membered monocyclic ring system or a 9- to 12-membered bicyclic ring system optionally substituted with 1-3 independent C1-C10 alkyl: 45 C2-C10 alkenyl: C2-C10 alkynyl: C3-C10 cycloalkyl: and C4-C10 cycloalkenyl; R.; halo: haloalkyl: CF; OR; SR': NR'R'; COOR': NO; CN: C(O)R': C(O)C(O)R': C(O) each R" is independently selected from H or optionally NR'R'': S(O).R.'; N(R')C(O)R'; N(R')(COOR"); N(R) substituted C-C straight or branched alkyl, wherein: S(O).R.'; S(O)NR'R'; OC(O)R'; NR'C(O)NR'R'; 50 when Het is a polycyclic heteroaryl, L is an optionally Sub NRC(O)C(O)R'; NRC(O)R'; NR'S(O)NR'R'; NR'S stituted phenylene, Q and Het are attached to L in a meta (O).R.'; NRC(O)C(O)NR'R'; C1-C10 alkyl substituted orientation, and Ar' is optionally Substituted phenyl; then Q is with 1-3 independent R', halo, CF, OR', SRs', NR'R''. not —NH CO)—. COOR, NO, CN, C(O)R', C(O)NR'R', NHC(O)R', NH(COOR), S(O)NR'R', OC(O)R'; C2-C10 alkenyl In certain embodiments, when Het is a polycyclic het substituted with 1-3 independent R', halo, CF, OR', SRs', 55 eroaryl, L is optionally Substituted phenylene, and Ar' is NR'R', COOR, NO, CN, C(O)R., C(O)NR'R', NHC optionally substituted phenyl; then Q is not NH COO)—. (O)R', NH(COOR), S(O)NR'R'', OC(O)R'; or R.'; and In certain embodiments (e.g., when the compound is a Ring B is substituted with at least one sirtuin activator), Het and Q are attached to L in a 1-, 2- or 60 1-3-configuration (e.g., when L is phenylene, Het and Q are attached in an ortho or a meta orientation). In certain embodi ments where Het and Q are attached to L in a 1-3-configu ration, if Het is benzoxazolyl, L is pyridylene and Q is —NH CO) NH, then Ar' is not 3.4 dioxymethlyene phe 65 nyl; if Het is methyl thiazolyl, L is phenylene and Q is —NH CO)—, then Ar" is not 3-dimethylamino phenyl; if Het is oxazolopyridyl, L is pyridylene and Q is —NH C US 8,178,536 B2 55 56 (O) NH, then Ar' is not 4-dimethylamino phenyl; if Het is -continued oxazolopyridyl or benzoxazolyl and L is N SN2N 5 OC) C)
\ / N / O N s 10 Na2NsCy s C4. s then Q is not —NH (SO) ; and if Het is oxazolopyridyl, L is 15 CN \ s O)^- N
O)4. N s OC)4No s and Q is —NH C(O)—, then Ar' is not 3.4 dimethoxyphe nyl or pyridyl. 25 When Het is substituted, it is typically substituted at up to CC)2NS s CC4Ns s 2 carbon atoms with a substituent independently selected from R, N(R), NH(R), OR2, C(O)—NH R. C(O)—N(R), N(R)—OR2, CH N(R), C(O)OR, 30 C(O)CH, 21 S s 2N s
N1 NH S N1 N 35 - |-C CH3 - 40 C7. X-. and \YN . s where each R is independently selected from optionally 45 where up to 2 ring carbons not immediately adjacent to the substituted C-C straight or branched alkyl. indicated attachment point are independently Substituted In certain embodiments, Het is selected from oxazolopy with optionally substituted C-C straight or branched alkyl, ridyl, benzothienyl, benzofuryl, indolyl, quinoxalinyl, ben phenyl, halo, N(R), NH(R), OR, C(O)—NH R. Zothiazolyl, benzoxazolyl, benzimidazolyl, quinolinyl, iso 50 C(O)—N(R), N(R)—OR, CH N(R), C(O)OR, quinolinyl or isoindolyl. In other embodiments, Het C(O)OH, comprises one ring N heteroatom and 1 to 2 additional ring heteroatoms independently selected from N, O or S, such as thiazolyl, triazolyl, oxadiazolyl, thiazolopyridyl, imida Zothiazolyl, benzoxazinonyl, or imidazopyridyl. 55 Particular examples of Het include: --- |-- N N-N N-N 60 21 4No N N N N 65 O) Cy wherein each R2 is independently selected from optionally substituted C-C straight or branched alkyl. US 8,178,536 B2 57 58 In certain embodiments, L is selected from In certain embodiments, Het is selected from
Z as EZ V
{Z-Z / s MZ-Z / , 10
Zs
OZ6 , or S4/O/. 15 wherein: each of Z, Z, Z and Z is independently selected from CH or N, wherein not more than three of said Z, Z, Z or Z. is N: each of Zs and Z is independently selected from C, N, O or S. provided that at least one of Zs and Z is N; and L is optionally substituted at 1 to 2 carbon atoms with a Substituent independently selected from R, N(R), 25 NH(R), OR2, C(O) NH R1, C(O) N(R), N(R)—OR, CH N(R), C(O)OR, C(O)OH,
30 N1 r - 1 Nu HC1 Nu 35
NO Nur 40 N N1 N - 1. - 1. N \ { N s , or CH In preferred embodiments, L is selected from phenylene or 45 pyridylene, such as unsubstituted phenylene or phenylene s^\s s substituted with a single substituent selected from C(O) OCH, C(O)OH, CHOH, N(CH), or CHN(CH), or unsubstituted pyridylene. 50 and wherein up to 2 ring carbons not immediately adjacent to In certain embodiments, Q is selected from —NH C the indicated attachment point are independently substituted (O) , -NH S(O) NH-C(O) NH-, -C(O) with optionally substituted C-C straight or branched alkyl, NH-, -CH , – N(CH) C(O) NH-, -NH C phenyl or halo; (O) N(CH) , or NH S(O). NH , particularly L is selected from unsubstituted phenylene, phenylene sub NH C(O) , C(O) NH-, NH NH C 55 stituted with a single substituent selected from C(O)OCH, (O) NH, or - NH S(O), . C(O)OH, CHOH, N(CH), or CHN(CH), or unsubsti In certain embodiments, Ar' is selected from optionally tuted pyridylene; substituted phenyl, benzothiazolyl, or benzoxazolyl. When Q is selected from NH CO)— —C(O)—NH , Ar' is phenyl, typical optional substituents are 1 to 3 substitu 60 NH-, -NH C(O) NH, or -NH-S(O) ; and ents independently selected from halo, (optionally Substi Ar' is selected from optionally substituted phenyl, ben tuted C-C straight or branched alkyl), O-(optionally substi Zothiazolyl, or benzoxazolyl, wherein said phenyl is option tuted C-C straight or branched alkyl), S-(optionally ally substituted with 1 to 3 substituents independently substituted C-C straight or branched alkyl), N(CH) or selected from chloro, methyl, O-methyl, S-methyl, N(CH), optionally substituted heterocyclyl, or wherein two substitu 65 morpholino, or 3.4 dioxymethylene. ents on adjacent ring atoms are taken together to form a In certain embodiments, Q is selected from —NH C dioxymethylene. (O) = C(O) NH-, - NH- or -NH C(O) NH. US 8,178,536 B2 59 60 In certain embodiments, the substituents on Arare selected alkyl, O—(C-C straight or branched alkyl), N(C-C, from chloro, methyl, O-methyl, S-methyl or N(CH). In straight or branched alkyl), or a 5 to 6-membered hetero certain embodiments, the only substituent on Ar' is an O-me cycle, thyl group, particularly an O-methyl group ortho or meta to Q. wherein when Rs is unsubstituted In certain embodiments, when there are two or more O-me thyl groups or Ar", at least one is ortho or meta to Q. In certain embodiments, L is pyridyl and Het and Q are at N N the 1.3- or 2.4-position with respect to the pyridyl nitrogen atom. In certain Such embodiments, Q is —NH-S(O) -. In certain embodiments where L is further substituted, the 10 CC)21 No s substituent is typically meta to both Het and Q. In certain embodiments, Q is —NH and Het is thiazolyl then ring A is: or oxazolopyridyl. a) not simultaneously substituted at the 2- and 6-positions In certain embodiments, Q is —NH and Aris benzothia with O—(C-C straight or branched alkyl); Zolyl or benzoxazolyl. 15 b) not simultaneously substituted at the 2-position with In certain embodiments, such as when the sirtuin modula C-C straight or branched alkyl or O—(C-C straight tor is a sirtuin activator, L is or branched alkyl) and at the 3-position with O (C-C, straight or branched alkyl); c) not substituted at the 4-position with O—(C-C straight or branched alkyl) unless simultaneously substituted at the 3-position with halo or O—(C-C straight or branched alkyl) and unsubstituted at all other positions; not substituted at the 4-position with N(C-C straight or s 25 branched alkyl), or said 5 to 6-membered heterocycle. In certain such embodiments, L is unsubstituted and/or Het is and Q is NH (SO) . In certain such embodiments, Het oxazolopyridyl. is oxazolopyridyl. When L. Q and optionally Het have these In yet another aspect, the invention provides novel sirtuin values, Ar is advantageously naphthyl or phenyl, where Ar' is modulating compounds of Formula (V): optionally substituted with 1 to 3 substituents independently 30 selected from CN, halo, (C-C straight or branched alkyl). O—(C-C straight or branched alkyl), N(C-C straight or (V) branched alkyl), or a 5 to 6-membered heterocycle. In certain embodiments, such as when the sirtuin modula tor is a sirtuin activator, L is 35 N Y2,
N 40
or a salt thereof, wherein: Ring A is optionally substituted with at least one R'group: Y.Y.Y.Y., and Ys are independently R'; and Q is —NH C(O)—. In certain such embodiments, Het 45 R. R. R. R. R. R. R. R. R. Ro', and Rare as is oxazolopyridyl. When L. Q and optionally Het have these defined above; values, Ar is advantageously pyridyl or phenyl optionally each haloalkyl is independently a C1-C10 alkyl substituted substituted with 1 to 3 substituents independently selected from CN, halo, (C1-C3 straight or branched alkyl). O—(C1 with one or more halogenatoms, selected from F, Cl, Br, or I, C3 straight or branched alkyl), N(C1-C3 straight or branched 50 wherein the number of halogen atoms may not exceed that alkyl)2, or a 5 to 6-membered heterocycle. number that results in a perhaloalkyl group; and In certain embodiments, such as when the sirtuin modula each aryl is independently a 5- to 7-membered monocyclic tory is a sirtuin inhibitor, Het comprises one N heteroatom ring system or a 9- to 12-membered bicyclic ring system and 1 to 2 additional heteroatoms independently selected optionally substituted with 1-3 independent C1-C10 alkyl: from N, O or S: 55 C2-C10 alkenyl: C2-C10 alkynyl: C3-C10 cycloalkyl: L is C4-C10 cycloalkenyl; R.; halo: haloalkyl: CF; OR'; SR': NR'R'; COOR': NO; CN: C(O)R': C(O)C(O)R': C(O) NR'R'': S(O).R.'; N(R')C(O)R'; N(R')(COOR); N(R') S(O).R.'; S(O)NR'R'; OC(O)R'; NR'C(O)NR'R'': 60 NR',C(O)C(O)R'; NRC(O)R'; NRS(O)NR'R'; NR'S(O).R.'; NR'C(O)C(O)NR'R'; C1-C10 alkyl substi tuted with 1-3 independent R', halo, CF OR', SRs', NR'R', COOR, NO, CN, C(O)R., C(O)NR'R', NHC and is optionally Substituted; (O)R', NH(COOR"), S(O)NR'R'', OC(O)R'; C2-C10 Q is —NH CO)—; and 65 alkenyl substituted with 1-3 independent R', halo, CF, OR.", Ar is phenyl substituted with 1 to 3 substituents indepen SR', NR'R'', COOR, NO, CN, C(O)R', C(O)NR'R'', dently selected from CN, halo, C-C straight or branched NHC(O)R', NH(COOR), S(O)NR'R'', OC(O)R'; or R'. US 8,178,536 B2 61 62 In a preferred embodiment of the above compound, zero to one R is a solubilizing group; either Y, or Y is Zero to one R is an optionally Substituted C-C straight or branched alkyl; and R’ is selected from NR' C(O) = NR, S(O) , - NR' C(O) NR' , - NR' C(S)-NR' , NR' C(S) NR' CR'R' , NR' C(O) CR'R' NR' , - NR' C(=NR") NR' , C(O) NR' , C(O) NR' S(O) , NR' , 10 —CR'R' , – NR' C(O) CR'—CR' NR' S X, X2, Xs, X, and Xs are independently selected from (O) NR' , – NR' C(O) NR' S(O) = NR, CR" and N; and CR'R' C(O) NR' , CR'R' C(O) NR' , X is selected from NR", O, and S. - NR' C(O) CR'—CR' CR'R' , - NR' C According to an even more preferred embodiment, X and (=N-CN) NR' , NR' C(O) CR'R' O , X- are N; X, X, and Xs are CR'; and X is O. 15 NR' C(O) CR'R' CR'R' O-, - NR' S(O), According to anothereven more preferred embodiment, X CR'R' , NR' S(O), CR 'R' CR'R' , or and X are N; X2, X, and Xs are CR'; and X is O. According to another even more preferred embodiment, X R" is selected from an optionally substituted monocyclic and X are N; X2, X, and Xs are CR'; and X is O. or bicyclic aryl, or an optionally Substituted monocyclic or According to another even more preferred embodiment, X bicyclic heteroaryl, with the provisos that said compound is and Xs are N; X, X, and X are CR'; and X is O. not: In another aspect, the invention provides sirtuin-modulat ing compounds of Structural Formula (VII): O 25 (VII) M R31 HN -( ) 2 x4'8 O i 2 O X-R 30 X10 N ? or a salt thereof, wherein: or that when R' is each of X7, Xs, X and Xo is independently selected from N, CR', or CR', wherein: 35 each R' is independently selected from Hora solubilizing group; each R" is independently selected from H or optionally substituted C-C straight or branched alkyl; one of X7, Xs, X and X is N and the others are selected 40 from CR' or CR.'; and Zero to one R is a solubilizing group: R" is selected from: and R is NHC(O) , R is not an optionally substituted phenyl. 45 In certain embodiments, compounds of Structural Formula (VII) have the following values: each of X, Xs, X and Xo is independently selected from N, CR, or CR", wherein: 50 Z13. a2 Z11. Z13, a2 Z11 s each R' is independently selected from Hora solubilizing Z2 Z2 group; each R" is independently selected from H or optionally substituted C-C straight or branched alkyl; 55 one of X, Xs, X and X is N and the others are selected ^s) O )Z14 s from CR or CR.'; and ZS ZNY Z15 Z15 zero to one R is a solubilizing group; R" is selected from: wherein: each Zo. Z, Z and Z is independently selected from 60 N, CR, or CR.'; and each Z, Zs and Z is independently selected from N. NR', S, O, CR, or CR", wherein: Z10 Zero to two of Zo. Z, Z or Z are N: rs. aS at least one of Z, Zs and Z is N, NR', S or O; 65 Z132 Z11: Z13, 27.11 s Zero to one of Z, Zs and Z is S or O. Zero to two of Z, Zs and Z are N or NR'; US 8,178,536 B2 64 -continued In certain embodiments, when Z, is CR" and R' is a solubilizing group, the solubilizing group is not —C(O) OCHCH-COOH, Z14 ZiS2.O) or ZSZOz, N N ~ N 1N1 N wherein: H each Zo. Z, Z and Z is independently selected from 10 Null O N, CR, or CR.'; and each Z, Zs and Z is independently selected from N. NR', S, O, CR, or CR", wherein: In certain embodiments, when Xs and X are eachindepen Zero to two of Zo. Z, Z or Z are N: dently selected from CR or CR'R'' is at least one of Z, Zs and Z is N, NR', S or O; 15 Zero to one of Za Zs and Z is S or O. Zero to two of Z, Zs and Z are N or NR'; zero to one R is a solubilizing group; Zero to one R is an optionally Substituted C-C straight or 20 branched alkyl; and R’ is selected from —NR' C(O)— —NR' S(O) , —NR' C(O) NR' , - NR' C(S)- NR' , NR' C(S)-NR' CR'R' , NR, C(O) and each of Zo. Z, Z and Z is independently selected CR'R' NR' , NR' C(=NR') NR' , 25 from CR, or CR', then: C(O) NR' , C(O) NR' S(O) , NR' , a) at least one of Xs and X is not CH; or —CR'R' , – NR' C(O) CR'—CR' , – NR' S b) at least one of Zo, Z, and Z is CR, wherein Risa (O) NR' , – NR' C(O) NR' S(O) = NR, Solubilizing group. CR'R' C(O) NR' , CR'R' C(O) NR' , In certain embodiments, when R' is —NR' C(O) CR'—CR' CR'R' , NR, C 30 (=N-CN) NR' , NR' C(O)—CR'R' O , NR' C(O) CR'R' CR'R' O. , NR S(O), CR'R' , NR' S(O), CR 'R' CR'R' , or —NR' C(O)—CR'R' ; and R is selected from an optionally substituted monocyclic or bicyclic aryl, or an optionally Substituted monocyclic or bicyclic heteroaryl, with the provisos that: said compound is not: and each of Zo. Z, Z2 and Zis is CR, or CR'; Xs and Xo 40 are CR' or CR'; R is NHC(O) ; and R is optionally O substituted phenyl, then R is a substituted phenyl, at least one R' in a CR' moiety is optionally substituted C-C, M straight or branched alkyl or at least one R' in a CR” is a HN -( ) solubilizing group, or a combination thereof. In certain embodiments, R' is selected from phenyl, e O 45 pyridyl, thienyl or furyl. S / In certain embodiments, R' is
50 and when Xs and X are each independently selected from CR20 or CR, R is
55 Z10 wherein each of Zo. Z, Z and Z is independently S selected from CR' or CR.'; and R’ is NH C(O) ; and Zisse/il 60 R" is a substituted phenyl. In certain such embodiments, when X is N, R is not 2.4 dimethoxyphenyl and/or when Xio is N, R is not halo sub and each of Zo. Z, Z and Z is independently selected stituted phenyl: 3,4-dioxoethylenephenyl; or 3,5-dimethox from CR'', or CR", then: yphenyl. a) at least one of Xs and X is not CH; or 65 In preferred embodiments, R is optionally substituted b) at least one of Zo. Z, Zand Z is CR, wherein R' with 1 to 3 substituents independently selected from is a solubilizing group. —OCH, —CH —N(CH), pyrazinoxy or a solubilizing US 8,178,536 B2 65 66 group. Suitable examples of R' include 3-methoxy-4-(4- (O) NR' , – NR' C(O) NR' S(O) = NR, methylpiperazin-1-yl)methyl)phenyl, 3-methoxy-4-mor CR'R' C(O) NR' , CR'R' C(O) NR' , pholinomethylphenyl, 3-methoxy-4-diaminomethylphenyl, - NR' C(O) CR'—CR' CR'R' , – NR' C 3-methoxy-4-((pyrrolidin-1-yl)methyl)phenyl, 3,4- (—N CN) NR' , NR' C(O) CR'R' O , dimethoxyphenyl, 3,5-dimethoxyphenyl, 2,3,4-trimethox NR' C(O) CR'R' CR'R' O-, - NR S(O), yphenyl, 3,4,5-trimethoxyphenyl, 2-dimethylaminophenyl, CR'R' NR' S(O). CR'R' CR'R' , or 3-dimethylaminophenyl, 4-dimethylaminophenyl, or 3.5- dimethylphenyl. R" is selected from an optionally substituted monocyclic In certain embodiments, R' is selected from or bicyclic aryl, or an optionally Substituted monocyclic or 10 bicyclic heteroaryl, with the provisos that: when R." is methyl, and R is NH-C(O) , R is not S Z10 aS Z O s 15 N C Z132Z2 Z11 O wherein one of Zo. Z, Z, and Z is N and the others are independently selected from CR' or CR'; R’ is selected from NH-, -NH C(O) , NH C 1-methoxynaphthyl, 2-methoxynaphthyl, or unsubstituted (O) NH, -NH C(S)- NH or -NH S(O) ; and R" is selected from an optionally substituted phenyl, an 2-thienyl: optionally substituted naphthyl, or an optionally Substituted when R is methyl, and R is NH-C(O)— heteroaryl. 25 CH-CH , R is not In certain Such embodiments, a) when R is NH S(O) , either: i) Zo is N, or ii) Z is N and R is halophenyl or 2-methoxy-5-me N C thylphenyl: 30 b) when R' is O
when R is methyl, and R is NH-C(O) CH-O-, S 35 R" is not unsubstituted naphthyl, 2-methoxy, 4-nitrophenyl, 4-chloro-2-methylphenyl, or 4-t-butylphenyl; and when R is NH C(O) , R is not optionally substi tuted phenyl. R" is not 4-dimethylaminophenyl, 2,3,4-trimethoxyphenyl, 40 In certain embodiments, R is NH CO) ; and R is or 3.5 dimethoxyphenyl; and/or phenyl optionally substituted with 1 to 3 substituents inde c) when R is NH-C(O) NH- and Zo is N, R is pendently selected from —OCH, —CH, N(CH), or a not 4-dimethylaminophenyl. solubilizing group. In certain such embodiments, R is selected from option In certain such embodiments, R is NH C(O)—and ally substituted phenyl, benzothiazolyl, or benzoxazolyl. 45 R" is selected from unsubstituted phenyl, 2-methoxyphenyl, In yet another embodiment, the invention provides sirtuin 3-methoxyphenyl, 2,3,4-trimethoxyphenyl, 3,4,5-trimethox modulating compounds of Structural Formula (VIII): yphenyl, 2,4-dimethoxyphenyl, 3.5-dimethoxyphenyl, 2-me thyl-3-methoxyphenyl, 2-morpholinophenyl, 2-methoxy-4- methylphenyl, 2-dimethylaminophenyl, (VIII) 50 4-dimethylaminophenyl, or R3 l, M R21 21N O 2 M 55 O. \ Ž, O CH3 or a salt thereof, wherein: R" is selected from H or optionally substituted C-C, 60 straight or branched alkyl; R’ is selected from NR particularly phenyl: 2-methoxyphenyl: 3-methoxyphenyl: 2,3,4-trimethoxyphenyl: 3,4,5-trimethoxyphenyl; 2.4- 65 dimethoxyphenyl: 3,5-dimethoxyphenyl: 2-methyl-3-meth oxyphenyl: 2-morpholinophenyl: 2-methoxy-4-methylphe nyl: 2-dimethylaminophenyl; or 4-dimethylaminophenyl. US 8,178,536 B2 67 68 In a further embodiment, the invention provides sirtuin In another aspect, the invention provides sirtuin-modulat modulating compounds of Structural Formula (IX): ing compounds of Structural Formula (XI): (IX) (XI) -R31 NH-6-R30, R22 21 O 2 M 2 M s N \ ŽSR 10 O)-(N N \ ŽSR or a salt thereof, wherein: or a salt thereof, wherein: R" is selected from H or optionally substituted C-C, R" is selected from H or optionally substituted C-C, straight or branched alkyl, and straight or branched alkyl, R" is selected from 2,3-dimethoxyphenyl, phenoxyphe 15 R’ is selected from NR’ C(O) = NR, S(O) , nyl, 2-methyl-3-methoxyphenyl, 2-methoxy-4-methylphe - NR' C(O) NR' , - NR' C(S)- NR' , nyl, or phenyl substituted with 1 to 3 substituents, wherein NR' C(S)- NR' CR'R' , NR, C(O) one of said Substituents is a solubilizing group; with the CR'R' NR' , - NR' C(=NR") NR' , provisos that R is not substituted simultaneously with a C(O) NR' , C(O) NR' S(O) , NR' , solubilizing group and a nitro group, and R' is not singly —CR'R' , – NR' C(O) CR'—CR' NR' S Substituted at the 4-position with cyclic Solubilizing group or (O) NR' , – NR' C(O) NR' S(O) = NR, at the 2-position with a morpholino group. CR'R' C(O) NR' , CR'R' C(O) NR' , In one aspect, the invention provides sirtuin-modulating - NR' C(O) CR'—CR' CR'R' , - NR' C compounds of Structural Formula (X): (=N-CN) NR' , NR' C(O) CR'R' O , 25 NR' C(O) CR'R' CR'R' O-, - NR' S(O), (X) CR'R' , NR' S(O). CR'R' CR'R' , or O NR' C(O)—CR'R' , wherein R is an optionally | Substituted C-C straight or branched alkyl, and NH-C-R1, R" is selected from an optionally substituted monocyclic 21 O o or bicyclic aryl, or an optionally Substituted monocyclic or 30 bicyclic heteroaryl, with the provisos that: 2 M when R’ is NH-C(O)-CH=CH-, R is not unsub nu? \ i?, stituted furyl, 5-(2-methyl-3-chlorophenyl)-furanyl. 2,4- dichlorophenyl, 3,5-dichloro-2-methoxyphenyl, 3-nitrophe or a salt thereof, wherein: nyl, 4-chlorophenyl, 4-chloro-3-nitrophenyl, R" is selected from H or optionally substituted C-C, 35 4-isopropylphenyl, 4-methoxyphenyl, 2-methoxy-5-bro straight or branched alkyl, and mophenyl, or unsubstituted phenyl: R" is selected from an optionally substituted monocyclic when R’ is -NH C(O)-CH , R is not 3,4- heteroaryl, an optionally Substituted bicyclic heteroaryl, oran dimethoxyphenyl, 4-chlorophenyl, or unsubstituted phenyl: optionally substituted naphthyl, wherein R is not chloro when R’ is NH C(O) CH-O-, R is not 2,4- benzo(b)thienyl, unsubstituted benzodioxolyl, unsubstituted 40 dimethyl-6-nitrophenyl, 2- or 4-nitrophenyl, 4-cyclohexy benzofuranyl, methyl-benzofuranyl, unsubstituted furanyl, lphenyl, 4-methoxyphenyl, unsubstituted naphthyl, or unsub phenyl-, bromo-, or nitro-furyl, chlorophenyl-isoxazolyl, stituted phenyl, or phenyl monosubstituted, disubstituted or oXobenzopyranyl, unsubstituted naphthyl, methoxy-, trisubstituted solely with substituents selected from straight methyl-, or halo-naphthyl, unsubstituted thienyl, unsubsti or branched-chain alkyl or halo: tuted pyridinyl, or chloropyridinyl. 45 when R’ is NH C(O) CH(CH) O-, R is not In certain embodiments, R is selected from pyrazolyl, 2,4-dichlorophenyl, 4-chlorophenyl, or unsubstituted phenyl: thiazolyl, oxazolyl pyrimidinyl, furyl, thienyl, pyridyl, isox and azolyl, indolyl, benzopyrazolyl, benzothiazolyl, benzox when R’ is NH S(O) , R is not unsubstituted phe azolyl, quinoxalinyl, benzofuranyl, benzothienyl, quinolinyl, nyl. benzoisoxazolyl, benzotriazinyl, triazinyl, naphthyl, or 50 In certain embodiments, R’ is selected from —C(O)— NH-, -NH , or -C(O) NH-CH. In certain embodiments, such as when R’ is selected from C(O) NH-, -NH , or - C(O) NH-CH R is selected from optionally substituted phenyl, benzothiazolyl, quinoxalinyl, or benzoxazolyl. 55 In yet another aspect, the invention provides sirtuin-modu O)- lating compounds of Structural Formula (XII): and wherein R is optionally substituted. In certain such embodiments, R is selected from pyrazolyl, thiazolyl, (XII) oxazolyl pyrimidinyl, indolyl pyrazinyl, triazinyl, or 60
65 or a salt thereof, wherein: O)- each of X7, Xs, X and X is independently selected from N. and R is optionally substituted. CR', or CR', wherein: US 8,178,536 B2 69 70 each R' is independently selected from Hora solubilizing each R" is independently selected from Horasolubilizing group; group; each R" is independently selected from H or optionally each R" is independently selected from H or optionally substituted C-C straight or branched alkyl: substituted C-C straight or branched alkyl: one of X, Xs, X and X is N and the others are selected 5 one of X, Xs, X and X is N and the others are selected from CR' or CR.'; and from CR' or CR.'; and zero to one R is a solubilizing group; zero to one R is a solubilizing group; R" is selected from: R" is selected from:
10 al,O *C, al, O *C, Zre/1 Zre/1 s 15 Zre/1 Ziezu s
^sZiS7.O)- or ZSZOz, 20 ^sZS2.IO) or ZSZOz, wherein: wherein: each Zo. Z, Z and Z is independently selected from 25 each Zo. Z, Z and Z is independently selected from N, CR, or CR.'; and N, CR, or CR.'; and each Z, Zs and Z is independently selected from N. each Z, Zs and Z is independently selected from N. NR', S, O, CR, or CR", wherein: NR', S, O, CR, or CR', wherein: Zero to two of Zo. Z, Z or Z are N: Zero to two of Zo. Z, Z or Z are N: at least one of Z, Zs and Z is N, NR', O or S. 30 at least one of Za Zs and Z is N, NR', S or O: Zero to one of Za Zs and Z is S or O. Zero to one of Z, Zs and Z is S or O. Zero to two of Z, Zs and Z are N or NR'; Zero to two of Z, Zs and Z are N or NR'; zero to one R is a solubilizing group; zero to one R is a solubilizing group; Zero to one R' is an optionally substituted C-C straight or Zero to one R is an optionally Substituted C-C straight or branched alkyl; and 35 branched alkyl, and R’ is selected from NR' C(O) = NR, S(O) , Ri' is selected from —NR' C(O)— —NR' S(O) , -NR' C(O) NR' , - NR' C(S)-NR' , NR, CO) NR, -NR, CS) N.B., CR'R' NR' NR, C( NR) NR, , , C. N. - NR' C(=NR") NR' , 'N S', ' ' ', 40 –C(O) NR' , —C(O) NR' S(O), , – NR' , C(O) NR' , C(O) NR' S(O)2 , NR' , —CR'R'al , – NR' C(O) CR'—CR' NR' S —CR'R' , – NR' C(O) CR'—CR' , – NR' S (O) NR NR' C(O) NR' S(O) NR' (O) NR' , – NR' C(O) NR' S(O) = NR, CRR, C(O) NR, CR. R. GO) NR CR'R' C(O) NR' , CR'R' C(O)NR' NR co, CR dR CRRF NR d —NR' C(O) CR'—CR' CR'R' , —NR' C 45 (—N CN) NR, s NR, C(O) CR'R'' O s (—N CN) NR' , NR' C(O) CR'R' O , NR' C(O) CR'R' CR'R' O-, - NR' S(O), NR' C(O) CR'R' CR'R' O-, - NR S(O), CR'R' NR' S(O). CR'R' CR'R' , or CR'R' , —NR'—S(O), CR'R' CR'R' , or NR' C(O) CR'R' ; and —NR' C(O)—CR'R' ; and R" is selected from an optionally Substituted monocyclic R" is selected from an optionally substituted monocyclic 50 or bicyclic aryl, or an optionally substituted monocyclic or or bicyclic aryl, or an optionally Substituted monocyclic or bicyclic heteroaryl, with the proviso that: bicyclic heteroaryl, with the proviso that when R' is when X, is N, R is
55
as nS
a'sZ Z a'sZ Z 13 Z2 13 zá 60 Zo. Z, Zia and Zis are each CH, and R is —NHC(O)—, and each of Zo. Z, Z and Z is independently selected R" is not an optionally substituted phenyl. from CR, or CR', then: In certain embodiments, the compounds of Structural For- a) at least one of Xs, X9 or Xo is C—(C-C straight or mula (XI) have the following values: 65 branched alkyl) or C-(solubilizing group); or each of X7, Xs, X and Xo is independently selected from b) at least one of Zo. Z, Zand Z is CR, wherein R' N, CR'', or CR", wherein: is a solubilizing group. US 8,178,536 B2 71 72 In certain embodiments, R is NH-C(O)—and R' is -continued selected from:
Z14
Z132rt Z11: ZS2.C. O 10 In certain such embodiments, R is not phenyl-substituted furyl. In certain embodiments, R' is selected from
15
O In certain embodiments, R' is selected from optionally NS Substituted phenyl, optionally Substituted pyridyl, optionally substituted thienyl or optionally substituted furyl. Z13, 27.11 In certain embodiments, R' is /.../ 2 each of Zo. Z, Z and Z is independently selected from CR, or CR'; 25 R’ is selected from NH-C(O) , NH C(O)– CH-CH(CH)–O, NH C(O) NH , NH C (S)- NH-, NH C(S) NH-CH , or NH S (O) ; and 30 R" is selected from an optionally substituted phenyl, an wherein each of Zo. Z, Z and Z is independently optionally substituted naphthyl, or an optionally Substituted selected from CR” or CR.'; and heteroaryl. R’ is selected from NH C(O) , NH CO) CH In certain such embodiments, R is selected from phenyl, (CH)—O— —NH CO)—CH2—O , or NH S(O) naphthyl, pyrazolyl, furyl, thienyl, pyridyl, isoxazolyl, ben —CH2—CH2—, and 35 Zopyrazolyl, benzofuryl, benzothienyl, quinolinyl, ben R" is selected from an optionally substituted aryl, or an Zoisoxazolyl, or optionally substituted heteroaryl. In certain such embodiments, R is optionally substituted with 1 to 3 substituents independently selected from H —OCH-CH —N(CH), phenyl, phenoxy, 3,4-dioxym 40 N ethylene, fluoro, or another solubilizing group. Suitable examples of R' include unsubstituted quinolinyl, 2.4- dimethoxyphenyl, 3,4-dimethoxyphenyl, 3,5-dimethoxyphe OX=0. nyl, 3,4,5-trimethoxyphenyl, 2,3,4-trimethoxyphenyl, 2-dimethylaminophenyl, 3-dimethylaminophenyl, 4-dim 45 ethylaminophenyl, 3,5-dimethylphenyl, 3,5-difluorophenyl, 3-trifluoromethoxyphenyl, unsubstituted quinoxalinyl, and R is optionally substituted (e.g., optionally substituted unsubstituted benzopyrimidinyl, with up to three substituents independently selected from —OCH, —CH, -N(CH), —O-phenyl, or another solu 50 bilizing group). Suitable examples of R' include unsubsti tuted phenyl, 3-methoxyphenyl, 4-methoxyphenyl, 2.3 dimethoxyphenyl, 2,4-dimethoxyphenyl, 2.5-bis(trifluorom ethyl)phenyl, 3,4-dimethoxyphenyl, 3,5-dimethoxyphenyl, 3,4,5-trimethoxyphenyl, 2,3,4-trimethoxyphenyl, 2-meth 55 oxy-4-methylphenyl, 2-phenoxyphenyl, 3-dimethylami nophenyl, 4-dimethylaminophenyl, unsubstituted 2-furanyl. unsubstituted 2-thienyl,
60 21 s 65 C. US 8,178,536 B2 73 -continued
N N N1 N CF
o 21 N —NR' C(O)—CR'R' ; and / R" is selected from an optionally substituted monocyclic s RN s or bicyclic aryl, or an optionally Substituted monocyclic or N-O bicyclic heteroaryl, with the provisos that: when R is NH C(O) , R is not unsubstituted 10 furyl, 5-bromofuryl, unsubstituted phenyl, phenyl monosub stituted with halo or methyl, 3- or 4-methoxyphenyl, 4-bu C N toxyphenyl, 4-t-butylphenyl, 3-trifluoromethylphenyl, 2-benzoylphenyl, 2- or 4-ethoxyphenyl, 2.3-, 2,4-, 3,4-, or 3,5-dimethoxyphenyl, 3,4,5-trimethoxyphenyl, 2,4- or 2-6 15 difluorophenyl, 3,4-dioxymethylene phenyl, 3,4- or 3.5- dimethlyphenyl, 2-chloro-5-bromophenyl, 2-methoxy-5- chlorophenyl, unsubstituted quinolinyl, thiazolyl substituted simultaneously with methyl and phenyl, or ethoxy-Substi In certain embodiments, one or more of the following tuted pyridinyl: conditions applies: when R is NH-C(O)-CH(CH-CH) , R is not when X is N, R is NH C(S)- NH-, and R' is unsubstituted phenyl: phenyl, R is not 2-methoxy-5-nitrophenyl, 2-S-methylphe when R is NH-C(O)—CH2—, R is not unsubsti nyl or 2-acetylphenyl: tuted phenyl, 3-methylphenyl, 4-chlorophenyl, 4-ethoxyphe when Xs is N, R is NH S(O) , and R' is phenyl, 25 nyl, 4-fluorophenyl or 4-methoxyphenyl: R" is not thiadiazole-substituted thienyl or 4-methylsulfo when R is NH C(O) CH-O-, R is not unsub nylphenyl: stituted phenyl or 4-chlorophenyl; and when X is N, R is NH CO. , and R' is phenyl, R' when R is NH S(O) , R is not 3,4-dioxymethyl is not 2,4-difluorophenyl, pyridyl-substituted thienyl, 3,4- ene phenyl, 2,4,5-trimethylphenyl, 2,4,6-trimethylphenyl, dichlorophenyl, 4-t-butylphenyl, or 3-benzyloxyphenyl: 30 2,4- or 3,4-dimethylphenyl, 2,5-difluorophenyl, 2.5- or 3,4- when X is N, R is NH C(O)—and R' is dimethoxyphenyl, fluorophenyl, 4-chlorophenyl, 4-bro mophenyl, 4-ethylphenyl, 4-methylphenyl, 3-methyl-4- methoxyphenyl, unsubstituted phenyl, unsubstituted pyridinyl, unsubstituted thienyl, chloro-substituted thienyl, 35 S or methyl-substituted benzothiazolyl. In certain embodiments, R." is selected from Hor option ally substituted C-C straight or branched alkyl: R’ is selected from NR' C(O) = NR, S(O) , - NR' C(O) NR' , - NR' C(S)- NR' , R" is not 2,3,4-trimethoxyphenyl or 3.5-dimethoxyphenyl: 40 NR' C(S) NR' CR'R' , NR' C(O) and CR'R' NR' , - NR' C(=NR) NR' , when X is N, R is NH-C(O)— and R' is phenyl, C(O) NR' , C(O) NR' S(O) , NR' , R" is not 3,5-dimethoxyphenyl. —CR'R' , – NR' C(O) CR'—CR' , —NR' S (O) NR' , – NR' C(O) NR' S(O) = NR, In a further embodiment, the invention provides com 45 CR'R' C(O) NR' , CR'R' C(O) NR' , pounds of Structural Formula (XIII): - NR' C(O) CR'—CR' CR'R' , - NR' C (—N CN) NR' , NR' C(O) CR'R' O , (XIII) NR' C(O) CR'R' CR'R' O-, - NR S(O), R31 CR'R' , NR' S(O). CR'R' CR'R' , or R21 50 R" is selected from a monocyclic or bicyclic aryl or a N monocyclic or bicyclic heteroaryl, and comprises a solubiliz A. ing group Substituent. S / \ ŽSR In certain embodiments, R is selected from phenyl, naph 55 thyl, pyrazolyl, furyl, thienyl, pyridyl, isoxazolyl, benzopy razolyl, benzofuryl, benzothienyl, quinolinyl, benzoisox or a salt thereof, wherein: azolyl, or R" is selected from H or optionally substituted C-C, straight or branched alkyl; R’ is selected from NR' C(O) = NR, S(O) , 60 H -NR' C(O) NR' , - NR' C(S)-NR' , N NR' C(S)- NR' CR'R' , NR' C(O) X=0. CR'R' NR' , - NR' C(=NR) NR' , O C(O) NR' , C(O) NR' S(O) , NR' , —CR'R' , – NR' C(O) CR'—CR' , – NR' S 65 (O) NR' , – NR' C(O) NR' S(O) = NR, CR'R' C(O) NR' , CR'R' C(O) NR' , and R is optionally substituted. US 8,178,536 B2 75 In certain embodiments, R is selected from NH-C -continued (O) , NH C(O) CH-CH(CH)–O, NH-C(O) NH-, -NH C(S) NH-, -NH C(S) NH-CH , Z10 or —NH S(O) ; and R" is selected from an optionally substituted phenyl, an A. X ^rs optionally substituted naphthyl, or an optionally Substituted Z13, a Z11 O heteroaryl. nzá , or 16-z, s In certain such embodiments, particularly when R is -NH C(O) , R is selected from R is selected from wherein: unsubstituted phenyl, 3-methoxyphenyl, 4-methoxyphenyl, 10 2.3 dimethoxyphenyl, 2,4-dimethoxyphenyl, 2.5-bis(trifluo each Zo. Z, Z and Z is independently selected from romethyl)phenyl, 3,4-dimethoxyphenyl, 3,5-dimethoxyphe N, CR, or CR.'; and nyl, 3,4,5-trimethoxyphenyl, 2,3,4-trimethoxyphenyl, each Z, Zs and Z is independently selected from N. 2-methoxy-4-methylphenyl, 2-phenoxyphenyl, 3-dimethy NR', S, O, CR'', or CR', wherein: laminophenyl, 4-dimethylaminophenyl, unsubstituted 15 2-furanyl, unsubstituted 2-thienyl, Zero to two of Zo. Z, Z or Z are N: at least one of Za Zs and Z is N, NR', O or S. Zero to one of Z, Zs and Z is S or O. 21 Zero to two of Z, Zs and Z are N or NR'; Zero to one R is a solubilizing group; and e -(- s Zero to one R is an optionally Substituted C-C straight or N/ s s branched alkyl: 25 each R' is independently selected from Hora solubilizing group; R’ is selected from —NR' C(O) , NR S(O) , - NR' C(O) NR' , - NR' C(S)- NR' , NR' C(S)- NR' CR'R' , NR, C(O) s Al-O= s 30 CR'R' NR' , - NR' C(=NR") NR' , C(O) NR' , C(O) NR' S(O) , NR' , O - —CR'R'', , – NR' C(O) CR'—CR' , —NR' S (O) NR' , – NR' C(O) NR' S(O) = NR, 35 CR'R' C(O) NR' , CR'R' C(O) NR' , - NR' C(O)—CR'—CR-CR'R' , - NR' C (—N CN) NR' , NR' C(O) CR'R'-O-, C 2's O NR' C(O) CR'R' CR'R'-O-, - NR' S(O), CR'R' , NR' S(O), CR'R' CR'R' , 40 NR' C(O) CR'R' CR'R' , NR' C(S) NR' CR'R' CR'R' , NR' C(O) O O In one aspect, the invention provides sirtuin-modulating compounds of Structural Formula (XIV): each R" is independently selected from H or optionally Substituted C-C straight or branched alkyl, and 45 R" is selected from an optionally substituted monocyclic 31 (XIV) or bicyclic aryl, or an optionally Substituted monocyclic or R23 2 Y R R bicyclic heteroaryl, 2\N2N wherein when R' is 50
R25 or a salt thereof, wherein: each of R and R is independently selected from H, 55 —CH or a solubilizing group: R’ is selected from Hora solubilizing group; and R" is selected from: 60 R’ is NH C(O) and R is -H, R is not an optionally Substituted phenyl group, and wherein said compound is not 2-chloro-N-3-3-(cyclohexylamino) imidazol-2-alpyridin-2-ylphenyl-4-nitrobenzamide. In certain embodiments, each of R and R is indepen 65 dently selected from H. —CH or a solubilizing group; R’ is selected from H, or a solubilizing group; and US 8,178,536 B2 77 78 R" is selected from: R" is selected from:
5 Z14
A-XZ - All IOI6 O nza O Zs s
10 wherein: wherein: each Zo. Z, Z and Z is independently selected from each Zo. Z, Z and Z is independently selected from N, CR, or CR.'; and N, CR, or CR.'; and each Z, Zs and Z is independently selected from N. each Z, Zs and Z is independently selected from N. NR', S, O, CR2, or CR', 15 NR', S, O, CR, or CR', wherein: wherein: Zero to two of Zo. Z, Z or Z are N: Zero to two of Zo. Z, Z or Z are N: at least one of Za Zs and Z is N, NR', O or S. at least one of Z, Zs and Z is N, NR', O or S. Zero to one of Z, Zs and Z is S or O; Zero to one of Z, Zs and Z is S or O. Zero to two of Z, Zs and Z is N or NR'; Zero to two of Z, Zs and Z are N or NR'; Zero to one R is a solubilizing group; and Zero to one R is a solubilizing group; and Zero to one R is an optionally Substituted C-C straight or Zero to one R is an optionally Substituted C-C straight or branched alkyl: branched alkyl: each R" is independently selected from Horasolubilizing each R' is independently selected from Hora solubilizing 25 group; group; R’ is selected from NR' C(O) = NR, S(O) , - NR' C(O) NR' , - NR' C(S)- NR' , NR' C(S) NR' CR'R' , NR' C(O) CR'R' NR' , - NR' C(=NR") NR' , 30 C(O) NR' , C(O) NR' S(O) , NR' , —CR'R' , – NR' C(O) CR'—CR' , —NR' S (O). NR = NR, C(O) NR' S(O), , – NR CR'R' C(O) NR' , CR'R' C(O) NR' , - NR' C(O) CR'—CR' CR'R' , - NR' C 35 (—N CN) NR' , NR' C(O) CR'R'-O-, NR' C(O) CR'R' CR'R'-O-, - NR' S(O), CR'R' , NR' S(O), CR'R' CR'R' , NR' C(O) CR'R' CR'R' , NR' C(S) NR, CR, 'R' CR'R', , NR, C(O) O O 40 —NR' C(O)—CR'R' (particularly —NH CO)—); —NR' C(O)—CR'R' (particularly —NH CO) ); and and each R" is independently selected from H or optionally each R" is independently selected from H or optionally Substituted C-C straight or branched alkyl, and Substituted C-C straight or branched alkyl, and R" is selected from an optionally substituted monocyclic 45 R" is selected from an optionally substituted monocyclic or bicyclic aryl, or an optionally Substituted monocyclic or or bicyclic aryl, or an optionally Substituted monocyclic or bicyclic heteroaryl. bicyclic heteroaryl, In certain such embodiments, R is not 2,4-dimethox wherein when R' is phenyl, at least one of R. R', or R yphenyl. is a solubilizing group and wherein said compound is not Typically, R is selected from H. —CH2 N(CH), or 50 2-chloro-N-3-3-(cyclohexylamino)imidazo[1,2-alpyridin 2-yl)phenyl-4-nitrobenzamide. Typically, R is selected from H. —CH, N(CH), or
55
Typically, Rand Rare H. Typically, R' is selected from phenyl, pyridyl, thienyl or 60 furyl, particularly optionally substituted phenyl. Preferably, a phenyl is optionally substituted with: Typically, Rand Rare H. a) up to three —O-CH groups; or Typically, R' is selected from phenyl, pyridyl, thienyl or b) one —N(CH) group. furyl, particularly optionally substituted phenyl. Preferably, a In certain embodiments, each of R and R is indepen 65 phenyl is optionally substituted with: dently selected from H. —CH or a solubilizing group; b) up to three —O—CH groups; or R’ is selected from H, or a solubilizing group; and b) one —N(CH) group. US 8,178,536 B2 79 In another aspect, the invention provides sirtuin-modulat ing compounds of Structural Formula (XV):
(XV) 5
10 —NR' C(O)—CR'R' (particularly —NH CO)—); and each R" is independently selected from H or optionally or a salt thereof, wherein: Substituted C-C straight or branched alkyl; and R’ is selected from NR C(O)— —NR' S(O) , 15 R is an optionally substituted phenyl, wherein: -NR' C(O) NR' , - NR' C(S)-NR' , when R is NH C(O) , R is a substituted phenyl NR' C(S)- NR' CR'R' , NR' C(O) other than phenyl singly substituted with halo, methyl, nitro CR'R' NR' , - NR' C(=NR) NR' , or methoxy: 2-carboxyphenyl: 4-n-pentylphenyl: 4-ethox C(O) NR' , —C(O)—NR' S(O)— —NR' . yphenyl: 2-carboxy-3-nitrophenyl: 2-chloro-4-nitrophenyl: —CR'R'', , – NR' C(O) CR'—CR' , —NR' S 2-methoxy-5-ethylphenyl; 2,4-dimethoxyphenyl: 3,4,5-tri (O) NR' , – NR' C(O) NR' S(O) = NR, methoxyphenyl; 2.4 dichlorophenyl; 2,6-difluorophenyl: 3.5- CR'R' C(O) NR' , CR'R' C(O) NR' , dinitrophenyl: or 3,4-dimethylphenyl: —NR' C(O) CR'—CR' CR'R' , —NR' C NH C (=N-CN) NR' , NR' C(O)—CR'R'-O-, when R is NR' C(O)—CR'R' or NR' C(O) CR'R' CR'R'-O-, - NR' S(O), (O)-CH(CH)—O, R is a substituted phenyl: CR'R'', , NR' S(O), CR 'R' CR'R' , 25 when R is NH-C(O)-CH, R is not unsubstituted NR' C(O) CR'R' CR'R' , NR' C(S) phenyl, 4-methoxyphenyl: 3,4-dimethoxyphenyl or 4-chlo NR, CR, 'R' CR'R' , NR, C(O) O O rophenyl: —NR' C(O)—CR'R' (particularly —NH CO) ); when R is NH C(O)-CH, O, R is not 2,4-bis(1, and 1-dimethylpropyl)phenyl: each R" is independently selected from H or optionally 30 when R is NH C(O) NH-, R is not 4-methox Substituted C-C straight or branched alkyl, and yphenyl; and R is selected from an optionally substituted bicyclic aryl, when R is NH-S(O) , R is a substituted phenyl or an optionally Substituted monocyclic or bicyclic het other than 3-methylphenyl, 3-trifluoromethylphenyl, 2,4,5- eroaryl, wherein: or 2,4,6-trimethylphenyl, 2,4- or 3,4-dimethylphenyl, 2.5- or when R is NH C(O) , R’ is not unsubstituted 2-fu 35 3,4-dimethoxyphenyl, 2,5-dimethoxy-4-chlorophenyl, 3.6- ryl, 2-(3-bromofuryl), unsubstituted 2-thienyl, unsubstituted dimethoxy, 4-methylphenyl, 2.5- or 3,4-dichlorophenyl, 2.5- 3-pyridyl, unsubstituted 4-pyridyl, diethoxyphenyl, 2-methyl -5-nitrophenyl, 2-ethoxy-5-bro mophenyl, 2-methoxy-5-bromophenyl, 2-methoxy-3,4-
dichlorophenyl, 2-methoxy-4-methyl-5-bromophenyl, 3.5- 40 dinitro-4-methylphenyl, 3-methyl-4-methoxyphenyl, 3-nitro-4-methylphenyl, 3-methoxy-4-halophenyl, 3-meth oxy-5-chlorophenyl, 4-n-butoxyphenyl, 4-halophenyl, 4-eth ylphenyl, 4-methylphenyl, 4-nitrophenyl, 4-ethoxyphenyl, 4-acetylaminophenyl, 4-methoxyphenyl, 4-t-butylphenyl, or 45 para-biphenyl. In certain embodiments, R is selected from NR’ C and when R is NR' S(O) , R’ is not unsubstituted 2-thienyl or unsubstituted naphthyl. In yet another aspect, the invention provides sirtuin-modu 50 lating compounds of Structural Formula (XVI):
(XVI) R33 55
21Ne.N. each R" is independently selected from H or optionally 60 substituted C-C straight or branched alkyl: N-N / R’ is an optionally substituted C-C straight or branched alkyl; and or a salt thereof, wherein: R is phenyl comprising a solubilizing group Substituent, R’ is selected from —NR' C(O)— —NR' S(O) , wherein: when R is NH S(O), said phenyl comprises an —NR' C(O) NR' , - NR' C(S)- NR' , 65 additional substituent. NR' C(S)-NR' CR'R' , NR, C(O) In certain embodiments, R is selected from NR’ C CR'R' NR' , - NR' C(=NR) NR' , (O) , – NR' C(S) NR' , NR' C(S) NR' US 8,178,536 B2 82 or a salt thereof, wherein R" is selected from:
5
NR' , 10 each R" is independently selected from H or optionally Substituted C-C straight or branched alkyl, and R’ is an optionally substituted C-C straight or branched alkyl. O In certain embodiments, R is optionally substituted on up 15 to three carbon atoms with a substituent independently selected from —O CH, CH, N(CH), —S(CH), or CN; or substituted on adjacent carbon atoms with wherein: each Zo. Z, Z and Z is independently selected from N, CR, or CR.'; and each Z, Zs and Z is independently selected from N. (). (J NR', S, O, CR, or CR', wherein: 25 Zero to two of Zo. Z, Z or Z are N: bridging said adjacent carbon atoms. at least one of Za Zs and Z is N, NR', S or O; In a further embodiment, the invention provides sirtuin Zero to one of Z, Zs and Z is S or O; modulating compounds of Structural Formula (XVII): Zero to two of Z, Zs and Z are N or NR'; 30 Zero to one R is a solubilizing group; and (XVII) Zero to one R' is an optionally substituted C-C straight or O 24 branched alkyl: R 5-( each R' is independently selected from Hora solubilizing R23 21 2N. R29, 35 group; R’ is selected from NR' C(O) = NR, S(O) , N-N / - NR' C(O) NR' , - NR' C(S)- NR' , NR' C(S)- NR' CR'R' , NR, C(O) CR'R' NR' , - NR' C(=NR") NR' , or a salt thereof, wherein: 40 C(O) NR' , C(O) NR' S(O) , NR' , each of R and R is independently selected from Hor —CR'R' , – NR' C(O) CR'—CR' NR' S —CH, wherein at least one of R and R is H; and (O) NR' , – NR' C(O) NR' S(O) = NR, R’ is phenyl substituted with: CR'R' C(O) NR' , CR'R' C(O) NR' , a) two —O—CH groups; - NR' C(O) CR'—CR' CR'R' , - NR' C b) three —O—CH groups located at the 2.3 and 4 posi 45 (=N-CN) NR' , NR' C(O) CR'R' O , tions; or NR' C(O) CR'R' CR'R' O-, - NR' S(O), c) one —N(CH) group; and; CR'R' , NR' S(O), CR'R' CR'R' , d) when R is CH, one —O-CH group at the 2 or 3 NR' C(O) CR'R' : NR' C(O) CR'R' position, CR'R' , NR' C(S) NR' CR'R' CR'R' , wherein R’ is optionally additionally substituted with a 50 - NR' C(O) O , solubilizing group. In certain embodiments, R is phenyl substituted with: a) three —O—CH groups located at the 2.3 and 4 posi tions; or b) one —N(CH) group. 55 In one aspect, the invention provides sirtuin-modulating compounds of Structural Formula (XVIII):
(XVIII) 60
-NR
65 wherein each R" is independently selected from H or option ally Substituted C-C straight or branched alkyl, and US 8,178,536 B2 83 84 R" is selected from an optionally substituted monocyclic -continued or bicyclic aryl, or an optionally Substituted monocyclic or O bicyclic heteroaryl, with the proviso that when R' is N
-NR) {\,
10 each R" is independently selected from H or optionally Substituted C-C straight or branched alkyl, and R" is selected from an optionally substituted monocyclic Zo. Z, Z and Z are each CH, R is H. and R is or bicyclic aryl, or an optionally Substituted monocyclic or —NHC(O) , R is not an optionally substituted phenyl. bicyclic heteroaryl. In certain embodiments, R' is selected from: 15 In certain Such embodiments, compounds of Structural Formula (XVIII) have the formula:
(XIX) R31 V R21 S N e R20 Sl s 25 - / wherein: each Zo. Z, Z and Z is independently selected from or a salt thereof, wherein N, CR, or CR.'; and R’ is selected from Hora solubilizing group; each Z, Zs and Z is independently selected from N. 30 R’ is selected from NH-C(O) , or - NH-C(O) NR', S, O, CR2, or CR', CH2—, and R is selected from an optionally substituted monocyclic wherein: or bicyclic aryl, or an optionally Substituted monocyclic or Zero to two of Zo. Z, Z or Z are N: bicyclic heteroaryl. at least one of Z, Zs and Z is N, NR', O or S. 35 Typically, R' in compounds of Structural Formula (XVIII) is selected from phenyl, pyridyl, thienyl or furyl, Zero to one of Z, Zs and Z is S or O. particularly optionally Substituted phenyl. Zero to two of Z, Zs and Z are N or NR'; Typically, R is selected from H. —CH N(CH), Zero to one R is a solubilizing group; and Zero to one R is an optionally Substituted C-C straight or 40 branched alkyl: each R' is independently selected from Hora solubilizing group; 45
50
55
60
65 US 8,178,536 B2 85 -continued or a salt thereof, wherein R" is selected from:
Z10 Z Typically, R is selected from phenyl, pyrazolyl, furyl, S O ^. 14 s pyridyl, pyrimidinyl, thienyl, naphthyl, benzopyrazolylben Z13, 2 Z11 O Zofuryl, quinolinyl, quinoxalinyl, or benzothienyl and 10 Sz2 Zs wherein R is optionally substituted. Typically, R is selected from NH-C(O)— or - NH C(O) CH-. wherein: In certain such embodiments, when R is NR' C each Zo. Z, Z and Z is independently selected from (O) , R is not 4-cyanophenyl or 15 N, CR, or CR.'; and each Z, Zs and Z is independently selected from N. NR', S, O, CR, or CR', wherein: N / N O Zero to two of Zo. Z, Z or Z are N: \ -2 N at least one of Za Zs and Z is N, NR', O or S. Zero to one of Z, Zs and Z is S or O. and/or when R is NR S(O) , R is not 4-methox Zero to two of Z, Zs and Z are N or NR'; yphenyl or 4-t-butylphenyl. 25 Zero to one R is a solubilizing group; and In certain such embodiments, when R' is Zero to one R is an optionally Substituted C-C straight or branched alkyl: each R' is independently selected from Hora solubilizing 30 group; Z14 A R' is independently selected from H or a solubilizing ZiS7.O) or ZS2.Oz, group; R’ is selected from NR' C(O) = NR, S(O) , 35 - NR' C(O) NR' , - NR' C(S)- NR' , and R is NR' C(O) , R is not 4-cyanophenyl or NR' C(S)- NR' CR'R' , NR, C(O) CR'R' NR' , - NR' C(=NR") NR' , C(O) NR' , C(O) NR' S(O) , NR' , N 40 —CR'R' , – NR' C(O) CR'—CR' , —NR' S / N (O) NR' , – NR' C(O) NR' S(O) = NR, O CR'R' C(O) NR' , CR'R' C(O) NR' , - NR' C(O)—CR'—CR-CR'R' , - NR' C (—N CN) NR' , NR' C(O) CR'R' O , and/or when Ris 45 NR' C(O) CR'R' CR'R' O-, - NR' S(O), CR'R' , NR' S(O), CR'R' CR'R' , NR' C(O) CR'R' : NR' C(O) CR'R' CR'R' , NR' C(S) NR' CR'R' CR'R' , - NR' C(O) O , Z 14 O 50 ( ) ( )Z14 Zs Z15 ZSZZ15 and R is NR' S(O) , R is not 4-methoxyphenyl or 55 4-t-butylphenyl. In another aspect, the invention provides sirtuin-modulat ing compounds of Structural Formula (XX):
60 (XX) R31 s -NR S N R21
wherein 20,\-N{ X-k/ 65 each R" is independently selected from H or optionally Substituted C-C straight or branched alkyl, and US 8,178,536 B2 87 88 R" is selected from an optionally substituted monocyclic NR' C(S) NR' CR'R' , NR' C(O) or bicyclic aryl, or an optionally Substituted monocyclic or CR'R' NR' , - NR' C(=NR) NR' , bicyclic heteroaryl, wherein when R' is C(O)—NR' , —C(O)—NR' S(O)— —NR' . —CR'R' , – NR' C(O) CR'—CR' NR' S (O). NR = NR, C(O) NR' S(O), , – NR Z10 S CR'R' C(O) NR' , CR'R' C(O) NR' , - NR' C(O) CR'—CR' CR'R' , - NR' C Sz22 (=N-CN) NR' , NR' C(O) CR'R' O , 10 NR' C(O) CR'R' CR'R' O-, - NR' S(O), and Zo. Z, Z and Z are each CH, R" is a solubilizing CR'R' , NR' S(O). CR'R' CR'R' , group. NR' C(O) CR'R' : NR, C(O) CR'R', Typically, R' incompounds of Structural Formula (XX) is CR'R' , NR' C(S) NR' CR'R' CR'R' , selected from phenyl, pyridyl, thienyl or furyl, particularly - NR' C(O) O , optionally substituted phenyl. 15 Typically, R' is selected from H. —CH N(CH), O
NN O s N Or R -NR \ O vo voH N 25 ) - Xu)\ duo -NR \
wherein 30 each R" is independently selected from H or optionally Substituted C-C straight or branched alkyl; and R is an optionally substituted monocyclic or bicyclic heteroaryl, or an optionally substituted bicyclic aryl, wherein: when R is NH-C(O)-CH , R is not unsubsti 35 tuted thien-2-yl: when R is NH C(O) , R’ is not furan-2-yl, 5-bro mofuran-2-yl, or 2-phenyl-4-methylthiazol-5-yl; when R is NH S(O) , R is not unsubstituted naphthyl or 5-chlorothien-2-yl. 40 In certain embodiments, R is selected from pyrrolyl, pyrazolyl pyrazinyl, furyl, pyridyl, pyrimidinyl, or thienyl, and R is optionally substituted and is optionally benzo fused.
45 In certain embodiments, R is selected from NR' C
Typically, R is selected from phenyl, pyrazolyl, furyl, pyridyl, pyrimidinyl, thienyl, naphthyl, benzopyrazolylben Zofuryl, quinolinyl, quinoxalinyl, or benzothienyl and 50 wherein R is optionally substituted. Typically, R is selected from NH-C(O)— or -NH C(O)-CH-. In yet another aspect, the invention provides sirtuin-modu lating compounds of Structural Formula (XXI): 55
(XXI) R21-R32, &S N N 60 or a salt thereof, wherein 65 R’ is selected from —NR' C(O)— —NR' S(O) , US 8,178,536 B2 89 90 -continued yphenyl, 2-methoxy-3,4-dichlorophenyl, 2-methoxy, 5-bro O mophenyl-3,4-dioxyethylenephenyl, 3,4-dimethoxyphenyl, 3,4-dichlorophenyl, 3,4-dimethylphenyl, 3- or 4-methylphe nyl, 4-alkoxyphenyl, 4-phenoxyphenyl, 4-halophenyl, 4-bi \ s phenyl, or 4-acetylaminophenyl. ) { Preferably, R is selected from NH C(O) or \ - NH C(O) CH-. -NR s In one aspect, the invention provides sirtuin-modulating compounds of Structural Formula (XXII): wherein 10 each R" is independently selected from H or optionally Substituted C-C straight or branched alkyl; and R33 (XXII) R is selected from benzofuryl, methylfuryl, benzothie- V 2 nyl, pyridyl, pyrazinyl, pyrimidinyl, pyrazolyl, wherein said R4, methylfuryl, pyridyl, pyrazinyl, pyrimidinyl or pyrazolyl is 15 S N optionally benzofused and wherein R is optionally substi- N tuted or further substituted. N N / In a further aspect, the invention provides sirtuin-modulat ing compounds of Structural Formula (XXII): 20 or a salt thereof wherein: R’ is selected from NH-C(O) , or - NH-C(O) (XXII) CH2—, and R33 R is phenyl substituted with Ye, e) one —N(CH) group; 25 f) one CN group at the 3 position; S- g) one —S(CH) group; or &J / or a salt thereof, wherein: 30 cyQ cy1No N R’ is selected from NR' C(O) = NR, S(O) , O —NR' C(O) NR' , - NR' C(S)- NR' , NR, CS), NR' CR'R' NR, CO) bridging the 3 and 4 positions. Cls.8. NS, —, C(O) SNR, N. 35 In another aspect, the invention provides sirtuin-modulat —CR'R' , – NR's C(O) CR'—CR'2 3, – NR' Ss ing compounds of Structural Formula (XXIII): (O). NR = NR, C(O) NR' S(O), , – NR CR'R' C(O) NR' , CR'R' C(O) NR' , (XXIII) —NR' C(O) CR'—CR' CR'R' , - NR' C R31 (—N CN) NR' , NR' C(O) CR'R' , it 4. —NR' C(O) CR'R' CR'R' O , —NR' C J. R (O) CR'R' O-, —NR' S(O), CR'R' , 21 N o y-R". NR' S(O). CR'R' CR'R' , -O / 7
O 45 Sl?R20a \/R'' N--> )(Y - Or or a salt thereof, wherein: R -NR \ 50 each R and R' is independently selected from H or a O solubilizing group; each R', R" and R." is independently selected from Hor optionally Substituted C-C straight or branched alkyl; \ R’ is selected from —NR' C(O) , NR S(O) , ) - 55 - NR' C(O) NR' , - NR' C(S)- NR' , -NR' \ NR' C(S)- NR' CR'R' , NR, C(O) CR'R' NR' , - NR' C(=NR") NR' , - NR' C(O) CR'—CR' , NR' S(O), NR' , wherein each R" is independently selected from H or option- NR' C(O) NR' S(O) , NR' CR'R' C ally Substituted C-C straight or branched alkyl, and 60 (O) NR' , – NR' C(O) CR'—CR' CR'R' , R is an optionally substituted phenyl, wherein: - NR' C(=N-CN) NR' , - NR' C(O)— when R is NR' C(O) , R." is not H: CR'R'-O-, -NR' C(O) CR'R' CR'R' O , when R is NH C(O)-CH, or -NH C(O) NR' S(O). CR'R' , NR' S(O), CR'R'' CH. O. , R is not unsubstituted phenyl or 4-halophenyl: CR'R' , or - NR' C(O) CR'R' ; and and 65 R is selected from an optionally substituted monocyclic when R is NH S(O) , R is not unsubstituted phe or bicyclic aryl, or an optionally Substituted monocyclic or nyl, 2,4- or 3,4-dimethylphenyl, 2,4-dimethyl-5-methox bicyclic heteroaryl, with the provisos that: US 8,178,536 B2 91 92 when R is -NH C(O) , R is not is not 3,5-dinitro -continued phenyl, 4-butoxyphenyl, s V F F HO O
e N F, N In certain embodiments, R is selected from NH-C (O) , or -NH C(O) NR'-. 10 In certain embodiments, R is selected from optionally Substituted phenyl, quinoxalinyl or quinolinyl. For example, R" is optionally substituted with up to 3 substituents inde pendently selected from —OCH, N(CH), or a solubiliz 15 ing group. Suitable examples of R' include 4-dimethylami nophenyl, 3,4-dimethoxyphenyl, 3,5-dimethoxyphenyl, 3.4. O 5-trimethoxyphenyl, 3-methoxy-4-((piperazin-1-yl)methyl) phenyl, 3-methoxy-4-((morpholino)methyl)phenyl, 3-methoxy-4-((pyrrolidin-1-yl)methyl)phenyl, unsubstituted rs2N phenyl, unsubstituted quinoxalinyl, and unsubstituted quino linyl. In a particular aspect, the invention provides sirtuin-modu lating compounds of Structural Formula (XXIII): when R is NH C(O) and each of R. R. R.", R" and R." is hydrogen, R is not 25 (XXIII) R31 M O R" R21 M 30 Y-, - s R'--O) ( ) N-NH R20a unsubstituted phenyl, 2- or 4-nitrophenyl, 2,4-dinitrophenyl, 35 2- or 4-chlorophenyl, 2-bromophenyl, 4-fluorophenyl, 2,4- or a salt thereof, wherein: dichlorophenyl, 2-carboxyphenyl, 2-azidophenyl, 2- or each R and R' is independently selected from Hora 4-aminophenyl, 2-acetamidophenyl, 4-methylphenyl, or solubilizing group; 4-methoxyphenyl: when R is NH-C(O) , R." is methyl; and each of each R', R" and R." is independently selected from Hor R. R. R." and R." is hydrogen, R is not 2-methylami 40 optionally substituted C-C straight or branched alkyl: nophenyl,
, or 45 O N Onn1 N
50 when R is -NH C(O) CH or NH C(S) —NR' C(O)—CR'R' ; and NH-, and each of R', R', R', R." and R." is hydrogen, R" is selected from an optionally substituted monocyclic R" is not unsubstituted phenyl: or bicyclic aryl, or an optionally Substituted monocyclic or when R is NH S(O) , R." is hydrogen or methyl, bicyclic heteroaryl, and each of R. R. R." and R." is hydrogen, R is not 55 wherein: 4-methylphenyl; and i) at least one R is a solubilizing group or at least one R." when R is -NH S(O) , R is hydrogen or is an optionally Substituted C-C straight or branched —CH N(CHCH), and each of R. R. R." and R." is alkyl or both; or hydrogen, R is not ii) R' is a solubilizing group other than CH N 60 (CH2CH). In certain embodiments, R is selected from NH-C (O) , or -NH C(O) NR' . In certain embodiments, R is selected from optionally Substituted phenyl, quinoxalinyl or quinolinyl. For example, O 65 R" is optionally substituted with up to 3 substituents inde /- O pendently selected from —OCH, N(CH), or a solubiliz ing group. Suitable examples of R' include 4-dimethylami US 8,178,536 B2 93 94 nophenyl, 3,4-dimethoxyphenyl, 3.5-dimethoxyphenyl, 3.4. R" is not unsubstituted naphthyl, 4-chlorophenyl, 4-nitro 5-trimethoxyphenyl, 3-methoxy-4-((piperazin-1-yl)methyl) phenyl, 4-methoxyphenyl, unsubstituted phenyl, unsubsti phenyl, 3-methoxy-4-((morpholino)methyl)phenyl, tuted thienyl 3-methoxy-4-((pyrrolidin-1-yl)methyl)phenyl, unsubstituted phenyl, unsubstituted quinoxalinyl, and unsubstituted quino 5 linyl. In yet another aspect, the invention provides sirtuin-modu N O lating compounds of Structural Formula (XXIV): A N CF, O A. o RN 10 R31 (XXIV) V R it R21 when R is NH CO)—CH2—, R is methyl, and each of R', R'.R.", and R." is hydrogen, R is not unsub 15 stituted phenyl: R'-- 7 when R is NH-C(O)-CH=CH, R." is methyl or (); —SR". hydrogen, and each of R', R', R', and R." is hydrogen, Nes?R20a \/R iii. R" is not unsubstituted furyl, nitrophenyl-substituted furyl, 2,4-dichlorophenyl, 3,5-dichloro-2-methoxyphenyl, 3- or 4-nitrophenyl, 4-methoxyphenyl, unsubstituted phenyl, or or a salt thereof, wherein: nitro-substituted thienyl: each R' and R' is independently selected from H or a when R is NH-C(O)-CH(CHCH) , and each of solubilizing group; R. R', R', R", and R." is hydrogen, R is not unsub each R', R." and R" is independently selected from Hor 25 stituted phenyl: optionally Substituted C-C straight or branched alkyl; when R is NH C(O)-CH(CH)-O-, R." is methyl or hydrogen, and each of R. R', R', and R." is hydrogen, R" is not 2,4-dichlorophenyl. In a particular aspect, the invention provides sirtuin-modu 30 lating compounds of Structural Formula (XXIV):
(XXIV)
35
R" is selected from an optionally substituted monocyclic or bicyclic aryl, or an optionally Substituted monocyclic or bicyclic heteroaryl, with the provisos that: 40 when R is NH-C(O)-CH , R is not 2-meth ylphenyl, or 3,4-dimethoxyphenyl: when R is NH-C(O)-CH=CH-, R is not or a salt thereof, wherein: 2-chlorophenyl: each R and R' is independently selected from Hora when R is NH-C(O) NH-, R is not unsubsti 45 solubilizing group and at least one of R and R' is a solu tuted benzimidazolyl: bilizing group; when R is NH S(O) , and each of R. R', R', each R', R" and R." is independently selected from Hor R" and R." is hydrogen, R is not unsubstituted phenyl, optionally substituted C-C straight or branched alkyl: 4-chlorophenyl, 4-methylphenyl, or 4-acetoamidophenyl: 50 R’ is selected from NR’ C(O) = NR, S(O) , when R is NH-S(O) , each of R and R." is - NR' C(O) NR' , - NR' C(S)- NR' , methyl or hydrogen, and each of R. R', and R." is hydro NR' C(S)- NR' CR'R' , NR, C(O) gen, R is not 4-nitrophenyl: CR'R' NR' , - NR' C(=NR") NR' , when R is NH C(O) CH-O-, R." is methyl or - NR' C(O) CR'—CR' , NR' S(O), NR' , hydrogen, and each of R', R', R', and R." is hydrogen, 55 NR' C(O) NR' S(O) , NR, CR, 'R' C R" is not 2.3-, 2.5-, 2,6-, 3,4- or 3,5-dimethylphenyl, 2.4- (O) NR' , – NR' C(O) CR'—CR' CR'R' , dichloromethyl, 2,4-dimethyl-6-bromophenyl, 2- or 4-chlo - NR' C(=N CN) NR' , - NR' C(O)– rophenyl, 2-(1-methylpropyl)phenyl, 5-methyl-2-(1-methyl CR'R' O-, -NR' C(O)—CR'R' CR'R' O-, ethyl)phenyl, 2- or 4-methylphenyl, 2,4-dichloro-6- NR' S(O). CR'R' , NR' S(O). CR'R'' methylphenyl, nitrophenyl, 2,4-dimethyl-6-nitrophenyl, 2- or 60 CR'R' , or NR' C(O) CR'R' , wherein R’ is an 4-methoxyphenyl, 4-acetyl-2-methoxyphenyl, 4-chloro-3,5- optionally substituted C-C straight or branched alkyl; and dimethylphenyl, 3-ethylphenyl. 4.-bromophenyl, 4-cyclo R" is selected from an optionally substituted monocyclic hexyphenyl, 4-(1-methylpropyl)phenyl, 4-(1-methylethyl) or bicyclic aryl, or an optionally Substituted monocyclic or phenyl, 4-(1,1-dimethylethyl)phenyl, or unsubstituted bicyclic heteroaryl. phenyl: 65 In certain embodiments, when R is NH-C(O)— when R is NH C(O)-CH , R." is methyl or CH. , Ris not 2-methylphenyl; or 3,4-dimethoxyphenyl: hydrogen, and each of R', R', R', and R." is hydrogen, when R is NH-C(O)-CH=CH-, R is not 2-chlo US 8,178,536 B2 95 96 rophenyl; and/or when R is NH C(O) NH-, R is or phenyl-substituted quinolinyl, chloro-, bromo- or nitro not unsubstituted benzimidazolyl. substituted thienyl, unsubstituted thienyl, or In a further aspect, the invention provides sirtuin-modulat ing compounds of Structural Formula (XXV):
O (XXV) 4N1
R" ins, 10 ON A R'-- In a particular aspect, the invention provides sirtuin-modu O)-( ) lating compounds of Structural Formula (XXVI): R20a 15 or a salt thereof, wherein: O (XXVI) each R and R' is independently selected from H or a solubilizing group, wherein at least one of R'' and R' is a solubilizing group; is each R', R." and R." is independently selected from Hor optionally Substituted C-C straight or branched alkyl, and R'-- R is an optionally substituted phenyl. () { } In certain embodiments, R is selected from 3,4- 25 N-x / \ 7 dimethoxyphenyl, 2,6-dimethoxyphenyl, or 2,4-dimethox yphenyl; wherein R’ is further optionally substituted with a solubilizing group. or a salt thereof, wherein: In certain embodiments, R is not unsubstituted thienyl: unsubstituted phenyl: 2-methylphenyl: 4-fluorophenyl: 30 each R and R' is independently selected from Hora 4-methoxyphenyl, 4-methylphenyl: 3,4-dioxyethylenephe solubilizing group, wherein at least one of R' or R' is nyl: 3-acetylamino-4-methylphenyl, 3-(6-amino-1-oxo a solubilizing group: hexyl)amino-4-methylphenyl, 3-amino-4-methylphenyl: each R', R" and R." is independently selected from Hor 3,5-dimethoxyphenyl: 3-halo-4-methoxyphenyl: 3-nitro-4- 35 optionally Substituted C-C straight or branched alkyl, methylphenyl; or 4-propoxyphenyl. and In one aspect, the invention provides sirtuin-modulating R is selected from an optionally substituted heteroaryl or compounds of Structural Formula (XXVI): an optionally substituted bicyclic aryl. In another aspect, the invention provides sirtuin-modulat 40 ing compounds of Structural Formula (XXVII): O (XXVI)
(XXVII) R" ins, R" 31 M 45 \ R211 R°, R'-- R" 21 X- R19A O) ( ) ls / Sld / \ V S. N R20a 50 or a salt thereof, wherein: wherein: each R and R' is independently selected from H or a solubilizing group; each R and R' is independently selected from Hora Solubilizing group; each R', R." and R" is independently selected from Hor 55 optionally substituted C-C straight or branched alkyl, and each R" and R." is independently selected from H or R is selected from an optionally substituted heteroarylor optionally Substituted C-C straight or branched alkyl, an optionally substituted bicyclic aryl, with the provisos that: R" is selected from: when each of R and R." is hydrogen or methyl and each of R", Ro and Ro, is hydrogen, R is not 5,6,7,8-tetrahy 60 dronaphthyl, unsubstituted benzofuryl, unsubstituted ben Zothiazolyl, chloro- or nitro-substituted benzothienyl, unsub stituted furyl, phenyl-, bromo- or nitro-substituted furyl, Z10 dimethyl-substituted isoxazolyl, unsubstituted naphthyl, Sz S 5-bromonaphthyl, 4-methylnaphthyl, 1- or 3-methoxynaph 65 Z132 Z11: Z132 Z11 s thyl, azo-substituted naphthyl, unsubstituted pyrazinyl, Z2 Z2 S-methyl-substituted pyridyl, unsubstituted pyridyl, thienyl US 8,178,536 B2 97 98 -continued R" is selected from:
O s Z14 Z14 Z Z15 A-X Z13, a Z11 Z13, 27.11 wherein: Z2 s Z2 s each Zo. Z, Z and Z is independently selected from 10 N, CR, or CR.'; and each Z, Zs and Z is independently selected from N. NR', S, O, CR, or CR", wherein: ^isIO / OZ14 Zero to two of Zo. Z, Z or Z are N: 16sz, O 16sz, s at least one of Z, Zs and Z is N, NR', S or O. 15 Zero to one of Z, Zs and Z is S or O. wherein: Zero to two of Z, Z and Z are N or NR'; Zero to one R is a solubilizing group: each Zo. Z, Z and Z is independently selected from Zero to one R is an optionally Substituted C-C straight or N, CR, or CR.'; and branched alkyl; and each Z, Zs and Z is independently selected from N. R’ is selected from —NR' C(O)— —NR' S(O) , NR', S, O, CR, or CR', wherein: NR' C(O) NR' , – NR' C(S)- NR' , Zero to two of Zo. Z, Z or Z are N: NR' C(S) NR' CR'R' , – NR' C(O) at least one of Za Zs and Z is N, NR', S or O; CR'R' NR' , – NR' C(=NR) NR' , 25 - C(O) NR' , —C(O) NR' S(O) , Zero to one of Z, Zs and Z is S or O; NR' , CR'R' , NR' C(O) Zero to two of Z, Zs and Z are N or NR'; CR'—CR' , – NR' S(O). NR ' , NR, C zero to one R is a solubilizing group; (O) NR' S(O) , NR' CR'R' C(O) NR' CR'R' C(O) NR' , – NR' C(O) 30 Zero to one R is an optionally Substituted C-C straight or CR'—CR, CR 'R' , NR' C(=N CN) branched alkyl; and NR' , NR' C(O) CR'R' O. , NR' C R’ is selected from NR' C(O) = NR, S(O) , (O) CR'R' CR'R' O , NR' S(O), NR' C(O) NR' , NR' C(S) NR' , CR'R' , NR' S(O). CR'R' CR'R' , or NR' C(S) NR' CR'R' , – NR' C(O) —NR' C(O)—CR'R' ; and 35 CR'R' NR' , – NR' C(=NR) NR' , R" is selected from an optionally substituted monocyclic - C(O) NR' , —C(O) NR' S(O) , or bicyclic aryl, or an optionally Substituted monocyclic or bicyclic heteroaryl,
provided that when R is NH C(O) and R' is 40
45 —NR' C(O)—CR'R' ; and R" is selected from an optionally substituted monocyclic or bicyclic aryl, oran optionally Substituted monocyclic or bicyclic heteroaryl, with the provisos that: R" is not unsubstituted pyridyl, 2,6-dimethoxyphenyl, 3,4,5-trimethoxyphenyl or unsubstituted furyl. 50 when R is NH C(O) , R is not pyrazolyl: In a particular aspect, the invention provides sirtuin-modu when R is NH , and R' is thiazolyl, R' is not lating compounds of Structural Formula (XXVII): optionally substituted phenyl or optionally substituted pyridyl; when R is NH C(O)—CH2—, and R' is pyrazolyl, (XXVII) 55 R" R" is not unsubstituted indolylor unsubstituted phenyl: R31, when R is NH C(O)—CH2—, and R' is 21 \ AR211 R'-- X- R19 sa N 60 R20a or a salt thereof, wherein: each R' and R' is independently selected from H or a Solubilizing group; 65 each R" and R." is independently selected from H or optionally Substituted C-C straight or branched alkyl, R" is not 2-methylphenyl or 3,4-dimethoxyphenyl: US 8,178,536 B2 99 100 when R is NH C(O)-CH=CH-, and R' is when R is NH-C(O)—and R' is
10 R" is not 2-chlorophenyl: when R is -NH C(O) NH-, and R is pyrazolyl, R" is not unsubstituted isoxazolyl, unsubstituted naph thyl, unsubstituted phenyl, 2,6-difluorophenyl, 2,5-dim ethylphenyl, 3,4-dichlorophenyl, or 4-chlorophenyl: 15 when R is NH C(O) NH-, and R' is
25 , or R" is not unsubstituted benzimidazolyl; when R is NH-, and R' is pyrazolyl, R is not unsubstituted pyridyl: when R' is a solubilizing group, R' is 1-methylpyrrolyl 30 and R is NH-C(O) , R is not unsubstituted phe nyl, unsubstituted furyl, unsubstituted pyrrolyl, unsub stituted pyrazolyl, unsubstituted isoquinolinyl, unsub In certain embodiments, R is selected from NH-C stituted benzothienyl, chloro-substituted benzothienyl, 35 (O) or - NH-C(O) NR' , preferably NH C 2-fluoro-4-chlorophenyl or phenyl singly substituted (O)—. with a solubilizing group; In certain embodiments, R is selected from optionally when R” is a solubilizing group, R' is thienyland R is Substituted phenyl, quinoxalinyl or quinolinyl; preferably NH CO) , R is not unsubstituted phenyl: optionally substituted phenyl. For example, R is optionally when R' is a solubilizing group, R' is methylimidazolyl 40 substituted with up to 3 substituents independently selected and R is NH-C(O) , R is not 1-methyl-4-(1,1- from —OCH, N(CH), or a solubilizing group. Suitable dimethylethyloxycarbonylamino)pyrrol-2-yl or phenyl examples of R' include 4-dimethylaminophenyl: 3,4- singly Substituted with a solubilizing group; dimethoxyphenyl: 3,5-dimethoxyphenyl: 3,4,5-trimethox when R is NH- and R' is pyridyl, oxadiazolyl or yphenyl: 3-methoxy-4-((piperazin-1-yl)methyl)phenyl: thiadiazolyl, R' is not unsubstituted phenyl, 3-methox 45 3-methoxy-4-((morpholino)methyl)phenyl: 3-methoxy-4- yphenyl or 4-methoxyphenyl: ((pyrrolidin-1-yl)methyl)phenyl; unsubstituted phenyl: when R is NH C(O)—and R' is thiazolyl or pyri unsubstituted quinoxalinyl; and unsubstituted quinolinyl. midinyl, R' is not unsubstituted phenyl: Preferred examples of R' include 3,4-dimethoxyphenyl; 2.6- when R is NH C(O) and R' is dimethoxyphenyl; or 2,4-dimethoxyphenyl; wherein R is 50 further optionally substituted with a solubilizing group. In preferred embodiments, R is NH C(O)—and R' is selected from 3-methoxyphenyl: 3,4-dimethoxyphenyl: 3,4,5-trimethoxyphenyl; or 4-dimethylaminophenyl. In certain embodiments, when R is NH C(O) , R' 55 is not
R" is not unsubstituted pyridyl, unsubstituted thienyl, unsub 60 stituted phenyl, 2-methylphenyl, 4-fluorophenyl, 4-methox yphenyl, 4-methylphenyl, 3,4-dioxyethylenephenyl, 3-acety lamino-4-methylphenyl, 3-(6-amino-1-oxohexyl)amino-4- O methylphenyl, 3-amino-4-methylphenyl, 2,6- dimethoxyphenyl, 3,5-dimethoxyphenyl, 3-halo-4- 65 In certain embodiments, when R is NH C(O) , R' methoxyphenyl, 3-nitro-4-methylphenyl, 4-propoxyphenyl, is not optionally Substituted pyrazolyl, thiazolyl, thienyl, pyr 3,4,5-trimethoxyphenyl or unsubstituted furyl; rolyl or pyrimidinyl; when R is NH C(O)-CH2- or US 8,178,536 B2 101 102 -NH C(O) NH-, R' is not pyrazolyl; and/or when R' when R is NH-C(O) NH , and R' is pyrazolyl, is NH R' is not optionally substituted pyridyl, thiaz R" is not unsubstituted isoxazolyl, unsubstituted naph thyl, unsubstituted phenyl, 2,6-difluorophenyl; 2.5-dim olyl pyrazolyl, thiadiazolyl, or oxadiazolyl. ethylphenyl: 3,4-dichlorophenyl; or 4-chlorophenyl: In a more particular aspect, the invention provides sirtuin when R is a solubilizing group, R' is 1-methylpyrrolyl modulating compounds of Structural Formula (XXVII): and R is NH C(O), R is not unsubstituted phe nyl; unsubstituted furyl; unsubstituted pyrrolyl; unsub stituted pyrazolyl; unsubstituted isoquinolinyl; unsub (XXVII) stituted benzothienyl: chloro-substituted benzothienyl: J." R31 2-fluoro-4-chlorophenyl or phenyl singly substituted R211 10 with a solubilizing group; 21 N A when R' is a solubilizing group, R is thienyl and R is R'-- X-R? NH-C(O) , Ris not unsubstituted phenyl: y-N. when R' is a solubilizing group, R is methylimidazolyl R20a and R is NH-C(O) , R is not 1-methyl-4-(1,1- dimethylethyloxycarbonylamino)pyrrol-2-yl or phenyl 15 singly Substituted with a solubilizing group; and or a salt thereof, wherein: when R is NH C(O) and R' is thiazolyl or pyri each R and R' is independently selected from H or a midinyl, R is not unsubstituted phenyl. Solubilizing group; In certain embodiments, R is selected from NH-C each R" and R." is independently selected from H or (O) or - NH-C(O) NR' , preferably NH C optionally substituted C-C straight or branched alkyl: (O)—. R" is selected from: In certain embodiments, R is selected from optionally Substituted phenyl, quinoxalinyl or quinolinyl; preferably optionally substituted phenyl. For example, R is optionally substituted with up to 3 substituents independently selected 25 from —OCH, N(CH), or a solubilizing group. Suitable examples of R' include 4-dimethylaminophenyl: 3,4- dimethoxyphenyl: 3,5-dimethoxyphenyl: 3,4,5-trimethox Z13, 2 Z11 Z13, 27.11 yphenyl: 3-methoxy-4-((piperazin-1-yl)methyl)phenyl: Z2 s Z2 s 3-methoxy-4-((morpholino)methyl)phenyl: 3-methoxy-4- 30 ((pyrrolidin-1-yl)methyl)phenyl; unsubstituted phenyl: unsubstituted quinoxalinyl; and unsubstituted quinolinyl. Preferred examples of R' include 3,4-dimethoxyphenyl; 2.6- dimethoxyphenyl; or 2,4-dimethoxyphenyl; wherein R is ^rsIO OZ14 further optionally substituted with a solubilizing group. 16sz, O 16sz, s In preferred embodiments, R is NH C(O)—and R' 35 is selected from 3-methoxyphenyl: 3,4-dimethoxyphenyl: wherein: 3,4,5-trimethoxyphenyl; or 4-dimethylaminophenyl. each Zo. Z, Z and Z is independently selected from In yet another aspect, the invention provides compounds of N, CR, or CR.'; and Structural Formula (XXVIII): each Z, Zs and Z is independently selected from N. 40 NR', S, O, CR'', or CR", wherein: (XXVII) one to two of Zo. Z11, Z12 or Z are N: ,, Y R31 s at least one of Z, Zs and Z is N, NR', S or O. 21 N R Zero to one of Z, Zs and Z is S or O. Zero to two of Z, Zs and Z are N or NR'; 45 R A R19 Zero to one R is a solubilizing group: y Zero to one R" is an optionally Substituted C-C straight R20a or branched alkyl; and or a salt thereof, wherein: 50 each R and R' is independently selected from Hora Solubilizing group; each R" and R." is independently selected from H or optionally Substituted C-C straight or branched alkyl, R’ is selected from: 55
Z10 60 A. S —NR' C(O)—CR'R' ; and Z13, 2 Z11 O Z132 Z11 R" is selected from an optionally substituted monocyclic nz3 Z2 s or bicyclic aryl, oran optionally Substituted monocyclic or bicyclic heteroaryl, with the provisos that: wherein: when R is NH-C(O) , R' is not pyrazolyl: 65 each Zo. Z, Z and Z is independently selected from when R is NH CO)—CH2—, and R' is pyrazolyl, N. CR', or CR", wherein one of Zo. Z, Z or Z is R" is not unsubstituted indolylor unsubstituted phenyl: N; and US 8,178,536 B2 103 104 zero to one R is a solubilizing group; group (e.g., —NR' C(O)—) is attached to a bivalent Zero to one R" is an optionally substituted C-C straight arylene or heteroarylene group (e.g., R') and the right hand or branched alkyl; and side of a bivalent group is attached to a monovalentaryl group R’ is selected from NR' C(O) = NR, S(O) , (e.g., R). NR' C(O) NR' , NR' C(S)- NR' , 5 Sirtuin-modulating compounds of the invention having NR' C(S) NR' CR'R' , – NR' C(O) hydroxyl Substituents, unless otherwise indicated, also CR'R' NR' , - NR' C(=NR) NR' , include the related secondary metabolites, such as phosphate, - NR' C(O)—CR'—CR' , —NR' S(O)— Sulfate, acyl (e.g., acetyl, fatty acid acyl) and Sugar (e.g., NR' , – NR' C(O) NR' S(O) , – NR' glucurondate, glucose) derivatives (e.g., of hydroxyl groups), CR'R' C(O) NR' , - NR' C(O)– 10 particularly the Sulfate, acyl and Sugar derivatives. In other CR'—CR, CR 'R' , NR, C(=N CN) words, substituent groups —OH also include —OSOM", NR' , – NR' C(O) CR'R'', O NR' C where M is a suitable cation (preferably H, NH, or an (O) CR'R' CR'R' O , NR' S(O), alkali metalion such as Na' or K) and Sugars such as CR'R'-, - NR' S(O). CR'R' CR'R' , or 15
—NR' C(O)—CR'R' ; and R" is selected from an optionally substituted monocyclic or bicyclic aryl, oran optionally Substituted monocyclic or bicyclic heteroaryl. In certain embodiments, R is optionally substituted phe nyl. Such as 3-methoxyphenyl, 3,4-dimethoxyphenyl, 3,4,5- trimethoxyphenyl, or 4-dimethylaminophenyl. and In certain embodiments, R is NH CO)—. In preferred embodiments, R is NH C(O)—and R' is an optionally Substituted phenyl. Such as 3-methoxyphenyl, 25 3,4-dimethoxyphenyl, 3,4,5-trimethoxyphenyl, or 4-dim ethylaminophenyl. In a further aspect, Such as when the sirtuin modulator is a sirtuin inhibitor, the invention provides novel sirtuin-modu lating compounds of Formula (VI): 30 These groups are generally cleavable to —OH by hydrolysis or by metabolic (e.g., enzymatic) cleavage. (VI) In certain embodiments, the compounds of the invention exclude one or more of the species disclosed in Tables 4-6. In 35 certain Such embodiments, the compounds of the invention exclude compound 7. Sirtuin-modulating compounds of the invention advanta geously modulate the leveland/or activity of a sirtuin protein, or a salt thereof, wherein: particularly the deacetylase activity of the sirtuin protein. Het is an optionally Substituted heterocyclic aryl group; 40 and Separately or in addition to the above properties, certain Ar is an optionally substituted carbocyclic or heterocyclic sirtuin-modulating compounds of the invention do not Sub aryl group. stantially have one or more of the following activities: inhi In certain embodiments, Het comprises one N heteroatom bition of PI3-kinase, inhibition of aldoreductase, inhibition of and 1 to 2 additional heteroatoms independently selected 45 tyrosine kinase, transactivation of EGFR tyrosine kinase, from N, O or S, such as oxazolopyridyl. coronary dilation, or spasmolytic activity, at concentrations In certain embodiments, Ar' is selected from optionally of the compound that are effective for modulating the substituted phenyl, benzothiazolyl, or benzoxazolyl. When deacetylation activity of a sirtuin protein (e.g., Such as a Ar' is substituted phenyl, typically it is substituted with I to 3 SIRT1 and/or a SIRT3 protein). substituents independently selected from halo, methyl, 50 An alkyl group is a straight chained, branched or cyclic O-methyl, S-methyl or N(CH), morpholino, or 3.4 dioxym non-aromatic hydrocarbon which is completely saturated. ethylene. Typically, a straight chained or branched alkyl group has from Compounds of the invention, including novel compounds 1 to about 20 carbonatoms, preferably from 1 to about 10, and of the invention, can also be used in the methods described a cyclic alkyl group has from 3 to about 10 carbon atoms, herein. 55 preferably from 3 to about 8. Examples of straight chained The compounds and salts thereof described herein also and branched alkyl groups include methyl, ethyl, n-propyl. include their corresponding hydrates (e.g., hemihydrate, iso-propyl. n-butyl, Sec-butyl, tert-butyl, pentyl, hexyl, pentyl monohydrate, dihydrate, trihydrate, tetrahydrate) and sol and octyl. A C1-C4 straight chained or branched alkyl group vates. Suitable solvents for preparation of solvates and is also referred to as a “lower alkyl group. hydrates can generally be selected by a skilled artisan. 60 An alkenyl group is a straight chained, branched or cyclic The compounds and salts thereof can be present in amor non-aromatic hydrocarbon which contains one or more phous or crystalline (including co-crystalline and poly double bonds. Typically, the double bonds are not located at morph) forms. the terminus of the alkenyl group, such that the double bond In the compounds described above, bivalent groups dis is not adjacent to another functional group. closed as possible values for variables can have either orien 65 An alkynyl group is a straight chained, branched or cyclic tation, provided that Such orientation results in a stable mol non-aromatic hydrocarbon which contains one or more triple ecule. Preferably, however, the left hand side of a bivalent bonds. Typically, the triple bonds are not located at the ter US 8,178,536 B2 105 106 minus of the alkynyl group, Such that the triple bond is not Combinations of substituents and variables envisioned by adjacent to another functional group. this invention are only those that result in the formation of A ring (e.g., 5- to 7-membered ring) or cyclic group stable compounds. As used herein, the term “stable' refers to includes carbocyclic and heterocyclic rings. Such rings can compounds that possess stability Sufficient to allow manufac be saturated or unsaturated, including aromatic. Heterocyclic 5 ture and that maintain the integrity of the compound for a rings typically contain 1 to 4 heteroatoms, although oxygen sufficient period of time to be useful for the purposes detailed and Sulfur atoms cannot be adjacent to each other. herein. Aromatic (aryl) groups include carbocyclic aromatic A hydrogen-bond donating group is a functional group groups such as phenyl, naphthyl, and anthracyl, and het having a partially positively-charged hydrogen atom (e.g., eroaryl groups such as imidazolyl, thienyl, furyl, pyridyl, 10 —OH, -NH2. —SH) or a group (e.g., an ester) that metabo pyrimidyl, pyranyl, pyrazolyl pyrroyl pyrazinyl, thiazolyl, lizes into a group capable of donating a hydrogen bond. oxazolyl, and tetrazolyl. As used herein, a “solubilizing group' is a moiety that has Aromatic groups also include fused polycyclic aromatic hydrophilic character sufficient to improve or increase the ring systems in which a carbocyclic aromatic ring or het water-solubility of the compound in which it is included, as eroaryl ring is fused to one or more other heteroaryl rings. 15 compared to an analog compound that does not include the Examples include benzothienyl, benzofuryl, indolyl, quino group. The hydrophilic character can be achieved by any linyl, benzothiazole, benzoxazole, benzimidazole, quinoli nyl, isoquinolinyl and isoindolyl. means, such as by the inclusion of functional groups that Non-aromatic heterocyclic rings are non-aromatic car ionize under the conditions of use to form charged moieties bocyclic rings which include one or more heteroatoms such as (e.g., carboxylic acids, Sulfonic acids, phosphoric acids, nitrogen, oxygen or Sulfur in the ring. The ring can be five, six, amines, etc.); groups that include permanent charges (e.g., seven or eight-membered. Examples include tetrahydrofuryl, quaternary ammonium groups); and/or heteroatoms or het tetrahyrothiophenyl, morpholino, thiomorpholino, pyrrolidi eroatomic groups (e.g., O, S, N, NH, N-(CH2), R. nyl, piperazinyl, piperidinyl, and thiazolidinyl, along with the N-(CH.), C(O)R’, N-(CH.), C(O)OR", N-(CH.), cyclic form of Sugars. —S(O).R", N-(CH), S(O).R", N-(CH.), C(O)NR'R'', A ring fused to a second ring shares at least one common 25 etc., wherein R is selected from hydrogen, lower alkyl, lower bond. cycloalkyl, (C6-C14) aryl, phenyl, naphthyl, (C7-C20) ary Suitable Substituents on an alkyl, alkenyl, alkynyl, aryl, lalkyl and benzyl, wherein R is optionally substituted; and y non-aromatic heterocyclic oraryl group (carbocyclic and het is an integer ranging from 0 to 6), optionally Substituted eroaryl) are those which do not substantially interfere with heterocyclic groups (e.g., -(CH2), R', -(CH2), C the ability of the disclosed compounds to have one or more of 30 (O) R, (CH), O-(CH) R', wherein R is the properties disclosed herein. A substituent substantially selected from an optionally Substituted Saturated monocyclic interferes with the properties of a compound when the mag heterocycle, an optionally substituted saturated bicyclic fused nitude of the property is reduced by more than about 50% in heterocycle, an optionally Substituted Saturated bicyclic spiro a compound with the Substituent compared with a compound heterocycle, an optionally Substituted heteroaryl and an without the substituent. Examples of suitable substituents 35 optionally substituted partially substituted non-aryl hetero include —OH, halogen ( Br. —Cl, —I and —F), —OR, - O COR, COR, C(O)R, CN, NO, cycle; and n is an integer ranging from 0 to 2). It should be -COOH,-COOR, OCOR', C(O)NR'R'', OC(O) understood that substituents present on R or R' need not NR'R', SOH, NH, NHR, N(RR), COOR, improve or increase water solubility over their unsubstituted CHO, CONH, CONHR, CONCRR), NH counterparts to be within the scope of this definition. All that COR, NRCOR, NHCONH, NHCONRH, NH 40 is required is that Such substituents do not significantly CONGRR), NRCONH, NRCONRH, NRCON reverse the improvement in water-solubility afforded by the (RR), —C(=NH) NH, C(-NH) NHR, unsubstituted R' or R moiety. C(-NH) N(RR), —C(NR) NH, In one embodiment, the solubilizing group increases the C(-NR). NHR, C(-NR) N(RR), NH C water-solubility of the corresponding compound lacking the (—NH) NH, -NH C(=NH) NHR", NH C 45 solubilizing group at least 5-fold, preferably at least 10-fold, (—NH) N(RR), NH C(=NR) NH, -NH C more preferably at least 20-fold and most preferably at least (—NR). NHR, NH C(-NR) N(RR), 50-fold. NRH C(-NH) NH, NR C(-NH) NHR, In one preferred embodiment, the Solubilizing group is a NR C(-NH) N(RR), NR C(—NR)NH, moiety of the formula: -(CH) R' N(R')(R'), NR C(-NR) NHR', NR C(—NR) N 50 wherein: (RR), NHNH, NHNHR, NHR'R', SONH, n is selected from 0, 1 or 2; SONHR', SONR'R''. CH-CHR, CH-CRR, R" is selected from a bond, —C(O)—, or O(CH), and CR-CRR, CR -CHR, CR-CRR, CCR, each R' is independently selected from: —SH, -SOR" (kis 0,1 or 2), S(O), OR" (kis 0, 1 or 2) and a. hydrogen; -NH C(-NH) NH. R. Rare each independently an b. C-C straight or branched alkyl, wherein said alkyl is aliphatic, substituted aliphatic, benzyl, substituted benzyl, 55 optionally substituted with halo, CN, OH, O (C-C, aromatic or Substituted aromatic group, preferably an alkyl, straight or branched alkyl), N(R')(R'), or—O; benzylic or aryl group. In addition, NR'R'', taken together, C. can also form a Substituted or unsubstituted non-aromatic heterocyclic group. A non-aromatic heterocyclic group, ben Zylic group or aryl group can also have an aliphatic or Sub 60 stituted aliphatic group as a Substituent. A Substituted ali phatic group can also have a non-aromatic heterocyclic ring, a Substituted a non-aromatic heterocyclic ring, benzyl, Sub stituted benzyl, aryl or Substituted aryl group as a Substituent. A Substituted aliphatic, non-aromatic heterocyclic group, 65 Substituted aryl, or Substituted benzyl group can have more than one Substituent. US 8,178,536 B2 107 108 any
10
ring structure is optionally benzofused or fused to a monocy 15 clic heteroaryl to produce a bicyclic ring. For clarity, the term “C to C alkylene, alkenylene or alkanediylidene bridge' means the multivalent structures f both R' moieties are taken together with the nitrogen CH , CH-CH , CH , —CH , atom to which they are bound to form a ring of the -CH=CH-, or=CH-CH=. The two R' moieties that Structure are optionally bound to one another can be either on the same carbon atom or different carbon atoms. The former produces a spiro bicyclic ring, while the latter produces a fused bicyclic ring. It will be obvious to those of skill in the art that when two 1434Z zZ30 25 R" are bound to one another to form a ring (whether directly Z35 N Ol or through one of the recited bridges), one or more terminal Zs,32. M hydrogen atoms on each R' will be lost. Accordingly, a “suitable non-cyclic R” moiety available for forming a ring is a non-cyclic R that comprises at least one terminal hydro gen atom. In another preferred embodiment, the solubilizing group is N a moiety of the formula: —(CH), O R', whereinn and R1 N , R'' are as defined above. 35 In another preferred embodiment, the solubilizing group is O a moiety of the formula: —(CH), C(O)—R', wherein n g. both R' moieties are taken together with the nitrogen and Rare as defined above. atom to which they are bound to form a 5-membered In a more preferred embodiment, a solubilizing group is heteroaryl ring containing 1 to 3 additional Natoms, selected from —(CH) R', wherein n is 0, 1 or 2; and wherein said heteroaryl ring is optionally substituted 40 R' is selected from with R'; wherein: each Z is independently selected from —O— —S—, NR' , or C(R)(R) , 45 wherein: O ) N N at least three of Zao, Z21, Z22, and Zas are-C(R)(R')–: O S at least three of Z24, Z2s. Z26, Z27, and Z2s are –C(R) (R) ; 50 R R at least four of Zao, Zai, Z2, and Zss are —C(R)(R')–: and N N at least four of Zsa Zss, Zsa Zs, and Zss are –C(R) (R) ; C N each R" is independently selected from hydrogen or a 55 C-C straight or branched alkyl optionally substituted with R N one or more substituent independently selected from halo, R1 YR, N R. CN, -OH,-OCH, -NH, -NH(CH), N(CH), or each R" is independently selected from R', halo, CN, OH, 60 O—(C-C straight or branched alkyl), N(R')(R), —CR', SR", =NR", =NOR', or =O; any two suitable non-cyclic Rare optionally bound to one another directly or via a C to C alkylene, alkenylene or 65 alkanediylidene bridge to produce a bicyclic fused or spiro ring; and US 8,178,536 B2 109 K)
SX
NRR ,
C US 8,178,536 B2 111 112 -continued -continued opo- or oro N s N
Cl O 10 \ N \- HO V R", OH,
15 O O
s N s R 1 OR
N O H YR, O 1. s OR O R O R1 O O 25 wherein R" are as defined above. In an even more preferred embodiment, a solubilizing group is selected from 2-dimethylaminoethylcarbamoyl, pip 30 erazin-1-ylcarbonyl, piperazinylmethyl, dimethylaminom ethyl, 4-methylpiperazin-1-ylmethyl, 4-aminopiperidin-1-yl -methyl, 4-fluoropiperidin-1-yl-methyl, morpholinomethyl, pyrrolidin-1-ylmethyl, 2-oxo-4-benzylpiperazin-1-ylmethyl, 35 4-benzylpiperazin-1-ylmethyl, 3-oxopiperazin-1-ylmethyl, piperidin-1-ylmethyl, piperazin-1-ylethyl, 2,3-dioxopropy laminomethyl, thiazolidin-3-ylmethyl, 4-acetylpiperazin-1- ylmethyl, 4-acetylpiperazin-1-yl, morpholino, 3.3-difluoro azetidin-1-ylmethyl, 2H-tetrazol-5-ylmethyl, 40 thiomorpholin-4-ylmethyl, 1-oxothiomorpholin-4-ylmethyl, 1,1-dioxothiomorpholin-4-ylmethyl, 1H -imidazol-1-ylm ethyl, 3,5-dimethylpiperazin-1ylmethyl, 4-hydroxypiperi N O din-1-ylmethyl, N-methyl(1-acetylpiperidin-4-yl)-aminom ethyl, N-methylcuinuclidin-3-ylaminomethyl, 1H-1,2,4- triazol-1-ylmethyl, 1-methylpiperidin-3-yl-oxymethyl, or 4-fluoropiperidin-1-yl. is To the extent not included within any of the definitions set R R forth above, the term “solubilizing group' also includes moi eties disclosed as being attached to the 7-position of 1-cyclo propyl-6-fluoro-1,4-dihydro-4-oxoquinoline-3-carboxylic 3, 50 acid (ciprofloxacin) and its derivatives, as disclosed in PCT publications WO 2005026165, WO 2005049602, and WO 2005033108, and European Patent publications EP 0343524, EP 068.8772, EP O153163, EP 0159174; as well as “water Ri'NN 55 -solubilizing groups' described in United States patent pub W s lication 2006/0035891. The disclosure of each of these patent R publications is incorporated herein by reference. Double bonds indicated in a structure as: 1N 60 \ { risri V R", are intended to include both the (E)- and (Z)-configuration. 65 Preferably, double bonds are in the (E)-configuration. A Sugar is an aldehyde or ketone derivative of a straight chain polyhydroxy alcohol, which contains at least three car US 8,178,536 B2 113 114 bon atoms. A Sugar can exist as a linear molecule or, prefer Thus, one embodiment relates to a method of making a ably, as a cyclic molecule (e.g., in the pyranose or furanose compound of the structure described herein using the follow form). Preferably, a Sugar is a monosaccharide such as glu ing synthesis scheme: cose or glucuronic acid. In embodiments of the invention where, for example, prolonged residence of a compound 5 derivatized with a Sugar is desired, the Sugar is preferably a O non-naturally occurring Sugar. For example, one or more N NH2 NH2 hydroxyl groups are substituted with another group, such as a n HO PPA -e- halogen (e.g., chlorine). The stereochemical configuration at 220° C. one or more carbonatoms can also be altered, as compared to 0 a naturally occurring Sugar. One example of a Suitable non 21 OH 93% naturally occurring Sugar is Sucralose. NH2 O A fatty acid is a carboxylic acid having a long-chained N N us hydrocarbon moiety. Typically, a fatty acid has an even num N R OH ber of carbon atoms ranging from 12 to 24, often from 14 to 15 HATU, HOAT 20. Fatty acids can be saturated or unsaturated and substituted 21 No DIPEA, DMF or unsubstituted, but are typically unsubstituted. Fatty acids t can be naturally or non-naturally occurring. In embodiments R of the invention where, for example, prolonged residence N time of a compound having a fatty acid moiety is desired, the N N-(O fatty acid is preferably non-naturally occurring. The acyl group of a fatty acid consists of the hydrocarbon moiety and s N the carbonyl moiety of the carboxylic acid functionality, but 21 No excludes the –OH moiety associated with the carboxylic acid functionality. 25 Also included in the present invention are salts, particularly One of skill in the art would recognize that this synthetic pharmaceutically acceptable salts, of the sirtuin-modulating scheme, or similar variants, usefully allows the incorporation compounds described herein. The compounds of the present of a variety of R groups into compounds falling within the invention that possess a sufficiently acidic, a Sufficiently Scope of the instant invention, for example, compounds of the tables below. basic, or both functional groups, can react with any of a 30 number of inorganic bases, and inorganic and organic acids, As can be appreciated by the skilled artisan, the above to form a salt. Alternatively, compounds that are inherently synthetic scheme is not intended to comprise a comprehen charged. Such as those with a quaternary nitrogen, can form a sive list of all means by which the compounds described and salt with an appropriate counterion (e.g., a halide such as claimed in this application may be synthesized. Further meth bromide, chloride, or fluoride, particularly bromide). 35 ods will be evident to those of ordinary skill in the art. Addi Acids commonly employed to form acid addition salts are tionally, the various synthetic steps described above may be inorganic acids such as hydrochloric acid, hydrobromic acid, performed in an alternate sequence or order to give the desired hydroiodic acid, Sulfuric acid, phosphoric acid, and the like, compounds. Synthetic chemistry transformations and meth and organic acids such as p-toluenesulfonic acid, methane odologies useful in synthesizing the sirtuin-modulating com Sulfonic acid, oxalic acid, p-bromophenyl-Sulfonic acid, car 40 pounds described herein are known in the art and include, for bonic acid, Succinic acid, citric acid, benzoic acid, acetic acid, example, those described in R. Larock, Comprehensive and the like. Examples of Such salts include the Sulfate, pyro Organic Transformations (1989); T. W. Greene and P. G. M. sulfate, bisulfate, sulfite, bisulfite, phosphate, monohydro Wuts, Protective Groups in Organic Synthesis, 2d. Ed. genphosphate, dihydrogenphosphate, metaphosphate, pyro (1991); L. Fieser and M. Fieser, Fieser and Fieser's Reagents phosphate, chloride, bromide, iodide, acetate, propionate, 45 for Organic Synthesis (1994); and L. Paquette, ed., Encyclo decanoate, caprylate, acrylate, formate, isobutyrate, caproate, pedia of Reagents for Organic Synthesis (1995). heptanoate, propiolate, oxalate, malonate. Succinate, Suber In an exemplary embodiment, a sirtuin-modulating com ate, sebacate, fumarate, maleate, butyne-1,4-dioate, hexyne pound may traverse the cytoplasmic membrane of a cell. For 1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dini example, a compound may have a cell-permeability of at least trobenzoate, hydroxybenzoate, methoxybenzoate, phthalate, 50 about 20%, 50%, 75%, 80%, 90% or 95%. Sulfonate, Xylenesulfonate, phenylacetate, phenylpropionate, Sirtuin-modulating compounds described herein may also phenylbutyrate, citrate, lactate, gamma-hydroxybutyrate, have one or more of the following characteristics: the com glycolate, tartrate, methanesulfonate, propanesulfonate, pound may be essentially non-toxic to a cell or subject; the naphthalene-1-sulfonate, naphthalene-2-sulfonate, mande sirtuin-modulating compound may be an organic molecule or late, and the like. 55 a small molecule of 2000 amu or less, 1000 amu or less; a Base addition salts include those derived from inorganic compound may have a half-life under normal atmospheric bases, such as ammonium or alkali or alkaline earth metal conditions of at least about 30 days, 60 days, 120 days, 6 hydroxides, carbonates, bicarbonates, and the like. Such months or 1 year, the compound may have a half-life in bases useful in preparing the salts of this invention thus solution of at least about 30 days, 60 days, 120 days, 6 months include Sodium hydroxide, potassium hydroxide, ammonium 60 or 1 year; a sirtuin-modulating compound may be more stable hydroxide, potassium carbonate, and the like. in solution than resveratrol by at least a factor of about 50%, According to another embodiment, the present invention 2 fold, 5 fold, 10 fold, 30 fold, 50 fold or 100 fold; a sirtuin provides methods of producing the above-defined sirtuin modulating compound may promote deacetylation of the modulating compounds. The compounds may be synthesized DNA repair factor Ku70; a sirtuin-modulating compound using conventional techniques. Advantageously, these com 65 may promote deacetylation of RelA/p65; a compound may pounds are conveniently synthesized from readily available increase general turnover rates and enhance the sensitivity of starting materials. cells to TNF-induced apoptosis. US 8,178,536 B2 115 116 In certain embodiments, a sirtuin-modulating compound 5 fold less than the EDso for modulating one or more of does not have any substantial ability to inhibit a histone human SIRT1, SIRT2, SIRT4, SIRT5, SIRT6, or SIRT7, and deacetylase (HDACs) class I, a HDAC class II, or HDACs I even more preferably at least 10 fold, 100 fold or even 1000 and II, at concentrations (e.g., in vivo) effective for modulat fold less. In one embodiment, a SIRT3 modulator does not ing the deacetylase activity of the sirtuin. For instance, in have any substantial ability to modulate a SIRT1 protein. preferred embodiments the sirtuin-modulating compound is a In certain embodiments, a sirtuin-modulating compound sirtuin-activating compound and is chosen to have an ECso for may have a binding affinity for a sirtuin protein of about activating sirtuin deacetylase activity that is at least 5 fold less 10M, 10'M, 10''M, 10'’M or less. A sirtuin-modulat than the ECs for inhibition of an HDAC I and/or HDAC II, ing compound may reduce (activator) or increase (inhibitor) and even more preferably at least 10 fold, 100 fold or even 10 the apparent Km of a sirtuin protein for its substrate or NAD+ 1000 fold less. Methods for assaying HDAC I and/or HDAC (or other cofactor) by a factor of at least about 2,3,4, 5, 10, 20, II activity are well known in the art and kits to perform such 30, 50 or 100. In certain embodiments, Kim values are deter assays may be purchased commercially. See e.g., BioVision, mined using the mass spectrometry assay described herein. Inc. (Mountain View, Calif.; world wide web at biovision. Preferred activating compounds reduce the Km of a sirtuin for com) and Thomas Scientific (Swedesboro, N.J.; world wide 15 its Substrate or cofactor to a greater extent than caused by web at tomas Sci.com). resveratrol at a similar concentration or reduce the Km of a In certain embodiments, a sirtuin-modulating compound sirtuin for its substrate or cofactor similar to that caused by does not have any substantial ability to modulate sirtuin resveratrol at a lower concentration. A sirtuin-modulating homologs. In one embodiment, an activator of a human sir compound may increase the Vmax of a sirtuin protein by a tuin protein may not have any Substantial ability to activate a factor of at least about 2, 3, 4, 5, 10, 20, 30, 50 or 100. A sirtuin protein from lower eukaryotes, particularly yeast or sirtuin-modulating compound may have an ED50 for modu human pathogens, at concentrations (e.g., in vivo) effective lating the deacetylase activity of a SIRT1 and/or SIRT3 pro for activating the deacetylase activity of human sirtuin. For tein of less than about 1nM, less than about 10 nM, less than example, a sirtuin-activating compound may be chosen to about 100 nM, less than about 1 uM, less than about 10 uM, have an ECso for activating a human sirtuin, Such as SIRT1 25 less than about 100 uM, or from about 1-10 nM, from about and/or SIRT3, deacetylase activity that is at least 5 fold less 10-100 nM, from about 0.1-1 uM, from about 1-10 uM or than the ECso for activating a yeast sirtuin, such as Sir2 (Such from about 10-100 uM. A sirtuin-modulating compound may as Candida, S. cerevisiae, etc.), and even more preferably at modulate the deacetylase activity of a SIRT1 and/or SIRT3 least 10 fold, 100 fold or even 1000 fold less. In another protein by a factor of at least about 5, 10, 20, 30, 50, or 100, embodiment, an inhibitor of a sirtuin protein from lower 30 as measured in a cellular assay or in a cell based assay. A eukaryotes, particularly yeast or human pathogens, does not sirtuin-activating compound may cause at least about 10%, have any substantial ability to inhibit a sirtuin protein from 30%, 50%, 80%, 2 fold, 5 fold, 10 fold, 50 fold or 100 fold humans at concentrations (e.g., in vivo) effective for inhibit greater induction of the deacetylase activity of a sirtuin pro ing the deacetylase activity of a sirtuin protein from a lower tein relative to the same concentration of resveratrol. A sir eukaryote. For example, a sirtuin-inhibiting compound may 35 tuin-modulating compound may have an ED50 for modulat be chosen to have an ICso for inhibiting a human sirtuin, Such ing SIRT5 that is at least about 10 fold, 20 fold, 30 fold, 50 as SIRT1 and/or SIRT3, deacetylase activity that is at least 5 fold greater than that for modulating SIRT1 and/or SIRT3. fold less than the ICso for inhibiting a yeast sirtuin, Such as 3. Exemplary Uses Sir2 (Such as Candida, S. cerevisiae, etc.), and even more In certain aspects, the invention provides methods for preferably at least 10 fold, 100 fold or even 1000 fold less. 40 modulating the level and/or activity of a sirtuin protein and In certain embodiments, a sirtuin-modulating compound methods of use thereof. may have the ability to modulate one or more sirtuin protein In certain embodiments, the invention provides methods homologs, such as, for example, one or more of human for using sirtuin-modulating compounds wherein the sirtuin SIRT1, SIRT2, SIRT3, SIRT4, SIRT5, SIRT6, or SIRT7. In modulating compounds activate a sirtuin protein, e.g., one embodiment, a sirtuin-modulating compound has the 45 increase the level and/or activity of a sirtuin protein. Sirtuin ability to modulate both a SIRT1 and a SIRT3 protein. modulating compounds that increase the level and/or activity In other embodiments, a SIRT1 modulator does not have of a sirtuin protein may be useful for a variety of therapeutic any substantial ability to modulate other sirtuin protein applications including, for example, increasing the lifespan homologs, such as, for example, one or more of human of a cell, and treating and/or preventing a wide variety of SIRT2, SIRT3, SIRT4, SIRT5, SIRT6, or SIRT7, at concen 50 diseases and disorders including, for example, diseases or trations (e.g., in vivo) effective for modulating the deacety disorders related to aging or stress, diabetes, obesity, neuro lase activity of human SIRT1. For example, a sirtuin-modu degenerative diseases, cardiovascular disease, blood clotting lating compound may be chosen to have an EDso for disorders, inflammation, cancer, and/or flushing, etc. The modulating human SIRT1 deacetylase activity that is at least methods comprise administering to a Subject in need thereof 5 fold less than the EDso for modulating one or more of 55 a pharmaceutically effective amount of a sirtuin-modulating human SIRT2, SIRT3, SIRT4, SIRT5, SIRT6, or SIRT7, and compound, e.g., a sirtuin-activating compound. even more preferably at least 10 fold, 100 fold or even 1000 In other embodiments, the invention provides methods for fold less. In one embodiment, a SIRT1 modulator does not using sirtuin-modulating compounds wherein the sirtuin have any substantial ability to modulate a SIRT3 protein. modulating compounds decrease sirtuin activity, e.g., In other embodiments, a SIRT3 modulator does not have 60 decrease the level and/or activity of a sirtuin protein. Sirtuin any substantial ability to modulate other sirtuin protein modulating compounds that decrease the level and/or activity homologs, such as, for example, one or more of human of a sirtuin protein may be useful for a variety of therapeutic SIRT1, SIRT2, SIRT4, SIRT5, SIRT6, or SIRT7, at concen application including, for example, increasing cellular sensi trations (e.g., in vivo) effective for modulating the deacety tivity to stress (including increasing radiosensitivity and/or lase activity of human SIRT3. For example, a sirtuin-modu 65 chemosensitivity), increasing the amount and/or rate of apo lating compound may be chosen to have an EDso for ptosis, treatment of cancer (optionally in combination modulating human SIRT3 deacetylase activity that is at least another chemotherapeutic agent), Stimulation of appetite, US 8,178,536 B2 117 118 and/or stimulation of weight gain, etc. The methods comprise homologs thereof. Increasing protein levels can be achieved administering to a subject in need thereof a pharmaceutically by introducing into a cell one or more copies of a nucleic acid effective amount of a sirtuin-modulating compound, e.g., a that encodes a sirtuin. For example, the level of a sirtuin can sirtuin-inhibiting compound. be increased in a mammalian cell by introducing into the While Applicants do not wish to be bound by theory, it is mammalian cell a nucleic acid encoding the sirtuin, e.g., believed that activators and inhibitors of the instant invention increasing the level of SIRT1 by introducing a nucleic acid may interact with a sirtuin at the same location within the encoding the amino acid sequence set forth in GenBank sirtuin protein (e.g., active site or site affecting the Km or Accession No. NP 036370 and/or increasing the level of Vmax of the active site). It is believed that this is the reason SIRT3 by introducing a nucleic acid encoding the amino acid why certain classes of sirtuin activators and inhibitors can 10 have Substantial structural similarity. sequence set forth in GenBank Accession No. AAHO1042. In certain embodiments, the sirtuin-modulating com The nucleic acid may be under the control of a promoter that pounds described herein may be taken alone or in combina regulates the expression of the SIRT1 and/or SIRT3 nucleic tion with other compounds. In one embodiment, a mixture of acid. Alternatively, the nucleic acid may be introduced into two or more sirtuin-modulating compounds may be admin 15 the cell at a location in the genome that is downstream of a istered to a subject in need thereof. In another embodiment, a promoter. Methods for increasing the level of a protein using sirtuin-modulating compound that increases the level and/or these methods are well known in the art. activity of a sirtuin protein may be administered with one or A nucleic acid that is introduced into a cell to increase the more of the following compounds: resveratrol, butein, fisetin, protein level of a sirtuin may encode a protein that is at least piceatannol, or quercetin. In an exemplary embodiment, a about 80%, 85%, 90%, 95%, 98%, or 99% identical to the sirtuin-modulating compound that increases the level and/or sequence of a sirtuin, e.g., SIRT1 (GenBank Accession No. activity of a sirtuin protein may be administered in combina NP 036370) and/or SIRT3 (GenBank Accession No. tion with nicotinic acid. In another embodiment, a sirtuin AAHO1042) protein. For example, the nucleic acid encoding modulating compound that decreases the level and/or activity the protein may be at least about 80%, 85%, 90%.95%.98%, of a sirtuin protein may be administered with one or more of 25 or 99% identical to a nucleic acid encoding a SIRT1 (e.g. the following compounds: nicotinamide (NAM), Suranim; GenBank Accession No. NM 012238) and/or SIRT3 (e.g., NF023 (a G-protein antagonist); NF279 (a purinergic recep GenBank Accession No. BC001042) protein. The nucleic tor antagonist); Trolox (6-hydroxy-2,5,7,8, tetramethylchro acid may also be a nucleic acid that hybridizes, preferably man-2-carboxylic acid); (-)-epigallocatechin (hydroxy on under Stringent hybridization conditions, to a nucleic acid sites 3.5.7.3',4',5'): (-)-epigallocatechin gallate (Hydroxy 30 encoding a wild-type sirtuin, e.g., SIRT1 (GenBank Acces sites 5.7.3',4',5' and gallate ester on 3): cyanidin choloride sion No. NM 012238) and/or SIRT3 (e.g., GenBank Acces (3,5,7,3',4'-pentahydroxyflavylium chloride); delphinidin sion No. BC001042) protein. Stringent hybridization condi chloride (3.5.7.3',4',5'-hexahydroxyflavylium chloride); tions may include hybridization and a wash in 0.2xSSC at 65° myricetin (cannabiscetin; 3.5.7.3',4',5'-hexahydroxyfla C. When using a nucleic acid that encodes a protein that is vone); 3.7.3', 4',5'-pentahydroxyflavone; gossypetin (3,5,7,8, 35 different from a wild-type sirtuin protein, such as a protein 3',4'-hexahydroxyflavone), sirtinol; and splitomicin (see e.g., that is a fragment of a wild-type sirtuin, the protein is prefer Howitz et al. (2003) Nature 425:191: Grozinger et al. (2001) ably biologically active, e.g., is capable of deacetylation. It is J. Biol. Chem. 276:38837; Dedalov et al. (2001) PNAS only necessary to express in a cell a portion of the sirtuin that 98:15113; and Hirao et al. (2003).J. Biol. Chem 278:52773). is biologically active. For example, a protein that differs from In yet another embodiment, one or more sirtuin-modulating 40 wild-type SIRT1 having GenBank Accession No. compounds may be administered with one or more therapeu NP 036370, preferably contains the core structure thereof. tic agents for the treatment or prevention of various diseases, The core structure sometimes refers to amino acids 62-293 of including, for example, cancer, diabetes, neurodegenerative GenBank Accession No. NP 036370, which are encoded by diseases, cardiovascular disease, blood clotting, inflamma nucleotides 237 to 932 of GenBank Accession No. tion, flushing, obesity, ageing, stress, etc. In various embodi 45 NM 012238, which encompasses the NAD binding as well ments, combination therapies comprising a sirtuin-modulat as the substrate binding domains. The core domain of SIRT1 ing compound may refer to (1) pharmaceutical compositions may also refer to about amino acids 261 to 447 of GenBank that comprise one or more sirtuin-modulating compounds in Accession No. NP 036370, which are encoded by nucle combination with one or more therapeutic agents (e.g., one or otides 834 to 1394 of GenBank Accession No. NM-012238; more therapeutic agents described herein); and (2) co-admin 50 to about amino acids 242 to 493 of GenBank Accession No. istration of one or more sirtuin-modulating compounds with NP 036370, which are encoded by nucleotides 777 to 1532 one or more therapeutic agents wherein the sirtuin-modulat of GenBank Accession No. NM 012238; or to about amino ing compound and therapeutic agent have not been formu acids 254 to 495 of GenBank Accession No. NP 036370, lated in the same compositions (but may be present within the which are encoded by nucleotides 813 to 1538 of GenBank same kit or package, such as a blister pack or other multi 55 Accession No. NM 012238. Whether a protein retains a chamber package; connected, separately sealed containers biological function, e.g., deacetylation capabilities, can be (e.g., foil pouches) that can be separated by the user; or a kit determined according to methods known in the art. where the sirtuin modulating compound(s) and other thera In certain embodiments, methods for reducing, preventing peutic agent(s) are in separate vessels). When using separate or treating diseases or disorders using a sirtuin-modulating formulations, the sirtuin-modulating compound may be 60 compound may also comprise decreasing the protein level of administered at the same, intermittent, staggered, prior to, a sirtuin, such as human SIRT1, SIRT2 and/or SIRT3, or Subsequent to, or combinations thereof, with the administra homologs thereof. Decreasing a sirtuin protein level can be tion of another therapeutic agent. achieved according to methods known in the art. For example, In certain embodiments, methods for reducing, preventing an siRNA, an antisense nucleic acid, or a ribozyme targeted to or treating diseases or disorders using a sirtuin-modulating 65 the sirtuin can be expressed in the cell. A dominant negative compound may also comprise increasing the protein level of sirtuin mutant, e.g., a mutant that is not capable of deacety a sirtuin, such as human SIRT1, SIRT2 and/or SIRT3, or lating, may also be used. For example, mutant H363Y of US 8,178,536 B2 119 120 SIRT1, described, e.g., in Luo et al. (2001) Cell 107:137 can (or donor/recipient individuals) may additionally be treated be used. Alternatively, agents that inhibit transcription can be with another therapeutic agent useful for prolonging graft used. Survival. Such as, for example, an immunosuppressive agent, Methods for modulating sirtuin protein levels also include a cytokine, an angiogenic factor, etc. methods for modulating the transcription of genes encoding In yet other embodiments, cells may be treated with a sirtuins, methods for Stabilizing/destabilizing the corre sirtuin-modulating compound that increases the level and/or sponding mRNAS, and other methods known in the art. activity of a sirtuin protein in Vivo, e.g., to increase their Aging/Stress lifespan or prevent apoptosis. For example, skin can be pro In one embodiment, the invention provides a method tected from aging (e.g., developing wrinkles, loss of elastic extending the lifespan of a cell, extending the proliferative 10 ity, etc.) by treating skin or epithelial cells with a sirtuin capacity of a cell, slowing ageing of a cell, promoting the modulating compound that increases the level and/or activity Survival of a cell, delaying cellular senescence in a cell, mim of a sirtuin protein. In an exemplary embodiment, skin is icking the effects of calorie restriction, increasing the resis contacted with a pharmaceutical or cosmetic composition tance of a cell to stress, or preventing apoptosis of a cell, by comprising a sirtuin-modulating compound that increases the contacting the cell with a sirtuin-modulating compound of the 15 level and/or activity of a sirtuin protein. Exemplary skin invention that increases the level and/or activity of a sirtuin afflictions or skin conditions that may be treated in accor protein. In an exemplary embodiment, the methods comprise dance with the methods described herein include disorders or contacting the cell with a sirtuin-activating compound. diseases associated with or caused by inflammation, Sundam The methods described herein may be used to increase the age or natural aging. For example, the compositions find amount of time that cells, particularly primary cells (i.e., cells utility in the prevention or treatment of contact dermatitis obtained from an organism, e.g., a human), may be kept alive (including irritant contact dermatitis and allergic contact der in a cell culture. Embryonic stem (ES) cells and pluripotent matitis), atopic dermatitis (also known as allergic eczema), cells, and cells differentiated therefrom, may also be treated actinic keratosis, keratinization disorders (including with a sirtuin-modulating compound that increases the level eczema), epidermolysis bullosa diseases (including penfi and/or activity of a sirtuin protein to keep the cells, or progeny 25 gus), exfoliative dermatitis, seborrheic dermatitis, erythemas thereof, in culture for longer periods of time. Such cells can (including erythema multiforme and erythema nodosum), also be used for transplantation into a subject, e.g., after ex damage caused by the Sun or other light Sources, discoid lupus vivo modification. erythematosus, dermatomyositis, psoriasis, skin cancer and In one embodiment, cells that are intended to be preserved the effects of natural aging. In another embodiment, sirtuin for long periods of time may be treated with a sirtuin-modu 30 modulating compounds that increase the level and/or activity lating compound that increases the level and/or activity of a of a sirtuin protein may be used for the treatment of wounds sirtuin protein. The cells may be in suspension (e.g., blood and/or burns to promote healing, including, for example, cells, serum, biological growth media, etc.) or in tissues or first-, second- or third-degree burns and/or a thermal, chemi organs. For example, blood collected from an individual for cal or electrical burns. The formulations may be administered purposes of transfusion may be treated with a sirtuin-modu 35 topically, to the skin or mucosal tissue, as an ointment, lotion, lating compound that increases the level and/or activity of a cream, microemulsion, gel, Solution or the like, as further sirtuin protein to preserve the blood cells for longer periods of described herein, within the context of a dosing regimen time. Additionally, blood to be used for forensic purposes effective to bring about the desired result. may also be preserved using a sirtuin-modulating compound Topical formulations comprising one or more sirtuin that increases the level and/or activity of a sirtuin protein. 40 modulating compounds that increase the level and/or activity Other cells that may be treated to extend their lifespan or of a sirtuin protein may also be used as preventive, e.g., protect against apoptosis include cells for consumption, e.g., chemopreventive, compositions. When used in a chemopre cells from non-human mammals (such as meat) or plant cells ventive method, susceptible skin is treated prior to any visible (such as Vegetables). condition in a particular individual. Sirtuin-modulating compounds that increase the level and/ 45 Sirtuin-modulating compounds may be delivered locally or activity of a sirtuin protein may also be applied during or systemically to a subject. In one embodiment, a sirtuin developmental and growth phases in mammals, plants, modulating compound is delivered locally to a tissue or organ insects or microorganisms, in order to, e.g., alter, retard or of a subject by injection, topical formulation, etc. accelerate the developmental and/or growth process. In another embodiment, a sirtuin-modulating compound In another embodiment, sirtuin-modulating compounds 50 that increases the level and/or activity of a sirtuin protein may that increase the level and/or activity of a sirtuin protein may be used for treating or preventing a disease or condition be used to treat cells useful for transplantation or cell therapy, induced or exacerbated by cellular senescence in a Subject; including, for example, Solid tissue grafts, organ transplants, methods for decreasing the rate of Senescence of a Subject, cell Suspensions, stem cells, bone marrow cells, etc. The cells e.g., after onset of senescence; methods for extending the or tissue may be an autograft, an allograft, a Syngraft or a 55 lifespan of a Subject; methods for treating or preventing a xenograft. The cells or tissue may be treated with the sirtuin disease or condition relating to lifespan; methods for treating modulating compound prior to administration/implantation, or preventing a disease or condition relating to the prolifera concurrently with administration/implantation, and/or post tive capacity of cells; and methods for treating or preventing administration/implantation into a subject. The cells or tissue a disease or condition resulting from cell damage or death. In may be treated prior to removal of the cells from the donor 60 certain embodiments, the method does not act by decreasing individual, ex vivo after removal of the cells or tissue from the the rate of occurrence of diseases that shorten the lifespan of donor individual, or post implantation into the recipient. For a subject. In certain embodiments, a method does not act by example, the donor or recipient individual may be treated reducing the lethality caused by a disease, such as cancer. systemically with a sirtuin-modulating compound or may In yet another embodiment, a sirtuin-modulating com have a subset of cells/tissue treated locally with a sirtuin 65 pound that increases the level and/or activity of a sirtuin modulating compound that increases the level and/or activity protein may be administered to a subject in order to generally of a sirtuin protein. In certain embodiments, the cells or tissue increase the lifespan of its cells and to protect its cells against US 8,178,536 B2 121 122 stress and/or against apoptosis. It is believed that treating a intervention, and disorders relating to an abnormal level of subject with a compound described herein is similar to sub high density and low density cholesterol. jecting the Subject to hormesis, i.e., mild stress that is benefi In one embodiment, a sirtuin-modulating compound that cial to organisms and may extend their lifespan. increases the level and/or activity of a sirtuin protein may be Sirtuin-modulating compounds that increase the level and/ administered as part of a combination therapeutic with or activity of a sirtuin protein may be administered to a another cardiovascular agent including, for example, an anti Subject to prevent aging and aging-related consequences or arrhythmic agent, an antihypertensive agent, a calcium chan diseases, such as stroke, heart disease, heart failure, arthritis, nel blocker, a cardioplegic solution, a cardiotonic agent, a high blood pressure, and Alzheimer's disease. Other condi fibrinolytic agent, a Sclerosing solution, a vasoconstrictor tions that can be treated include ocular disorders, e.g., asso 10 agent, a vasodilator agent, a nitric oxide donor, a potassium ciated with the aging of the eye. Such as cataracts, glaucoma, channel blocker, a sodium channel blocker, statins, or a natu and macular degeneration. Sirtuin-modulating compounds riuretic agent. that increase the level and/or activity of a sirtuin protein can In one embodiment, a sirtuin-modulating compound that also be administered to Subjects for treatment of diseases, increases the level and/or activity of a sirtuin protein may be e.g., chronic diseases, associated with cell death, in order to 15 administered as part of a combination therapeutic with an protect the cells from cell death. Exemplary diseases include anti-arrhythmia agent. Anti-arrhythmia agents are often orga those associated with neural cell death, neuronal dysfunction, nized into four main groups according to their mechanism of or muscular cell death or dysfunction, such as Parkinson's action: type I, Sodium channel blockade; type II, beta-adren disease, Alzheimer's disease, multiple Sclerosis, amniotropic ergic blockade; type III, repolarization prolongation; and type lateral Sclerosis, and muscular dystrophy: AIDS; fulminant IV, calcium channel blockade. Type I anti-arrhythmic agents hepatitis; diseases linked to degeneration of the brain, Such as include lidocaine, moricizine, mexiletine, tocainide, Creutzfeld-Jakob disease, retinitis pigmentosa and cerebellar procainamide, encainide, flecanide, tocainide, phenytoin, degeneration; myelodysplasis Such as aplastic anemia; propafenone, quinidine, disopyramide, and flecainide. Type ischemic diseases Such as myocardial infarction and stroke; II anti-arrhythmic agents include propranolol and esmolol. hepatic diseases such as alcoholic hepatitis, hepatitis B and 25 Type III includes agents that act by prolonging the duration of hepatitis C; joint-diseases such as osteoarthritis; atheroscle the action potential, such as amiodarone, artilide, bretylium, rosis; alopecia; damage to the skin due to UV light; lichen clofilium, isobutilide, Sotalol, azimilide, dolfetilide, dronedar planus; atrophy of the skin; cataract; and graft rejections. Cell one, ersentilide, ibutilide, tedisamil, and trecetilide. Type IV death can also be caused by Surgery, drug therapy, chemical anti-arrhythmic agents include Verapamil, dilitaizem, digi exposure or radiation exposure. 30 talis, adenosine, nickel chloride, and magnesium ions. Sirtuin-modulating compounds that increase the level and/ In another embodiment, a sirtuin-modulating compound or activity of a sirtuin protein can also be administered to a that increases the level and/or activity of a sirtuin protein may Subject suffering from an acute disease, e.g., damage to an be administered as part of a combination therapeutic with organ or tissue, e.g., a subject Suffering from stroke or myo another cardiovascular agent. Examples of cardiovascular cardial infarction or a subject Suffering from a spinal cord 35 agents include vasodilators, for example, hydralazine; angio injury. Sirtuin-modulating compounds that increase the level tensin converting enzyme inhibitors, for example, captopril; and/or activity of a sirtuin protein may also be used to repair anti-anginal agents, for example, isosorbide nitrate, glyceryl an alcoholic’s liver. trinitrate and pentaerythritol tetranitrate; anti-arrhythmic Cardiovascular Disease agents, for example, quinidine, procainaltide and lignocaine; In another embodiment, the invention provides a method 40 cardioglycosides, for example, digoxin and digitoxin; cal for treating and/or preventing a cardiovascular disease by cium antagonists, for example, Verapamil and nifedipine; administering to a Subject in need thereof a sirtuin-modulat diuretics, such as thiazides and related compounds, for ing compound that increases the level and/or activity of a example, bendrofluazide, chlorothiazide, chlorothalidone, sirtuin protein. hydrochlorothiazide and other diuretics, for example, Cardiovascular diseases that can be treated or prevented 45 fursemide and triamterene, and sedatives, for example, using the sirtuin-modulating compounds that increase the nitrazepam, flurazepam and diazepam. level and/or activity of a sirtuin protein include cardiomyopa Other exemplary cardiovascular agents include, for thy or myocarditis; such as idiopathic cardiomyopathy, meta example, a cyclooxygenase inhibitor Such as aspirin or bolic cardiomyopathy, alcoholic cardiomyopathy, drug-in indomethacin, a platelet aggregation inhibitor Such as clopi duced cardiomyopathy, ischemic cardiomyopathy, and 50 dogrel, ticlopidene or aspirin, fibrinogen antagonists or a hypertensive cardiomyopathy. Also treatable or preventable diuretic such as chlorothiazide, hydrochlorothiazide, flume using compounds and methods described herein are athero thiazide, hydroflumethiazide, bendroflumethiazide, methyl matous disorders of the major blood vessels (macrovascular chlorthiazide, trichloromethiazide, polythiazide or benzthi disease) Such as the aorta, the coronary arteries, the carotid azide as well as ethacrynic acid tricrynafen, chlorthalidone, arteries, the cerebrovascular arteries, the renal arteries, the 55 furosemide, musolimine, bumetanide, triamterene, amiloride iliac arteries, the femoral arteries, and the popliteal arteries. and Spironolactone and salts of such compounds, angiotensin Other vascular diseases that can be treated or prevented converting enzyme inhibitors such as captopril. Zofenopril, include those related to platelet aggregation, the retinal arte fosinopril, enalapril, ceranopril, cilaZopril, delapril, pento rioles, the glomerular arterioles, the Vasa nervorum, cardiac pril, quinapril, ramipril, lisinopril, and salts of Such com arterioles, and associated capillary beds of the eye, the kidney, 60 pounds, angiotensin II antagonists such as losartan, irbesartan the heart, and the central and peripheral nervous systems. The or Valsartan, thrombolytic agents such as tissue plasminogen sirtuin-modulating compounds that increase the level and/or activator (tPA), recombinant tRA, streptokinase, urokinase, activity of a sirtuin protein may also be used for increasing prourokinase, and anisoylated plasminogen streptokinase HDL levels in plasma of an individual. activator complex (APSAC, Eminase, Beecham Laborato Yet other disorders that may be treated with sirtuin-modu 65 ries), or animal salivary gland plasminogen activators, cal lating compounds that increase the level and/or activity of a cium channel blocking agents such as Verapamil, nifedipine sirtuin protein include restenosis, e.g., following coronary or diltiazem, thromboxane receptor antagonists Such as US 8,178,536 B2 123 124 ifetroban, prostacyclin mimetics, or phosphodiesterase Examples of the anti anginal drug include nitrate drugs (such inhibitors. Such combination products if formulated as a fixed as amyl nitrite, nitroglycerin, and isosorbide), B-adrenaline dose employ the compounds of this invention within the dose receptor blocking drugs (such as propranolol, pindolol, inde range described above and the other pharmaceutically active nolol, carteolol, bunitrolol, atenolol, acebutolol, metoprolol, agent within its approved dose range. timolol, nipradillol, penbutolol, nadolol, tilisolol, carvedilol, Yet other exemplary cardiovascular agents include, for bisoprolol, betaxolol, celiprolol, bopindolol, bevantolol, example, Vasodilators, e.g., bencyclane, cinnarizine, citi labetalol, alprenolol, amosulalol, arotinolol, befunolol, bucu coline, cyclandelate, cyclonicate, ebumamonine, phenox molol, bufetolol, buferalol, buprandolol, butylidine, buto eZyl, flunarizine, ibudilast, ifenprodil, lomerizine, naphlole, filolol, carazolol, cetamolol, cloranolol, dilevalol, epanolol. nikamate, nosergoline, nimodipine, papaverine, pentifylline, 10 levobunolol, mepindolol, metipranolol, moprolol, nadoxolol. nofedoline, Vincamin, Vinpocetine, Vichizyl, pentoxifylline, nevibolol, Oxprenolol, practol, pronetalol, Sotalol, Sufinalol, prostacyclin derivatives (such as prostaglandin E1 and pros talindolol, tertalol, toliprolol, and xybenolol), calcium chan taglandin I2), an endothelin receptor blocking drug (Such as nel blocking drugs (such as aranidipine, efonidipine, nicar bosentan), diltiazem, nicorandil, and nitroglycerin. Examples dipine, bamidipine, benidipine, manidipine, cilnidipine, of the cerebral protecting drug include radical scavengers 15 nisoldipine, nitrendipine, nifedipine, nilvadipine, felodipine, (such as edaravone, Vitamin E, and vitamin C), glutamate amlodipine, diltiazem, bepridil, clentiazem, phendiline, galo antagonists, AMPA antagonists, kainate antagonists, NMDA pamil, mibefradil, prenylamine, Semotiadil, terodiline, Vera antagonists, GABA agonists, growth factors, opioid antago pamil, cilnidipine, elgodipine, isradipine, lacidipine, lercani nists, phosphatidylcholine precursors, serotonin agonists, dipine, nimodipine, cinnarizine, flunarizine, lidoflazine, Na"/Ca" channel inhibitory drugs, and K" channel opening lomerizine, bencyclane, etafenone, and perhexiline) tri drugs. Examples of the brain metabolic stimulants include metazidine, dipyridamole, etafenone, dilaZep, trapidil, nic amantadine, tiapride, and gamma-aminobutyric acid. orandil, enoxaparin, and aspirin. Examples of the diuretic Examples of the anticoagulant include heparins (such as hep include thiazide diuretics (such as hydrochlorothiazide, arin Sodium, heparin potassium, dalteparin Sodium, dalte methyclothiazide, trichlormethiazide, benzylhydrochlorothi parin calcium, heparin calcium, pamaparin Sodium, reviparin 25 azide, and penflutizide), loop diuretics (such as furosemide, Sodium, and danaparoid sodium), warfarin, enoxaparin, arga etacrynic acid, bumetanide, piretanide, azosemide, and troban, batroXobin, and Sodium citrate. Examples of the anti torasemide), K" sparing diuretics (spironolactone, triam platelet drug include ticlopidine hydrochloride, dipy terene, and potassium can renoate), osmotic diuretics (such as ridamole, ciloStaZol, ethyl icosapentate, Sarpogrelate isosorbide, D-mannitol, and glycerin), nonthiazide diuretics hydrochloride, dilazep hydrochloride, trapidil, a nonsteroidal 30 (such as meticrane, tripamide, chlorthalidone, and mefru antiinflammatory agent (such as aspirin), beraprostSodium, side), and acetazolamide. Examples of the cardiotonic iloprost, and indobufene. Examples of the thrombolytic drug include digitalis formulations (such as digitoxin, digoxin, include urokinase, tissue-type plasminogen activators (such methyldigoxin, deslanoside, Vesnarinone, lanatoside C, and as alteplase, tisokinase, nateplase, pamiteplase, monteplase, proscillaridin), Xanthine formulations (such as aminophyl and rateplase), and nasaruplase. Examples of the antihyper 35 line, choline theophylline, diprophylline, and proxyphylline), tensive drug include angiotensin converting enzyme inhibi catecholamine formulations (such as dopamine, dobutamine, tors (such as captopril, alacepril, lisinopril, imidapril, and docarpamine), PDE III inhibitors (such as amrinone, quinapril, temocapril, delapril, benazepril, cilaZapril, tran olprinone, and milrinone), denopamine, ubidecarenone, dollapril, enalapril, ceronapril, fosinopril, imadapril, mobert pimobendan, levosimendan, aminoethylsulfonic acid, pril, perindopril, ramipril, spirapril, and randolapril), angio 40 Vesnarinone, carperitide, and colforsin daropate. Examples of tensin II antagonists (such as losartan, candesartan, Valsartan, the antiarrhythmic drug include ajmaline, pirmenol, eprosartan, and irbesartan), calcium channel blocking drugs procainamide, cibenzoline, disopyramide, quinidine, aprind (such as aranidipine, efonidipine, nicardipine, bamidipine, ine, mexiletine, lidocaine, phenyloin, pilsicainide, pro benidipine, manidipine, cilnidipine, niSoldipine, nitren pafenone, flecainide, atenolol, acebutolol, Sotalol, propra dipine, nifedipine, nilvadipine, felodipine, amlodipine, dilt 45 nolol, metoprolol, pindolol, amiodarone, nifekalant, iazem, bepridil, clentiazem, phendilin, galopamil, milbe diltiazem, bepridil, and Verapamil. Examples of the antihy fradil, prenylamine, semotiadil, terodiline, Verapamil, perlipidemic drug include atorvastatin, simvastatin, pravas cilnidipine. elgodipine, isradipine, lacidipine, lercanidipine, tatin Sodium, fluvastatin Sodium, clinofibrate, clofibrate, sim nimodipine, cinnarizine, flunarizine, lidoflazine, lomerizine, fibrate, fenofibrate, bezafibrate, colestimide, and bencyclane, etafenone, and perhexiline), B-adrenaline recep 50 colestyramine. Examples of the immunosuppressant include tor blocking drugs (propranolol, pindolol, indenolol, car azathioprine, mizoribine, cyclosporine, tacrolimus, gusperi teolol, bunitrolol, atenolol, acebutolol, metoprolol, timolol. mus, and methotrexate. nipradillol, penbutolol, nadolol, tilisolol, carvedilol, biso Cell Death/Cancer prolol, betaxolol, celiprolol, bopindolol, bevantolol, labe Sirtuin-modulating compounds that increase the level and/ talol, alprenolol, amoSulalol, arotinolol, befunolol, bucu 55 or activity of a sirtuin protein may be administered to Subjects molol, bufetolol, buferalol, buprandolol, butylidine, who have recently received or are likely to receive a dose of butofilolol, carazolol, cetamolol, cloranolol, dilevalol, radiation or toxin. In one embodiment, the dose of radiation or epanolol, levobunolol, mepindolol, metipranolol, moprolol. toxin is received as part of a work-related or medical proce nadoxolol, nevibolol, Oxprenolol, practol, pronetalol, Sotalol, dure, e.g., working in a nuclear power plant, flying an air Sufinalol, talindolol, tertalol, toliprolol, xybenolol, and 60 plane, an X-ray, CAT scan, or the administration of a radio esmolol), C.-receptor blocking drugs (such as amoSulalol, active dye for medical imaging; in Such an embodiment, the prazosin, teraZosin, doxazosin, bunaZosin, urapidil, phento compound is administered as a prophylactic measure. In lamine, arotinolol, dapiprazole, fenspiride, indoramin, labe another embodiment, the radiation or toxin exposure is talol, naftopidil, nicergoline, tamsulosin, tolazoline, trima received unintentionally, e.g., as a result of an industrial acci Zosin, and yohimbine), sympathetic nerve inhibitors (such as 65 dent, habitation in a location of natural radiation, terrorist act, clonidine, guanfacine, guanabenz, methyldopa, and reser or act of war involving radioactive or toxic material. In Such pine), hydralazine, todralazine, budralazine, and cadralazine. a case, the compound is preferably administered as soon as US 8,178,536 B2 125 126 possible after the exposure to inhibit apoptosis and the sub thioguanine, thiotepa, titanocene dichloride, topotecan, tras sequent development of acute radiation syndrome. tuZumab, tretinoin, vinblastine, Vincristine, Vindesine, and Sirtuin-modulating compounds may also be used for treat vinorelbine. ing and/or preventing cancer. In certain embodiments, sirtuin These chemotherapeutic agents may be categorized by modulating compounds that increase the level and/or activity their mechanism of action into, for example, following of a sirtuin protein may be used for treating and/or preventing groups: anti-metabolites/anti-cancer agents, such as pyrimi cancer. Calorie restriction has been linked to a reduction in dine analogs (5-fluorouracil, floXuridine, capecitabine, gem the incidence of age-related disorders including cancer (see citabine and cytarabine) and purine analogs, folate antago e.g., Bordone and Guarente, Nat. Rev. Mol. Cell Biol. (2005 nists and related inhibitors (mercaptopurine, thioguanine, epub): Guarente and Picard, Cell 120: 473-82 (2005); Berri 10 pentostatin and 2-chlorodeoxyadenosine(cladribine)); anti gan, et al., Carcinogenesis 23: 817-822 (2002); and Heilbronn proliferative/antimitotic agents including natural products and Ravussin, Am. J. Clin. Nutr. 78: 361-369 (2003)). Addi Such as Vinca alkaloids (vinblastine, Vincristine, and vinorel tionally, the Sir2 protein from yeast has been shown to be bine), microtubule disruptors such as taxane (paclitaxel, doc required for lifespan extension by glucose restriction (see etaxel), Vincristin, vinblastin, nocodazole, epothilones and e.g., Lin et al., Science 289: 2126-2128 (2000); Anderson et 15 navelbine, epidipodophyllotoxins(teniposide), DNA damag al., Nature 423: 181-185 (2003)), a yeast model for calorie ing agents (actinomycin, amsacrine, anthracyclines, bleomy restriction. Accordingly, an increase in the level and/or activ cin, buSulfan, camptothecin, carboplatin, chlorambucil, cis ity of a sirtuin protein may be useful for treating and/or platin, cyclophosphamide, cytoxan, dactinomycin, preventing the incidence of age-related disorders, such as, for daunorubicin, docetaxel, doxorubicin, epirubicin, hexameth example, cancer. In other embodiments, sirtuin-modulating ylmelamineoxaliplatin, iphosphamide, melphalan, merchlo compounds that decrease the level and/or activity of a sirtuin rethamine, mitomycin, mitoxantrone, nitrosourea, paclitaxel, protein may be used for treating or preventing cancer. For plicamycin, procarbazine, teniposide, triethylenethiophos example, inhibitory compounds may be used to stimulate phoramide and etoposide (VP16)); antibiotics such as dacti acetylation of substrates such as p53 and thereby increase nomycin (actinomycin D), daunorubicin, doxorubicin (adria apoptosis, as well as to reduce the lifespan of cells and organ 25 mycin), idarubicin, anthracyclines, mitoxantrone, isms, render them more sensitive to stress, and/or increase the bleomycins, plicamycin (mithramycin) and mitomycin; radiosensitivity and/or chemosensitivity of a cellor organism. enzymes (L-asparaginase which systemically metabolizes Thus, inhibitory compounds may be used, e.g., for treating L-asparagine and deprives cells which do not have the capac cancer. Exemplary cancers that may be treated using a sirtuin ity to synthesize their own asparagine); antiplatelet agents; modulating compound are those of the brain and kidney; 30 antiproliferative/antimitotic alkylating agents such as nitro hormone-dependent cancers including breast, prostate, tes gen mustards (mechlorethamine, cyclophosphamide and ana ticular, and ovarian cancers; lymphomas, and leukemias. In logs, melphalan, chlorambucil), ethylenimines and meth cancers associated with solid tumors, a modulating com ylmelamines (hexamethylmelamine and thiotepa), alkyl sulfonates-busulfan, nitrosoureas (carmustine (BCNU) and pound may be administered directly into the tumor. Cancer of analogs, streptozocin), traZenes—dacarbazinine (DTIC); blood cells, e.g., leukemia, can be treated by administering a 35 antiproliferative/antimitotic antimetabolites such as folic modulating compound into the blood stream or into the bone acid analogs (methotrexate); platinum coordination com marrow. Benign cell growth can also be treated, e.g., warts. plexes (cisplatin, carboplatin), procarbazine, hydroxyurea, Other diseases that can be treated include autoimmune dis mitotane, aminoglutethimide; hormones, hormone analogs eases, e.g., systemic lupus erythematosus, Scleroderma, and (estrogen, tamoxifen, goserelin, bicalutamide, nilutamide) arthritis, in which autoimmune cells should be removed. Viral 40 and aromatase inhibitors (letrozole, anastrozole); anticoagu infections such as herpes, HIV, adenovirus, and HTLV-1 asso lants (heparin, synthetic heparin salts and other inhibitors of ciated malignant and benign disorders can also be treated by thrombin); fibrinolytic agents (such as tissue plasminogen administration of sirtuin-modulating compound. Alterna activator, streptokinase and urokinase), aspirin, COX-2 tively, cells can be obtained from a subject, treated ex vivo to inhibitors, dipyridamole, ticlopidine, clopidogrel, abcix remove certain undesirable cells, e.g., cancer cells, and 45 imab; antimigratory agents; antisecretory agents (breveldin); administered back to the same or a different subject. immunosuppressives (cyclosporine, tacrolimus (FK-506), Chemotherapeutic agents that may be coadministered with Sirolimus(rapamycin), azathioprine, mycophenolate modulating compounds described herein as having anti-can mofetil); anti-angiogenic compounds (TNP-470, genistein) cer activity (e.g., compounds that induce apoptosis, com and growth factor inhibitors (vascular endothelial growth fac pounds that reduce lifespan or compounds that render cells 50 tor (VEGF) inhibitors, fibroblast growth factor (FGF) inhibi sensitive to stress) include: aminoglutethimide, amsacrine, tors, epidermal growth factor (EGF) inhibitors); angiotensin anastroZole, asparaginase, bcg, bicalutamide, bleomycin, receptor blocker, nitric oxide donors; anti-sense oligonucle buserelin, buSulfan, campothecin, capecitabine, carboplatin, otides; antibodies (trastuzumab); cell cycle inhibitors and carmustine, chlorambucil, cisplatin, cladribine, clodronate, differentiation inducers (tretinoin); mTOR inhibitors, topoi colchicine, cyclophosphamide, cyproterone, cytarabine, dac Somerase inhibitors (doxorubicin (adriamycin), atmsacrine, arbazine, dactinomycin, daunorubicin, dienestrol, diethylstil 55 camptothecin, daunorubicin, dactinomycin, eniposide, epiru bestrol, docetaxel, doxorubicin, epirubicin, estradiol, estra bicin, etoposide, idarubicin, irinotecan (CPT-11) and mitox mustine, etoposide, exemestane, filgrastim, fludarabine, antrone, topotecan, irinotecan), corticosteroids (cortisone, fludrocortisone, fluorouracil, fluoxymesterone, flutamide, dexamethasone, hydrocortisone, methylpednisolone, pred gemcitabine, genistein, goserelin, hydroxyurea, idarubicin, nisone, and prenisolone); growth factor signal transduction ifosfamide, imatinib, interferon, irinotecan, ironotecan, letro 60 kinase inhibitors; mitochondrial dysfunction inducers and Zole, leucovorin, leuprolide, levamisole, lomustine, mechlo caspase activators; chromatin disruptors. rethamine, medroxyprogesterone, megestrol, melphalan, These chemotherapeutic agents may be used by them mercaptopurine, mesna, methotrexate, mitomycin, mitotane, selves with a sirtuin-modulating compound described herein mitoxantrone, nilutamide, nocodazole, octreotide, oxalipl as inducing cell death or reducing lifespan or increasing sen atin, paclitaxel, pamidronate, pentostatin, plicamycin, por 65 sitivity to stress and/or in combination with other chemo fimer, procarbazine, raltitrexed, rituximab, Streptozocin, therapeutics agents. Many combinatorial therapies have been Suramin, tamoxifen, temozolomide, teniposide, testosterone, developed, including but not limited to those listed in Table 1. US 8,178,536 B2 127 128 TABLE 1. Exemplary combinatorial therapies for the treatment of cancer. Name Therapeutic agents ABV Doxorubicin, Bleomycin, Vinblastine ABVD Doxorubicin, Bleomycin, Vinblastine, Dacarbazine AC (Breast) Doxorubicin, Cyclophosphamide AC (Sarcoma) Doxorubicin, Cisplatin AC (Neuroblastoma) Cyclophosphamide, Doxorubicin ACE Cyclophosphamide, Doxorubicin, Etoposide ACe Cyclophosphamide, Doxorubicin AD Doxorubicin, Dacarbazine AP Doxorubicin, Cisplatin ARAC-DNR Cytarabine, Daunorubicin B-CAWe Bleomycin, Lomustine, Doxorubicin, Vinblastine BCVPP Carmustine, Cyclophosphamide, Vinblastine, Procarbazine, Prednisone BEACOPP Bleomycin, Etoposide, Doxorubicin, Cyclophosphamide, Vincristine, Procarbazine, Prednisone, Filgrastim BEP Bleomycin, Etoposide, Cisplatin BIP Bleomycin, Cisplatin, Ifosfamide, Mesna BOMP Bleomycin, Vincristine, Cisplatin, Mitomycin CA Cytarabine, Asparaginase CABO Cisplatin, Methotrexate, Bleomycin, Vincristine CAF Cyclophosphamide, Doxorubicin, Fluorouracil CAL-G Cyclophosphamide, Daunorubicin, Vincristine, Prednisone, Asparaginase CAMP Cyclophosphamide, Doxorubicin, Methotrexate, Procarbazine CAP Cyclophosphamide, Doxorubicin, Cisplatin CaT Carboplatin, Paclitaxel CAV Cyclophosphamide, Doxorubicin, Vincristine CAVE ADD CAV and Etoposide CA-VP16 Cyclophosphamide, Doxorubicin, Etoposide CC Cyclophosphamide, Carboplatin CDDPVP-16 Cisplatin, Etoposide CEF Cyclophosphamide, Epirubicin, Fluorouracil CEPP(B) Cyclophosphamide, Etoposide, Prednisone, with or without Bleomycin CEV Cyclophosphamide, Etoposide, Vincristine CF Cisplatin, Fluorouracil or Carboplatin Fluorouracil CHAP Cyclophosphamide or Cyclophosphamide, Altretamine, Doxorubicin, Cisplatin ChIVPP Chlorambucil, Vinblastine, Procarbazine, Prednisone CHOP Cyclophosphamide, Doxorubicin, Vincristine, Prednisone CHOP-BLEO Add Bleomycin to CHOP CISCA Cyclophosphamide, Doxorubicin, Cisplatin CLD-BOMP Bleomycin, Cisplatin, Vincristine, Mitomycin CMF Methotrexate, Fluorouracil, Cyclophosphamide CMFP Cyclophosphamide, Methotrexate, Fluorouracil, Prednisone CMFVP Cyclophosphamide, Methotrexate, Fluorouracil, Vincristine, Prednisone CMV Cisplatin, Methotrexate, Vinblastine CNF Cyclophosphamide, Mitoxantrone, Fluorouracil CNOP Cyclophosphamide, Mitoxantrone, Vincristine, Prednisone COB Cisplatin, Vincristine, Bleomycin CODE Cisplatin, Vincristine, Doxorubicin, Etoposide COMLA Cyclophosphamide, Vincristine, Methotrexate, Leucovorin, Cytarabine COMP Cyclophosphamide, Vincristine, Methotrexate, Prednisone Cooper Regimen Cyclophosphamide, Methotrexate, Fluorouracil, Vincristine, Prednisone COP Cyclophosphamide, Vincristine, Prednisone COPE Cyclophosphamide, Vincristine, Cisplatin, Etoposide COPP Cyclophosphamide, Vincristine, Procarbazine, Prednisone CP(Chronic lymphocytic Chlorambucil, Prednisone leukemia) CP (Ovarian Cancer) Cyclophosphamide, Cisplatin CT Cisplatin, Paclitaxel CVD Cisplatin, Vinblastine, Dacarbazine CVI Carboplatin, Etoposide, Ifosfamide, Mesna CVP Cyclophosphamide, Vincristine, Prednisome CVPP Lomustine, Procarbazine, Prednisone CYVADIC Cyclophosphamide, Vincristine, Doxorubicin, Dacarbazine DA Daunorubicin, Cytarabine DAT Daunorubicin, Cytarabine, Thioguanine DAV Daunorubicin, Cytarabine, Etoposide DCT Daunorubicin, Cytarabine, Thioguanine DHAP Cisplatin, Cytarabine, Dexamethasone DI Doxorubicin, Ifosfamide US 8,178,536 B2 129 130 TABLE 1-continued Exemplary combinatorial therapies for the treatment of cancer. Name Therapeutic agents DTIC, Tamoxifen Dacarbazine, Tamoxifen DVP Daunorubicin, Vincristine, Prednisone EAP Etoposide, Doxorubicin, Cisplatin EC Etoposide, Carboplatin EFP Etoposie, Fluorouracil, Cisplatin ELF Etoposide, Leucovorin, Fluorouracil EMA 86 Mitoxantrone, Etoposide, Cytarabine EP Etoposide, Cisplatin EVA Etoposide, Vinblastine FAC Fluorouracil, Doxorubicin, Cyclophosphamide FAM Fluorouracil, Doxorubicin, Mitomycin FAMTX Methotrexate, Leucovorin, Doxorubicin FAP Fluorouracil, Doxorubicin, Cisplatin F-CL, Fluorouracil, Leucovorin FEC Fluorouracil, Cyclophosphamide, Epirubicin FED Fluorouracil, Etoposide, Cisplatin FL Flutamide, Leuprolide FZ Flutamide, Goserelin acetate implant HDMTX Methotrexate, Leucovorin Hexa-CAF Altretamine, Cyclophosphamide, Methotrexate, Fluorouracil CE-T fosfamide, Carboplatin, Etoposide, Paclitaxel, Mesna DMTXF6-MP Methotrexate, Mercaptopurine, Leucovorin E fosfamide, Etoposie, Mesna foVP fosfamide, Etoposide, Mesna PA fosfamide, Cisplatin, Doxorubicin M-2 Vincristine, Carmustine, Cyclophosphamide, Prednisone, Melphalan MAC-III Methotrexate, Leucovorin, Dactinomycin, Cyclophosphamide MACC Methotrexate, Doxorubicin, Cyclophosphamide, Lomustine MACOP-B Methotrexate, Leucovorin, Doxorubicin, Cyclophosphamide, Vincristine, Bleomycin, Prednisone MAID Mesna, Doxorubicin, Ifosfamide, Dacarbazine m-BACOD Bleomycin, Doxorubicin, Cyclophosphamide, Vincristine, Dexamethasone, Methotrexate, Leucovorin MBC Methotrexate, Bleomycin, Cisplatin MC Mitoxantrone, Cytarabine MF Methotrexate, Fluorouracil, Leucovorin MICE Ifosfamide, Carboplatin, Etoposide, Mesna MINE Mesna, Ifosfamide, Mitoxantrone, Etoposide mini-BEAM Carmustine, Etoposide, Cytarabine, Melphalan MOBP Bleomycin, Vincristine, Cisplatin, Mitomycin MOP Mechlorethamine, Vincristine, Procarbazine MOPP Mechlorethamine, Vincristine, Procarbazine, Prednisone MOPPABV Mechlorethamine, Vincristine, Procarbazine, Prednisone, Doxorubicin, Bleomycin, Vinblastine MP (multiple myeloma) Melphalan, Prednisone MP (prostate cancer) Mitoxantrone, Prednisone MTXF6-MO Methotrexate, Mercaptopurine MTX6-MPVP Methotrexate, Mercaptopurine, Vincristine, Prednisone MTX-CDDPAdr Methotrexate, Leucovorin, Cisplatin, Doxorubicin MV (breast cancer) Mitomycin, Vinblastine MV (acute myelocytic Mitoxantrone, Etoposide leukemia) M-WAC Methotrexate Vinblastine, Doxorubicin, Cisplatin MVP Mitomycin Vinblastine, Cisplatin MVPP Mechlorethamine, Vinblastine, Procarbazine, Prednisone NFL Mitoxantrone, Fluorouracil, Leucovorin NOVP Mitoxantrone, Vinblastine, Vincristine OPA Vincristine, Prednisone, Doxorubicin OPPA Add Procarbazine to OPA. PAC Cisplatin, Doxorubicin PAC-I Cisplatin, Doxorubicin, Cyclophosphamide PA-CI Cisplatin, Doxorubicin PC Paclitaxel, Carboplatin or Paclitaxel, Cisplatin PCV Lomustine, Procarbazine, Vincristine PE Paclitaxel, Estramustine PFL Cisplatin, Fluorouracil, Leucovorin POC Prednisone, Vincristine, Lomustine ProMACE Prednisone, Methotrexate, Leucovorin, Doxorubicin, Cyclophosphamide, Etoposide ProMACE cytaBOM Prednisone, Doxorubicin, Cyclophosphamide, Etoposide, Cytarabine, Bleomycin, Vincristine, Methotrexate, Leucovorin, Cotrimoxazole US 8,178,536 B2 131 132 TABLE 1-continued Exemplary combinatorial therapies for the treatment of cancer. Name Therapeutic agents PROMACEAMOPP Prednisone, Doxorubicin, Cyclophosphamide, Etoposide, Mechlorethamine, Vincristine, Procarbazine, Methotrexate, Leucovorin Pt? VM Cisplatin, Teniposide PVA Prednisone, Vincristine, Asparaginase PVB Cisplatin, Vinblastine, Bleomycin PWDA Prednisone, Vincristine, Daunorubicin, Asparaginase SMF Streptozocin, Mitomycin, Fluorouracil TAD Mechlorethamine, Doxorubicin, Vinblastine, Vincristine, Bleomycin, Etoposide, Prednisone TCF Paclitaxel, Cisplatin, Fluorouracil TIP Paclitaxel, Ifosfamide, Mesna, Cisplatin TTT Methotrexate, Cytarabine, Hydrocortisone Topo/CTX Cyclophosphamide, Topotecan, MeSna VAB-6 Cyclophosphamide, Dactinomycin, Vinblastine, Cisplatin, Bleomycin WAC Vincristine, Dactinomycin, Cyclophosphamide WACAdr Vincristine, Cyclophosphamide, Doxorubicin, Dactinomycin, Vincristine WAD Vincristine, Doxorubicin, Dexamethasone WATH Vinblastine, Doxorubicin, Thiotepa, Flouxymesterone WBAP Vincristine, Carmustine, Doxorubicin, Prednisone VBCMP Vincristine, Carmustine, Melphalan, Cyclophosphamide, Prednisone VC Vinorelbine, Cisplatin VCAP Vincristine, Cyclophosphamide, Doxorubicin, Prednisone VD Vinorelbine, Doxorubicin WeIP Vinblastine, Cisplatin, Ifosfamide, Mesna VIP Etoposide, Cisplatin, Ifosfamide, Mesna VM Mitomycin, Vinblastine VMCP Vincristine, Melphalan, Cyclophosphamide, Prednisone VP Etoposide, Cisplatin WTAD Etoposide. Thioguanine, Daunorubicin, Cytarabine 5 - 2 Cytarabine, Daunorubicin, Mitoxantrone 7 - 3 Cytarabine with, Daunorubicin or Idarubicin or Mitoxantrone 8 in 1 Methylprednisolone, Vincristine, Lomustine, Procarbazine, Hydroxyurea, Cisplatin, Cytarabine, Dacarbazine
In addition to conventional chemotherapeutics, the sirtuin used to treat patients suffering from neurodegenerative dis modulating compounds described hereinas capable of induc 40 eases, and traumatic or mechanical injury to the central ner ing cell death or reducing lifespan can also be used with Vous system (CNS), spinal cord or peripheral nervous system antisense RNA, RNAi or other polynucleotides to inhibit the (PNS). Neurodegenerative disease typically involves reduc expression of the cellular components that contribute to tions in the mass and Volume of the human brain, which may unwanted cellular proliferation that are targets of conven be due to theatrophy and/or death of brain cells, which are far tional chemotherapy. Such targets are, merely to illustrate, 45 more profound than those in a healthy person that are attrib growth factors, growth factor receptors, cell cycle regulatory utable to aging. Neurodegenerative diseases can evolve proteins, transcription factors, or signal transduction kinases. gradually, after a long period of normal brain function, due to Combination therapies comprising sirtuin-modulating progressive degeneration (e.g., nerve cell dysfunction and compounds and a conventional chemotherapeutic agent may death) of specific brain regions. Alternatively, neurodegen be advantageous over combination therapies known in the art 50 erative diseases can have a quick onset, Such as those associ because the combination allows the conventional chemo ated with trauma or toxins. The actual onset of brain degen therapeutic agent to exert greater effect at lower dosage. In a eration may precede clinical expression by many years. preferred embodiment, the effective dose (EDs) for a che Examples of neurodegenerative diseases include, but are not motherapeutic agent, or combination of conventional chemo limited to, Alzheimer's disease (AD), Parkinson's disease therapeutic agents, when used in combination with a sirtuin 55 (PD), Huntington's disease (HD), amyotrophic lateral scle modulating compound is at least 2 fold less than the EDso for rosis (ALS: Lou Gehrig's disease), diffuse Lewy body dis the chemotherapeutic agent alone, and even more preferably ease, chorea-acanthocytosis, primary lateral Sclerosis, ocular at 5 fold, 10 fold or even 25 fold less. Conversely, the thera diseases (ocular neuritis), chemotherapy-induced neuropa peutic index (TI) for Such chemotherapeutic agent or combi thies (e.g., from Vincristine, paclitaxel, bortezomib), diabe nation of Such chemotherapeutic agent when used in combi 60 tes-induced neuropathies and Friedreich's ataxia. Sirtuin nation with a sirtuin-modulating compound described herein modulating compounds that increase the level and/or activity can be at least 2 fold greater than the TI for conventional of a sirtuin protein can be used to treat these disorders and chemotherapeutic regimen alone, and even more preferably others as described below. at 5 fold, 10 fold or even 25 fold greater. AD is a chronic, incurable, and unstoppable CNS disorder Neuronal Diseases/Disorders 65 that occurs gradually, resulting in memory loss, unusual In certain aspects, sirtuin-modulating compounds that behavior, personality changes, and a decline in thinking abili increase the level and/or activity of a sirtuin protein can be ties. These losses are related to the death of specific types of US 8,178,536 B2 133 134 brain cells and the breakdown of connections and their sup related glycolipids.Substrates for B-hexosaminidase accumu porting network (e.g. glial cells) between them. AD has been late in the nervous system and trigger acute neurodegenera described as childhood development in reverse. In most tion. In the most severe forms, the onset of symptoms begins people with AD, symptoms appear after the age 60. The in early infancy. A precipitous neurodegenerative course then earliest symptoms include loss of recent memory, faulty judg ensues, with affected infants exhibiting motor dysfunction, ment, and changes in personality. Later in the disease, those seizure, visual loss, and deafness. Death usually occurs by 2-5 with AD may forget how to do simple tasks like washing their years of age. Neuronal loss through an apoptotic mechanism hands. Eventually people with AD lose all reasoning abilities has been demonstrated (Huang et al., Hum. Mol. Genet. 6: and become dependent on other people for their everyday 1879-1885, 1997). care. Finally, the disease becomes so debilitating that patients 10 It is well-known that apoptosis plays a role in AIDS patho are bedridden and typically develop coexisting illnesses. genesis in the immune system. However, HIV-1 also induces PD is a chronic, incurable, and unstoppable CNS disorder neurological disease. Shietal. (J. Clin. Invest. 98: 1979-1990, that occurs gradually and results in uncontrolled body move 1996) examined apoptosis induced by HIV-1 infection of the ments, rigidity, tremor, and dyskinesia. These motor system CNS in an in vitro model and in brain tissue from AIDS problems are related to the death of brain cells in an area of the 15 patients, and found that HIV-1 infection of primary brain brain that produces dopamine, a chemical that helps control cultures induced apoptosis in neurons and astrocytes in vitro. muscle activity. In most people with PD, symptoms appear Apoptosis of neurons and astrocytes was also detected in after age 50. The initial symptoms of PD are a pronounced brain tissue from 10/11 AIDS patients, including 5/5 patients tremor affecting the extremities, notably in the hands or lips. with HIV-1 dementia and 4/5 nondemented patients. Subsequent characteristic symptoms of PD are stiffness or There are four main peripheral neuropathies associated slowness of movement, a shuffling walk, Stooped posture, and with HIV, namely sensory neuropathy, AIDP/CIPD, drug impaired balance. There are wide ranging secondary symp induced neuropathy and CMV-related. toms Such as memory loss, dementia, depression, emotional The most common type of neuropathy associated with changes, Swallowing difficulties, abnormal speech, sexual AIDS is distal symmetrical polyneuropathy (DSPN). This dysfunction, and bladder and bowel problems. These symp 25 syndrome is a result of nerve degeneration and is character toms will begin to interfere with routine activities, such as ized by numbness and a sensation of pins and needles. DSPN holding a fork or reading a newspaper. Finally, people with causes few serious abnormalities and mostly results in numb PD become so profoundly disabled that they are bedridden. ness or tingling of the feet and slowed reflexes at the ankles. ALS (motor neuron disease) is a chronic, incurable, and It generally occurs with more severe immunosuppression and unstoppable CNS disorder that attacks the motor neurons, 30 is steadily progressive. Treatment with tricyclic antidepres components of the CNS that connect the brain to the skeletal sants relieves symptoms but does not affect the underlying muscles. In ALS, the motor neurons deteriorate and eventu nerve damage. ally die, and though a person's brain normally remains fully A less frequent, but more severe type of neuropathy is functioning and alert, the command to move never reaches the known as acute or chronic inflammatory demyelinating poly muscles. Most people who get ALS are between 40 and 70 35 neuropathy (AIDP/CIDP). In AIDP/CIDP there is damage to years old. The first motor neurons that weaken are those the fatty membrane covering the nerve impulses. This kind of controlling the arms or legs. Those with ALS may have neuropathy involves inflammation and resembles the muscle trouble walking, they may drop things, fall, slur their speech, deterioration often identified with long-term use of AZT. It and laugh or cry uncontrollably. Eventually the muscles in the can be the first manifestation of HIV infection, where the limbs begin to atrophy from disuse. This muscle weakness 40 patient may not complain of pain, but fails to respond to will become debilitating and a person will need a wheelchair standard reflex tests. This kind of neuropathy may be associ or become unable to function out of bed. ated with seroconversion, in which case it can sometimes The causes of these neurological diseases have remained resolve spontaneously. It can serve as a sign of HIV infection largely unknown. They are conventionally defined as distinct and indicate that it might be time to considerantiviral therapy. diseases, yet clearly show extraordinary similarities in basic 45 AIDP/CIDP may be auto-immune in origin. processes and commonly demonstrate overlapping Symp Drug-induced, or toxic, neuropathies can be very painful. toms far greater than would be expected by chance alone. Antiviral drugs commonly cause peripheral neuropathy, as do Current disease definitions fail to properly deal with the issue other drugs e.g. Vincristine, dilantin (an anti-seizure medica of overlap and a new classification of the neurodegenerative tion), high-dose vitamins, isoniazid, and folic acid antago disorders has been called for. 50 nists. Peripheral neuropathy is often used in clinical trials for HD is another neurodegenerative disease resulting from antivirals as a dose-limiting side effect, which means that genetically programmed degeneration of neurons in certain more drugs should not be administered. Additionally, the use areas of the brain. This degeneration causes uncontrolled of Such drugs can exacerbate otherwise minor neuropathies. movements, loss of intellectual faculties, and emotional dis Usually, these drug-induced neuropathies are reversible with turbance. HD is a familial disease, passed from parent to child 55 the discontinuation of the drug. through a dominant mutation in the wild-type gene. Some CMV causes several neurological syndromes in AIDS, early symptoms of HD are mood Swings, depression, irrita including encephalitis, myelitis, and polyradiculopathy. bility or trouble driving, learning new things, remembering a Neuronal loss is also a salient feature of prion diseases, fact, or making a decision. As the disease progresses, concen such as Creutzfeldt-Jakob disease in human, BSE in cattle tration on intellectual tasks becomes increasingly difficult 60 (mad cow disease), Scrapie Disease in sheep and goats, and and the patient may have difficulty feeding himself or herself feline spongiform encephalopathy (FSE) in cats. Sirtuin and Swallowing. modulating compounds that increase the level and/or activity Tay-Sachs disease and Sandhoff disease are glycolipid of a sirtuin protein may be useful for treating or preventing storage diseases caused by the lack of lysosomal B-hex neuronal loss due to these prior diseases. osaminidase (Gravel et al., in The Metabolic Basis of Inher 65 In another embodiment, a sirtuin-modulating compound ited Disease, eds. Scriver et al., McGraw-Hill, New York, pp. that increases the level and/or activity of a sirtuin protein may 2839-2879, 1995). In both disorders, GM2 ganglioside and be used to treat or prevent any disease or disorder involving US 8,178,536 B2 135 136 axonopathy. Distal axonopathy is a type of peripheral neur As such, MS is now a common and well-known neurologi opathy that results from Some metabolic or toxic derange cal disorder that is characterized by episodic patches of ment of peripheral nervous system (PNS) neurons. It is the inflammation and demyelination which can occur anywhere most common response of nerves to metabolic or toxic dis in the CNS. However, almost always without any involve turbances, and as Such may be caused by metabolic diseases ment of the peripheral nerves associated therewith. Demyeli Such as diabetes, renal failure, deficiency syndromes Such as nation produces a situation analogous to that resulting from malnutrition and alcoholism, or the effects of toxins or drugs. cracks or tears in an insulator Surrounding an electrical cord. The most common cause of distal axonopathy is diabetes, and That is, when the insulating sheath is disrupted, the circuit is the most common distal axonopathy is diabetic neuropathy. “short circuited' and the electrical apparatus associated The most distal portions of axons are usually the first to 10 therewith will function intermittently or nor at all. Such loss degenerate, and axonal atrophy advances slowly towards the of myelin Surrounding nerve fibers results in short circuits in nerve’s cell body. If the noxious stimulus is removed, regen nerves traversing the brain and the spinal cord that thereby eration is possible, though prognosis decreases depending on result in symptoms of MS. It is further found that such demy the duration and severity of the stimulus. Those with distal 15 elination occurs in patches, as opposed to along the entire axonopathies usually present with symmetrical glove-stock CNS. In addition, such demyelination may be intermittent. ing sensori-motor disturbances. Deep tendon reflexes and Therefore, such plaques are disseminated in both time and autonomic nervous system (ANS) functions are also lost or Space. diminished in affected areas. It is believed that the pathogenesis involves a local disrup Diabetic neuropathies are neuropathic disorders that are tion of the blood brain barrier which causes a localized associated with diabetes mellitus. These conditions usually immune and inflammatory response, with consequent dam result from diabetic microvascular injury involving Small age to myelin and hence to neurons. blood vessels that supply nerves (vasa nervorum). Relatively Clinically, MS exists in both sexes and can occur at any common conditions which may be associated with diabetic age. However, its most common presentation is in the rela neuropathy include third nerve palsy, mononeuropathy; 25 tively young adult, often with a single focal lesion Such as a mononeuritis multiplex; diabetic amyotrophy; a painful poly damage of the optic nerve, an area of anesthesia (loss of neuropathy; autonomic neuropathy; and thoracoabdominal sensation), or paraesthesia (localize loss offeeling), or mus neuropathy. Clinical manifestations of diabetic neuropathy cular weakness. In addition, Vertigo, double vision, localized include, for example, sensorimotor polyneuropathy Such as pain, incontinence, and pain in the arms and legs may occur numbness, sensory loss, dysesthesia and nighttime pain; 30 upon flexing of the neck, as well as a large variety of less autonomic neuropathy Such as delayed gastric emptying or common symptoms. gastroparesis; and cranial neuropathy such as oculomotor An initial attack of MS is often transient, and it may be (3rd) neuropathies or Mononeuropathies of the thoracic or weeks, months, or years before a further attack occurs. Some lumbar spinal nerves. individuals may enjoy a stable, relatively event free condition Peripheral neuropathy is the medical term for damage to 35 for a great number of years, while other less fortunate ones nerves of the peripheral nervous system, which may be may experience a continual downhill course ending in com caused either by diseases of the nerve or from the side-effects plete paralysis. There is, most commonly, a series of remis of systemic illness. Peripheral neuropathies vary in their pre sion and relapses, in which each relapse leaves a patient sentation and origin, and may affect the nerve or the neuro Somewhat worse than before. Relapses may be triggered by muscular junction. Major causes of peripheral neuropathy 40 stressful events, viral infections or toxins. Therein, elevated include seizures, nutritional deficiencies, and HIV, though body temperature, i.e., a fever, will make the condition worse, diabetes is the most likely cause. Mechanical pressure from or as a reduction oftemperature by, for example, a cold bath, staying in one position for too long, a tumor, intraneural may make the condition better. hemorrhage, exposing the body to extreme conditions such as In yet another embodiment, a sirtuin-modulating com radiation, cold temperatures, or toxic Substances can also 45 pound that increases the level and/or activity of a sirtuin cause peripheral neuropathy. protein may be used to treat trauma to the nerves, including, In an exemplary embodiment, a sirtuin-modulating com trauma due to disease, injury (including Surgical interven pound that increases the level and/or activity of a sirtuin tion), or environmental trauma (e.g., neurotoxins, alcohol protein may be used to treat or prevent multiple Sclerosis ism, etc.). (MS), including relapsing MS and monosymptomatic MS, 50 Sirtuin-modulating compounds that increase the level and/ and other demyelinating conditions, such as, for example, or activity of a sirtuin protein may also be useful to prevent, chromic inflammatory demyelinating polyneuropathy treat, and alleviate symptoms of various PNS disorders, such (CIDP), or symptoms associated therewith. as the ones described below. The PNS is composed of the MS is a chronic, often disabling disease of the central nerves that lead to or branch off from the spinal cord and nervous system. Various and converging lines of evidence 55 CNS. The peripheral nerves handle a diverse array of func point to the possibility that the disease is caused by a distur tions in the body, including sensory, motor, and autonomic bance in the immune function, although the cause of this functions. When an individual has a peripheral neuropathy, disturbance has not been established. This disturbance per nerves of the PNS have been damaged. Nerve damage can mits cells of the immune system to “attack’ myelin, the fat arise from a number of causes, such as disease, physical containing insulating sheath that Surrounds the nerve axons 60 injury, poisoning, or malnutrition. These agents may affect located in the central nervous system (“CNS). When myelin either afferent or efferent nerves. Depending on the cause of is damaged, electrical pulses cannot travel quickly or nor damage, the nerve cell axon, its protective myelin sheath, or mally along nerve fiber pathways in the brain and spinal cord. both may be injured or destroyed. This results in disruption of normal electrical conductivity The term “peripheral neuropathy' encompasses a wide within the axons, fatigue and disturbances of vision, strength, 65 range of disorders in which the nerves outside of the brain and coordination, balance, sensation, and bladder and bowel spinal cord peripheral nerves—have been damaged. function. Peripheral neuropathy may also be referred to as peripheral US 8,178,536 B2 137 138 neuritis, or if many nerves are involved, the terms polyneur nerve impulses, due to, for example, myelin sheath dysfunc opathy or polyneuritis may be used. tion, or axonal loss. The nerve and nerve sheath injuries may Peripheral neuropathy is a widespread disorder, and there be caused by ischemia; inflammation; or a direct mechanical are many underlying causes. Some of these causes are com effect; Neuritis (a general term indicating inflammation of a mon, such as diabetes, and others are extremely rare, such as peripheral or cranial nerve). Clinical manifestation may acrylamide poisoning and certain inherited disorders. The include pain; paresthesias; paresis; or hyperesthesia; Poly most common worldwide cause of peripheral neuropathy is neuropathies (diseases of multiple peripheral nerves). The leprosy. Leprosy is caused by the bacterium Mycobacterium various forms are categorized by the type of nerve affected leprae, which attacks the peripheral nerves of affected people. (e.g., sensory, motor, or autonomic), by the distribution of Leprosy is extremely rare in the United States, where dia 10 nerve injury (e.g., distal VS. proximal), by nerve component betes is the most commonly known cause of peripheral neu primarily affected (e.g., demyelinating vs. axonal), by etiol ropathy. It has been estimated that more than 17 million ogy, or by pattern of inheritance. people in the United States and Europe have diabetes-related In another embodiment, a sirtuinactivating compound may polyneuropathy. Many neuropathies are idiopathic; no known be used to treat or prevent chemotherapeutic induced neur cause can be found. The most common of the inherited 15 opathy. The sirtuin modulating compounds may be adminis peripheral neuropathies in the United States is Charcot tered prior to administration of the chemotherapeutic agent, Marie-Tooth disease, which affects approximately 125,000 concurrently with administration of the chemotherapeutic persons. Another of the better known peripheral neuropathies is drug, and/or after initiation of administration of the chemo Guillain-Barré syndrome, which arises from complications therapeutic drug. If the sirtuin activating compound is admin associated with viral illnesses. Such as cytomegalovirus, istered after the initiation of administration of the chemo Epstein-Barr virus, and human immunodeficiency virus therapeutic drug, it is desirable that the sirtuin activating (HIV), or bacterial infection, including Campylobacterjejuni compound be administered prior to, or at the first signs, of and Lyme disease. The worldwide incidence rate is approxi chemotherapeutic induced neuropathy. mately 1.7 cases per 100,000 people annually. Other well Chemotherapy drugs can damage any part of the nervous known causes of peripheral neuropathies include chronic 25 system. Encephalopathy and myelopathy are fortunately very alcoholism, infection of the varicella-zoster virus, botulism, rare. Damage to peripheral nerves is much more common and and poliomyelitis. Peripheral neuropathy may develop as a can be a side effect of treatment experienced by people with primary symptom, or it may be due to another disease. For cancers, such as lymphoma. Most of the neuropathy affects example, peripheral neuropathy is only one symptom of dis sensory rather than motor nerves. Thus, the common Symp eases such as amyloid neuropathy, certain cancers, or inher 30 toms are tingling, numbness or a loss of balance. The longest ited neurologic disorders. Such diseases may affect the PNS nerves in the body seem to be most sensitive hence the fact and the CNS, as well as other body tissues. that most patients will report numbness or pins and needles in Other PNS diseases treatable with sirtuin-modulating their hands and feet. compounds that increase the level and/or activity of a sirtuin The chemotherapy drugs which are most commonly asso protein include: Brachial Plexus Neuropathies (diseases of 35 ciated with neuropathy, are the Vinca alkaloids (anti-cancer the cervical and first thoracic roots, nerve trunks, cords, and drugs originally derived from a member of the periwinkle peripheral nerve components of the brachial plexus. Clinical the Vinca plant genus) and a platinum-containing drug called manifestations include regional pain, paresthesia; muscle Cisplatin. The Vinca alkaloids include the drugs vinblastine, weakness, and decreased sensation in the upper extremity. Vincristine and vindesine. Many combination chemotherapy These disorders may be associated with trauma, including treatments for lymphoma for example CHOP and CVP con birth injuries; thoracic outlet syndrome; neoplasms, neuritis, 40 tain Vincristine, which is the drug known to cause this prob radiotherapy; and other conditions. See Adams et al., Prin lem most frequently. Indeed, it is the risk of neuropathy that ciples of Neurology, 6th ed., pp 1351-2); Diabetic Neuropa limits the dose of Vincristine that can be administered. thies (peripheral, autonomic, and cranial nerve disorders that Studies that have been performed have shown that most are associated with diabetes mellitus). These conditions usu patients will lose some reflexes in their legs as a result of ally result from diabetic microvascular injury involving Small 45 treatment with Vincristine and many will experience some blood vessels that supply nerves (vasa nervorum). Relatively degree oftingling (paresthesia) in their fingers and toes. The common conditions which may be associated with diabetic neuropathy does not usually manifest itself right at the start of neuropathy include third nerve palsy, mononeuropathy; the treatment but generally comes on over a period of a few mononeuritis multiplex; diabetic amyotrophy; a painful poly weeks. It is not essential to stop the drug at the first onset of neuropathy; autonomic neuropathy; and thoracoabdominal 50 symptoms, but if the neuropathy progresses this may be nec neuropathy (see Adams et al., Principles of Neurology, 6th ed, essary. It is very important that patients should report Such p1325); mononeuropathies (disease or trauma involving a symptoms to their doctors, as the nerve damage is largely single peripheral nerve in isolation, or out of proportion to reversible if the drug is discontinued. Most doctors will often evidence of diffuse peripheral nerve dysfunction). Mononeu reduce the dose of Vincristine or switch to another form of ritis multiplex refers to a condition characterized by multiple isolated nerve injuries. Mononeuropathies may result from a 55 Vinca alkaloid such as vinblastine or vindesine if the symp wide variety of causes, including ischemia; traumatic injury; toms are mild. Occasionally, the nerves Supplying the bowel compression; connective tissue diseases; cumulative trauma are affected causing abdominal pain and constipation. disorders; and other conditions; Neuralgia (intense or aching In another embodiment, a sirtuinactivating compound may pain that occurs along the course or distribution of a periph be used to treat or prevent a polyglutamine disease. Hunting eral or cranial nerve); Peripheral Nervous System Neoplasms 60 ton's Disease (HD) and Spinocerebellarataxia type 1 (SCA1) (neoplasms which arise from peripheral nerve tissue). This are just two examples of a class of genetic diseases caused by includes neurofibromas; Schwannomas; granular cell dynamic mutations involving the expansion of triplet tumors; and malignant peripheral nerve sheath tumors (see sequence repeats. In reference to this common mechanism, DeVita Jr et al., Cancer: Principles and Practice of Oncology, these disorders are called trinucleotide repeat diseases. At 5th ed., pp.1750-1); and Nerve Compression Syndromes (me 65 least 14 Such diseases are knownto affect human beings. Nine chanical compression of nerves or nerve roots from internal of them, including SCA1 and Huntington's disease, have or external causes). These may result in a conduction block to CAG as the repeated sequence (see Table 2 below). Since US 8,178,536 B2 139 140 CAG codes for an amino acid called glutamine, these nine Many transcription factors have also been found in neu trinucleotide repeat disorders are collectively known as poly ronal inclusions in different diseases. It is possible that these glutamine diseases. transcription factors interact with the polyglutamine-contain Although the genes involved in different polyglutamine ing proteins and then become trapped in the neuronal inclu diseases have little in common, the disorders they cause fol sions. This in turn might keep the transcription factors from low a strikingly similar course. Each disease is characterized turning genes on and off as needed by the cell. Another by a progressive degeneration of a distinct group of nerve observation is hypoacetylation of histones in affected cells. cells. The major symptoms of these diseases are similar, This has led to the hypothesis that Class I/II Histone Deacety although not identical, and usually affect people in midlife. lase (HDAC I/II) inhibitors, which are known to increase Given the similarities in Symptoms, the polyglutamine dis 10 histone acetylation, may be a novel therapy for polyglutamine eases are hypothesized to progress via common cellular diseases (U.S. patent application Ser. No. 10/476,627; mechanisms. In recent years, scientists have made great "Method of treating neurodegenerative, psychiatric, and strides in unraveling what the mechanisms are. other disorders with deacetylase inhibitors'). Above a certain threshold, the greater the number of 15 In yet another embodiment, the invention provides a glutamine repeats in a protein, the earlier the onset of disease method for treating or preventing neuropathy related to and the more severe the symptoms. This suggests that abnor ischemic injuries or diseases, such as, for example, coronary mally long glutamine tracts render their host protein toxic to heart disease (including congestive heart failure and myocar nerve cells. dial infarctions), stroke, emphysema, hemorrhagic shock, To test this hypothesis, scientists have generated geneti peripheral vascular disease (upper and lower extremities) and cally engineered mice expressing proteins with long poly transplant related injuries. In certain embodiments, the invention provides a method to glutamine tracts. Regardless of whether the mice express treat a central nervous system cell to prevent damage in full-length proteins or only those portions of the proteins response to a decrease in blood flow to the cell. Typically the containing the polyglutamine tracts, they develop symptoms severity of damage that may be prevented will depend in large of polyglutamine diseases. This suggests that a long poly 25 part on the degree of reduction in blood flow to the cell and the glutamine tract by itself is damaging to cells and does not duration of the reduction. By way of example, the normal have to be part of a functional protein to cause its damage. amount of perfusion to brain gray matter in humans is about For example, it is thought that the symptoms of SCA1 are 60 to 70 mL/100 g of brain tissue/min. Death of central not directly caused by the loss of normal ataxin-1 function but 30 nervous system cells typically occurs when the flow of blood falls below approximately 8-10 mL/100g of brain tissue/min, rather by the interaction between ataxin-1 and another protein while at slightly higher levels (i.e. 20-35 mL/100g of brain called LANP. LANP is needed for nerve cells to communicate tissue/min) the tissue remains alive but notable to function. In with one another and thus for their survival. When the mutant one embodiment, apoptotic or necrotic cell death may be ataxin-1 protein accumulates inside nerve cells, it “traps the prevented. In still a further embodiment, ischemic-mediated LANP protein, interfering with its normal function. After a 35 damage. Such as cytoxic edema or central nervous system while, the absence of LANP function appears to cause nerve tissue anoxemia, may be prevented. In each embodiment, the cells to malfunction. central nervous system cell may be a spinal cellora brain cell. TABLE 2 Summary of Polyglutamine Diseases. Normal Disease Gene Chromosomal Pattern of repeat repeat Disease l8le location inheritance Protein length length Spinobulbar muscular AR Xq13-21 X-linked androgen 9-36 38-62 atrophy (Kennedy recessive receptor (AR) disease) Huntington's HD 4p16.3 autosomal huntingtin 6-35 36-121 disease ominan Dentatorubral- DRPLA 12p13.31 autosomal atrophin-1 6-35 49-88 pallidoluysian ominan atrophy (Haw River syndrome) Spinocerebellar SCA1 6p23 autosomal ataxin-1 6-44 39-82 ataxia type 1 ominan Spinocerebellar SCA2 12q24.1 autosomal ataxin-2 15-31 36-63 ataxia type 2 ominan Spinocerebellar SCA3 14q32.1 autosomal ataxin-3 12-40 SS-84 ataxia type 3 ominan (Machado Joseph disease) Spinocerebellar SCA6 19p13 autosomal C1- 4-18 21-33 ataxia type 6 ominan voltage-dependent calcium channel Subunit Spinocerebellar SCA7 3p12-13 autosomal ataxin-7 4-35 37-306 ataxia type 7 ominan Spinocerebellar SCA17 6q27 autosomal TATA binding 25-42 45-63 ataxia type 17 ominan protein US 8,178,536 B2 141 142 Another aspect encompasses administrating a sirtuin acti due to arteriosclerosis. In yet another embodiment, the Vating compound to a subject to treat a central nervous system ischemic condition results from an injury to the brain or spinal ischemic condition. A number of central nervous system cord. ischemic conditions may be treated by the sirtuin activating In yet another aspect, a sirtuinactivating compound may be compounds described herein. In one embodiment, the 5 administered to reduce infarct size of the ischemic core fol ischemic condition is a stroke that results in any type of lowing a central nervous system ischemic condition. More ischemic central nervous system damage. Such as apoptotic or over, a sirtuin activating compound may also be beneficially necrotic cell death, cytoxic edema or central nervous system administered to reduce the size of the ischemic penumbra or tissue anoxia. The stroke may impact any area of the brain or transitional Zone following a central nervous systemischemic be caused by any etiology commonly known to result in the 10 condition. occurrence of a stroke. In one alternative of this embodiment, In one embodiment, a combination drug regimen may the stroke is a brain stem stroke. Generally speaking, brain include drugs or compounds for the treatment or prevention stem strokes strike the brain stem, which control involuntary of neurodegenerative disorders or secondary conditions asso life-support functions such as breathing, blood pressure, and ciated with these conditions. Thus, a combination drug regi heartbeat. In another alternative of this embodiment, the 15 men may include one or more sirtuin activators and one or stroke is a cerebellar stroke. Typically, cerebellar strokes more anti-neurodegeneration agents. For example, one or impact the cerebellum area of the brain, which controls bal more sirtuin-activating compounds can be combined with an ance and coordination. In still another embodiment, the effective amount of one or more of L-DOPA; a dopamine stroke is an embolic stroke. In general terms, embolic strokes agonist; an adenosine AA receptor antagonist; a COMT may impact any region of the brain and typically result from inhibitor; a MAO inhibitor; an N-NOS inhibitor, a sodium the blockage of an artery by a vaso-occlusion. In yet another channel antagonist; a selective N-methyl D-aspartate alternative, the stroke may be a hemorrhagic stroke. Like (NMDA) receptor antagonist; an AMPA/kainate receptor ischemic strokes, hemorrhagic stroke may impact any region antagonist; a calcium channel antagonist; a GABA-A recep of the brain, and typically result from a ruptured blood vessel toragonist; an acetyl-choline esterase inhibitor, a matrix met characterized by a hemorrhage (bleeding) within or Surround 25 alloprotease inhibitor; a PARP inhibitor; an inhibitor of p38 ing the brain. In a further embodiment, the stroke is a throm MAP kinase or c-jun-N-terminal kinases: TPA; NDA antago botic stroke. Typically, thrombotic strokes result from the nists; beta-interferons; growth factors; glutamate inhibitors; blockage of a blood vessel by accumulated deposits. and/or as part of a cell therapy. In another embodiment, the ischemic condition may result Exemplary N-NOS inhibitors include 4-(6-amino-pyridin from a disorder that occurs in a part of the subjects body 30 2-yl)-3-methoxyphenol 6-4-(2-dimethylamino-ethoxy)-2- outside of the central nervous system, but yet still causes a methoxy-phenyl-pyridin-2-yl-amine, 6-4-(2-dimethy reduction in blood flow to the central nervous system. These lamino-ethoxy)-2,3-dimet-hyl-phenyl-pyridin-2-yl-amine, disorders may include, but are not limited to a peripheral 6-4-(2-pyrrolidinyl-ethoxy)-2,3-dimethyl-p-henyl-pyri vascular disorder, a venous thrombosis, a pulmonary embo din-2-yl-amine, 6-4-(4-(n-methyl)piperidinyloxy)-2,3-dim lus, arrhythmia (e.g. atrial fibrillation), a myocardial infarc 35 ethyl-p-henyl-pyridin-2-yl-amine, 6-4-(2-dimethylamino tion, a transient ischemic attack, unstable angina, or sickle ethoxy)-3-methoxy-phenyl-pyridin-2-yl-amine, 6-4-(2- cell anemia. Moreover, the central nervous system ischemic pyrrolidinyl-ethoxy)-3-methoxy-phenyl-pyridin-2-yl condition may occur as result of the Subject undergoing a amine, 6-4-2-(6,7-dimethoxy-3,4-dihydro-1h-isoquinolin Surgical procedure. By way of example, the Subject may be 2-yl)-ethoxy-3-methoxy-phenyl-pyridin-2-yl-amine, 6-3- undergoing heart Surgery, lung Surgery, spinal Surgery, brain 40 methoxy-4-2-(4-phenethyl-piper-azin-1-yl)-ethoxy Surgery, vascular Surgery, abdominal Surgery, or organ trans phenyl-pyridin-2-yl-amine, 6-3-methoxy-4-2-(4-methyl plantation Surgery. The organ transplantation Surgery may piperazin-1-yl)-ethoxyl-phenyl-pyridin-2-yl-amine, 6-4- include heart, lung, pancreas, kidney or liver transplantation 2-(4-dimethylamin-o-piperidin-1-yl)-ethoxy-3-methoxy Surgery. Moreover, the central nervous system ischemic con phenyl-pyridin-2-yl -amine, 6-4-(2-dimethylamino dition may occur as a result of a trauma or injury to a part of 45 ethoxy)-3-ethoxy-phenyl-pyridin-2-yl-amine, 6-4-(2- the subject’s body outside the central nervous system. By way pyrrolidinyl-ethoxy)-3-ethoxy-phenyl-pyridin-2-yl-amine, of example, the trauma or injury may cause a degree of 6-4-(2-dimethylamino -ethoxy)-2-isopropyl-phenyl-pyri bleeding that significantly reduces the total volume of blood din-2-yl-amine, 4-(6-amino-pyridin-yl)-3-cyclopropyl-phe in the subject’s body. Because of this reduced total volume, nol 6-2-cyclopropyl-4-(2-dimethy-lamino-ethoxy)-phenyl the amount of blood flow to the central nervous system is 50 pyridin-2-yl -amine, 6-2-cyclopropyl-4-(2-pyrrolidin-1-yl concomitantly reduced. By way of further example, the ethoxy)-phenyl-pyridin-2-yl-amine, 3-3-(6-amino-pyridin trauma or injury may also result in the formation of a vaso 2yl)-4-cycl-opropyl-phenoxy-pyrrolidine-1-carboxylic acid occlusion that restricts blood flow to the central nervous sys tert-butyl ester 6-2-cyclopropyl-4-(1-methyl-pyrrolidin-3- tem. yl-oxy)-phenyl-pyridin-2-yl-amine, 4-(6-amino-pyridin-2- Of course it is contemplated that the sirtuin activating 55 yl)-3-cyclobutyl-phenol 6-2-cyclobutyl-4-(2-dime-thy compounds may be employed to treat the central nervous lamino -ethoxy)-phenyl-pyridin-2-yl-amine, 6-2- system ischemic condition irrespective of the cause of the cyclobutyl-4-(2-pyrrolid-in-1-yl-ethoxy) -phenyl-pyridin condition. In one embodiment, the ischemic condition results 2-yl-amine, 6-2-cyclobutyl-4-(1-methyl-pyr-rollidin-3-yl from a vaso-occlusion. The vaso-occlusion may be any type oxy) -phenyl-pyridin-2-yl-amine, 4-(6-amino-pyridin-2- of occlusion, but is typically a cerebral thrombosis or an 60 yl)-3-cy-clopentyl-phenol 6-2-cyclopentyl-4-(2- embolism. In a further embodiment, the ischemic condition dimethylamino-ethoxy)-phenyl-pyrid-in-2-yl-amine, 6-2- may result from a hemorrhage. The hemorrhage may be any cyclopentyl-4-(2-pyrrolidin-1yl-ethoxy)-phenyl-pyridin-2- type of hemorrhage, but is generally a cerebral hemorrhage or y1-amine, 3-4-(6-amino-pyridin-2yl)-3-methoxy-phenoxy a Subararachnoid hemorrhage. In still another embodiment, pyrrolidine-1-ca-rboxylic acid tert butyl ester 6-4-(1- the ischemic condition may result from the narrowing of a 65 methyl-pyrrolidin-3-yl-oxy)-2-metho-Xy-phenyl-pyridin-2- vessel. Generally speaking, the vessel may narrow as a result y1-amine, 4-4-(6-amino-pyridin-2yl)-3-methoxy of a vasoconstriction Such as occurs during vasospasms, or phenoxy--piperidine-1-carboxylic acid tert butyl ester 6-2-
US 8,178,536 B2 147 148 makalim, PCO-400 and SKP-450 (2-2"(1",3'-dioxolone)-2- Examples of hydroxamic acids and hydroxamic acid methyl-4-(2-oxo-1'-pyrrolidinyl)-6-nitro-2H-1-benzopyra derivatives, but are not limited to, trichostatin A (TSA), sub n). eroylanilide hydroxamic acid (SAHA), oxamflatin, suberic Exemplary AMPA/kainate receptor antagonists include bishydroxamic acid (SBHA), m-carboxy-cinnamic acid 6-cyano-7-nitroquinoxalin-2,3-di-one (CNOX); 6-nitro-7- 5 bishydroxamic acid (CBHA), valproic acid and pyroxamide. sulphamoylbenzof duinoxaline-2,3-dione (NBQX); 6.7- TSA was isolated as an antifungiantibiotic (Tsuji etal (1976) dinitroquinoxaline-2,3-dione (DNQX): 1-(4-aminophenyl)- J. Antibiot (Tokyo) 29:1-6) and found to be a potent inhibitor 4-methyl-7,8-m-ethylenedioxy-5H-2,3-benzodiazepine of mammalian HDAC (Yoshida et al. (1990).J. Biol. Chem. hydrochloride; and 2,3-dihydroxy-6-nitro-7-sulfamoyl 265:17174-17179). The finding that TSA-resistant cell lines benzo-fcquinoxaline. 10 have an altered HDAC evidences that this enzyme is an Exemplary Sodium channel antagonists include ajmaline, important target for TSA. Other hydroxamic acid-based procainamide, flecainide and riluzole. HDAC inhibitors, SAHA, SBHA, and CBHA are synthetic Exemplary matrix-metalloprotease inhibitors include compounds that are able to inhibit HDAC at micromolar 4-4-(4-fluorophenoxy)benzenesulfon-ylaminoltetrahydro concentration or lower in vitro or in vivo. Glicket al. (1999) 15 Cancer Res. 59:4392-4399. These hydroxamic acid-based pyran-4-carboxylic acid hydroxyamide: 5-Methyl-5-(4-(4- HDAC inhibitors all possess an essential structural feature: a fluorophenoxy)-phenoxy)-pyrimidine-2,4,6-trione: 5-n-Bu polar hydroxamic terminal linked through a hydrophobic tyl-5-(4-(4'-fluorophenoxy)-phenoxy)-pyrimidine-2,4,6- methylene spacer (e.g. 6 carbon at length) to another polar site trione and prinomistat. which is attached to a terminal hydrophobic moiety (e.g., Poly(ADP ribose) polymerase (PARP) is an abundant benzene ring). Compounds developed having Such essential nuclear enzyme which is activated by DNA strand single features also fall within the scope of the hydroxamic acids breaks to synthesize poly (ADP ribose) from NAD. Under that may be used as HDAC inhibitors. normal conditions, PARP is involved in base excision repair Cyclic peptides used as HDAC inhibitors are mainly cyclic caused by oxidative stress via the activation and recruitment tetrapeptides. Examples of cyclic peptides include, but are not of DNA repair enzymes in the nucleus. Thus, PARP plays a 25 limited to, trapoxin A, apicidin and depsipeptide. Trapoxin A role in cell necrosis and DNA repair. PARP also participates is a cyclic tetrapeptide that contains a 2-amino-8-oxo-9,10 in regulating cytokine expression that mediates inflamma epoxy-decanoyl (AOE) moiety. Kijima et al. (1993) J. Biol. tion. Under conditions where DNA damage is excessive (such Chem. 268:22429-22435. Apicidin is a fungal metabolite that as by acute excessive exposure to a pathological insult), exhibits potent, broad-spectrum antiprotozoal activitity and PARP is over-activated, resulting in cell-based energetic fail 30 inhibits HDAC activity at nanomolar concentrations. Darkin ure characterized by NAD depletion and leading to ATP con Rattray et al. (1996) Proc. Natl. Acad. Sci. USA. 93:13143 Sumption, cellular necrosis, tissue injury, and organ damage? 13147. Depsipeptide is isolated from Chromobacterium vio failure. PARP is thought to contribute to neurodegeneration laceum, and has been shown to inhibit HDAC activity at by depleting nicotinamide adenine dinucleotide (NAD+) micromolar concentrations. which then reduces adenosine triphosphate (ATP; Cosi and 35 Examples of benzamides include but are not limited to Marien, Ann. N.Y. Acad. Sci., 890:227, 1999) contributing to MS-27-275. Saito et al. (1990) Proc. Natl. Acad. Sci. USA. cell death which can be prevented by PARP inhibitors. Exem 96:4592-4597. Examples of short-chain fatty acids include plory PARP inhibitors can be found in Southan and Szabo, but are not limited to butyrates (e.g., butyric acid, arginine Current Medicinal Chemistry, 10:321, 2003. butyrate and phenylbutyrate (PB)). Newmark et al. (1994) Exemplary inhibitors of p38 MAP kinase and c-jun-N- 40 Cancer Lett. 78:1-5; and Carducci et al. (1997) Anticancer terminal kinases include pyridyl imidazoles, such as PD Res. 17:3972-3973. In addition, depudecin which has been 169316, isomeric PD 169316, SB 203580, SB 202190, SB shown to inhibit HDAC at micromolar concentrations (Kwon 220026, and RWJ 67657. Others are described in U.S. Pat. et al. (1998) Proc. Natl. Acad. Sci. USA.95:3356-3361) also No. 6,288,089, and incorporated by reference herein. falls within the scope of histone deacetylase inhibitor as In an exemplary embodiment, a combination therapy for 45 described herein. treating or preventing MS comprises a therapeutically effec Blood Coagulation Disorders tive amount of one or more sirtuin-modulating compounds In other aspects, sirtuin-modulating compounds that that increase the level and/or activity of a sirtuin protein and increase the level and/or activity of a sirtuin protein can be one or more of AvoneXOR) (interferon beta-1a). Tysabri R (na used to treat or prevent blood coagulation disorders (or hemo talizumab), or Fumaderm R) (BG-12/Oral Fumarate). 50 static disorders). As used interchangeably herein, the terms In another embodiment, a combination therapy for treating "hemostasis”, “blood coagulation, and “blood clotting refer or preventing diabetic neuropathy or conditions associated to the control of bleeding, including the physiological prop therewith comprises atherapeutically effective amount of one erties of vasoconstriction and coagulation. Blood coagulation or more sirtuin-modulating compounds that increase the level assists in maintaining the integrity of mammalian circulation and/or activity of a sirtuin protein and one or more of tricyclic 55 after injury, inflammation, disease, congenital defect, dys antidepressants (TCAS) (including, for example, imipramine, function or other disruption. After initiation of clotting, blood amytriptyline, desipramine and nortriptyline), serotonin coagulation proceeds through the sequential activation of reuptake inhibitors (SSRIs) (including, for example, fluoxet certain plasma proenzymes to their enzyme forms (see, for ine, paroxetine, Sertralene, and citalopram) and antiepileptic example, Coleman, R. W. etal. (eds.) Hemostasis and Throm drugs (AEDs) (including, for example, gabapentin, carbam 60 bosis, Second Edition, (1987)). These plasma glycoproteins, azepine, and topimirate). including Factor XII, Factor XI, Factor IX, Factor X, Factor In another embodiment, the invention provides a method VII, and prothrombin, are Zymogens of serine proteases. for treating or preventing a polyglutamine disease using a Most of these blood clotting enzymes are effective on a physi combination comprising at least one sirtuin activating com ological scale only when assembled in complexes on mem pound and at least one HDAC I/II inhibitor. Examples of 65 brane surfaces with protein cofactors such as Factor VIII and HDAC I/II inhibitors include hydroxamic acids, cyclic pep Factor V. Other blood factors modulate and localize clot for tides, benzamides, short-chain fatty acids, and depudecin. mation, or dissolve blood clots. Activated protein C is a spe US 8,178,536 B2 149 150 cific enzyme that inactivates procoagulant components. Cal combined with an effective amount of one or more of aspirin, cium ions are involved in many of the component reactions. heparin, and oral Warfarin that inhibits Vit K-dependent fac Blood coagulation follows either the intrinsic pathway, where tors, low molecular weight heparins that inhibit factors X and all of the protein components are present in blood, or the II, thrombininhibitors, inhibitors of platelet GPIb IIIa recep extrinsic pathway, where the cell-membrane protein tissue tors, inhibitors of tissue factor (TF), inhibitors of human von factor plays a critical role. Clot formation occurs when Willebrand factor, inhibitors of one of more factors involved fibrinogen is cleaved by thrombin to form fibrin. Blood clots in hemostasis (in particular in the coagulation cascade). In are composed of activated platelets and fibrin. addition, sirtuin-modulating compounds that increase the Further, the formation of blood clots does not only limit leveland/or activity of a sirtuin protein can be combined with bleeding in case of an injury (hemostasis), but may lead to 10 thrombolytic agents, such as t-PA, Streptokinase, reptilase, serious organ damage and death in the context of atheroscle rotic diseases by occlusion of an important artery or vein. TNK-t-PA, and staphylokinase. Thrombosis is thus blood clot formation at the wrong time Weight Control and place. It involves a cascade of complicated and regulated In another aspect, sirtuin-modulating compounds that biochemical reactions between circulating blood proteins 15 increase the level and/or activity of a sirtuin protein may be (coagulation factors), blood cells (in particular platelets), and used for treating or preventing weight gain or obesity in a elements of an injured vessel wall. Subject. For example, sirtuin-modulating compounds that Accordingly, the present invention provides anticoagula increase the level and/or activity of a sirtuin protein may be tion and antithrombotic treatments aiming at inhibiting the used, for example, to treat or prevent hereditary obesity, formation of blood clots in order to prevent or treat blood dietary obesity, hormone related obesity, obesity related to coagulation disorders, such as myocardial infarction, stroke, the administration of medication, to reduce the weight of a loss of a limb by peripheral artery disease or pulmonary Subject, or to reduce or prevent weight gain in a Subject. A embolism. Subject in need of Such a treatment may be a subject who is As used interchangeably herein, "modulating or modula obese, likely to become obese, overweight, or likely to tion of hemostasis' and “regulating or regulation of hemosta 25 become overweight. Subjects who are likely to become obese sis' includes the induction (e.g., stimulation or increase) of or overweight can be identified, for example, based on family hemostasis, as well as the inhibition (e.g., reduction or history, genetics, diet, activity level, medication intake, or decrease) of hemostasis. various combinations thereof. In one aspect, the invention provides a method for reducing In yet other embodiments, sirtuin-modulating compounds or inhibiting hemostasis in a subject by administering a sir 30 that increase the level and/or activity of a sirtuin protein may tuin-modulating compound that increases the level and/or be administered to subjects suffering from a variety of other activity of a sirtuin protein. The compositions and methods diseases and conditions that may be treated or prevented by disclosed herein are useful for the treatment or prevention of promoting weight loss in the Subject. Such diseases include, thrombotic disorders. As used herein, the term “thrombotic for example, high blood pressure, hypertension, high blood disorder includes any disorder or condition characterized by 35 cholesterol, dyslipidemia, type 2 diabetes, insulin resistance, excessive or unwanted coagulation or hemostatic activity, or glucose intolerance, hyperinsulinemia, coronary heart dis a hypercoagulable state. Thrombotic disorders include dis ease, angina pectoris, congestive heart failure, stroke, gall eases or disorders involving platelet adhesion and thrombus stones, cholescystitis and cholelithiasis, gout, osteoarthritis, formation, and may manifest as an increased propensity to obstructive sleep apnea and respiratory problems. Some types form thromboses, e.g., an increased number of thromboses, 40 of cancer (such as endometrial, breast, prostate, and colon), thrombosis at an early age, a familial tendency towards complications of pregnancy, poor female reproductive health thrombosis, and thrombosis at unusual sites. Examples of (such as menstrual irregularities, infertility, irregular ovula thrombotic disorders include, but are not limited to, throm tion), bladder control problems (such as stress incontinence); boembolism, deep vein thrombosis, pulmonary embolism, uric acid nephrolithiasis; psychological disorders (such as stroke, myocardial infarction, miscarriage, thrombophilia 45 depression, eating disorders, distorted body image, and low associated with anti-thrombin III deficiency, protein C defi self esteem). Stunkard A. J. Wadden T.A. (Editors) Obesity: ciency, protein S deficiency, resistance to activated protein C, theory and therapy, Second Edition. New York: Raven Press, dysfibrinogenemia, fibrinolytic disorders, homocystinuria, 1993. Finally, patients with AIDS can develop lipodystrophy pregnancy, inflammatory disorders, myeloproliferative disor or insulin resistance in response to combination therapies for ders, arteriosclerosis, angina, e.g., unstable angina, dissemi 50 AIDS. nated intravascular coagulation, thrombotic thrombocy In another embodiment, sirtuin-modulating compounds topenic purpura, cancer metastasis, sickle cell disease, that increase the level and/or activity of a sirtuin protein may glomerular nephritis, and drug induced thrombocytopenia be used for inhibiting adipogenesis or fat cell differentiation, (including, for example, heparin induced thrombocytopenia). whether in vitro or in vivo. In particular, high circulating In addition, sirtuin-modulating compounds that increase the 55 levels of insulin and/or insulin like growth factor (IGF) 1 will level and/or activity of a sirtuin protein may be administered be prevented from recruiting preadipocytes to differentiate to prevent thrombotic events or to prevent re-occlusion during into adipocytes. Such methods may be used for treating or or after therapeutic clot lysis or procedures such as angio preventing obesity. plasty or Surgery. In other embodiments, sirtuin-modulating compounds that In another embodiment, a combination drug regimen may 60 increase the level and/or activity of a sirtuin protein may be include drugs or compounds for the treatment or prevention used for reducing appetite and/or increasing Satiety, thereby of blood coagulation disorders or secondary conditions asso causing weight loss or avoidance of weight gain. A subject in ciated with these conditions. Thus, a combination drug regi need of such a treatment may be a Subject who is overweight, men may include one or more sirtuin-modulating compounds obese or a subject likely to become overweight or obese. The that increase the level and/or activity of a sirtuin protein and 65 method may comprise administering daily or, every other day, one or more anti-coagulation or anti-thrombosis agents. For or once a week, a dose, e.g., in the form of a pill, to a Subject. example, one or more sirtuin-modulating compounds can be The dose may be an “appetite reducing dose.” US 8,178,536 B2 151 152 In other embodiments, a sirtuin-modulating compound in combination with one or more anti-obesity agents. Exem that decreases the level and/or activity of a sirtuin protein may plary anti-obesity agents include, for example, phenylpro be used to stimulate appetite and/or weight gain. A method panolamine, ephedrine, pseudoephedrine, phentermine, a may comprise administering to a subject, Such as a subject in cholecystokinin-A agonist, a monoamine reuptake inhibitor need thereof, a pharmaceutically effective amount of a sir (such as Sibutramine), a sympathomimetic agent, a seroton tuin-modulating agent that decreases the level and/or activity ergic agent (such as dexfenfluramine or fenfluramine), a of a sirtuin protein, such as SIRT1 and/or SIRT3. A subject in dopamine agonist (Such as bromocriptine), a melanocyte need of Such a treatment may be a Subject who has cachexia stimulating hormone receptor agonist or mimetic, a melano or may be likely to develop cachexia. A combination of agents cyte-stimulating hormone analog, a cannabinoid receptor may also be administered. A method may further comprise 10 antagonist, a melanin concentrating hormone antagonist, the monitoring in the Subject the state of the disease or of activa OB protein (leptin), a leptin analog, a leptin receptoragonist, tion of sirtuins, for example, in adipose tissue. agalaninantagonist or a GIlipase inhibitor or decreaser (Such Methods for stimulating fat accumulation in cells may be as orlistat). Other anorectic agents include bombesin ago used in vitro, to establish cell models of weight gain, which nists, dehydroepiandrosterone or analogs thereof glucocor may be used, e.g., for identifying other drugs that prevent 15 ticoid receptor agonists and antagonists, orexin receptor Weight gain. antagonists, urocortin binding protein antagonists, agonists Also provided are methods for modulating adipogenesis or of the glucagon-like peptide-1 receptor Such as Exendin and fat cell differentiation, whether in vitro or in vivo. In particu ciliary neurotrophic factors such as AXokine. lar, high circulating levels of insulin and/or insulin like In another embodiment, sirtuin-modulating compounds growth factor (IGF) 1 will be prevented from recruiting that increase the level and/or activity of a sirtuin protein may preadipocytes to differentiate into adipocytes. Such methods be administered to reduce drug-induced weight gain. For may be used to modulate obesity. A method for stimulating example, a sirtuin-modulating compound that increases the adipogenesis may comprise contacting a cell with a sirtuin level and/or activity of a sirtuin protein may be administered modulating agent that decreases the level and/or activity of a as a combination therapy with medications that may stimulate sirtuin protein. 25 appetite or cause weight gain, in particular, weight gain due to In another embodiment, the invention provides methods of factors other than water retention. Examples of medications decreasing fat or lipid metabolism in a Subject by administer that may cause weight gain, include for example, diabetes ing a sirtuin-modulating compound that decreases the level treatments, including, for example, Sulfonylureas (such as and/or activity of a sirtuin protein. The method includes glipizide and glyburide), thiazolidinediones (such as piogli administering to a Subject an amount of a sirtuin-modulating 30 taZone and rosiglitaZone), meglitinides, nateglinide, repa compound, e.g., in an amount effective to decrease mobiliza glinide, Sulphonylurea medicines, and insulin; anti-depres tion offat to the blood from WAT cells and/or to decrease fat sants, including, for example, tricyclic antidepressants (such burning by BAT cells. as amitriptyline and imipramine), irreversible monoamine Methods for promoting appetite and/or weight gain may oxidase inhibitors (MAOIs), selective serotonin reuptake include, for example, prior identifying a Subject as being in 35 inhibitors (SSRIs), bupropion, paroxetine, and mirtazapine; need of decreased fat or lipid metabolism, e.g., by weighing steroids, such as, for example, prednisone; hormonetherapy: the subject, determining the BMI of the subject, or evaluating lithium carbonate; Valproic acid; carbamazepine; chlorprom fat content of the subject or sirtuin activity in cells of the azine; thiothixene; beta blockers (such as propranolo); alpha Subject. The method may also include monitoring the Subject, blockers (such as clonidine, prazosin and teraZosin); and con e.g., during and/or after administration of a sirtuin-modulat 40 traceptives including oral contraceptives (birth control pills) ing compound. The administering can include one or more or other contraceptives containing estrogen and/or progester dosages, e.g., delivered in boluses or continuously. Monitor one (Depo-Provera, Norplant, Ortho), testosterone or Mege ing can include evaluating a hormone or a metabolite. Exem strol. In another exemplary embodiment, sirtuin-modulating plary hormones include leptin, adiponectin, resistin, and compounds that increase the level and/or activity of a sirtuin insulin. Exemplary metabolites include triglyercides, choles 45 protein may be administered as part of a Smoking cessation terol, and fatty acids. program to prevent weight gain or reduce weight already In one embodiment, a sirtuin-modulating compound that gained. decreases the level and/or activity of a sirtuin protein may be Metabolic Disorders/Diabetes used to modulate (e.g., increase) the amount of subcutaneous In another aspect, sirtuin-modulating compounds that fat in a tissue, e.g., in facial tissue or in other surface-associ 50 increase the level and/or activity of a sirtuin protein may be ated tissue of the neck, hand, leg, or lips. The sirtuin-modu used for treating or preventing a metabolic disorder. Such as lating compound may be used to increase the rigidity, water insulin-resistance, a pre-diabetic state, type II diabetes, and/ retention, or Support properties of the tissue. For example, the or complications thereof. Administration of a sirtuin-modu sirtuin-modulating compound can be applied topically, e.g., lating compounds that increases the level and/or activity of a in association with another agent, e.g., for Surface-associated 55 sirtuin protein may increase insulin sensitivity and/or tissue treatment. The sirtuin-modulating compound may also decrease insulin levels in a subject. A Subject in need of Such be injected Subcutaneously, e.g., within the region where an a treatment may be a Subject who has insulin resistance or alteration in Subcutaneous fat is desired. other precursor symptom of type II diabetes, who has type II A method for modulating weight may further comprise diabetes, or who is likely to develop any of these conditions. monitoring the weight of the subject and/or the level of modu 60 For example, the Subject may be a subject having insulin lation of sirtuins, for example, in adipose tissue. resistance, e.g., having high circulating levels of insulin and/ In an exemplary embodiment, sirtuin-modulating com or associated conditions, such as hyperlipidemia, dyslipogen pounds that increase the level and/or activity of a sirtuin esis, hypercholesterolemia, impaired glucose tolerance, high protein may be administered as a combination therapy for blood glucose Sugar level, other manifestations of syndrome treating or preventing weight gain or obesity. For example, 65 X, hypertension, atherosclerosis and lipodystrophy. one or more sirtuin-modulating compounds that increase the In an exemplary embodiment, sirtuin-modulating com level and/or activity of a sirtuin protein may be administered pounds that increase the level and/or activity of a sirtuin US 8,178,536 B2 153 154 protein may be administered as a combination therapy for treat autoimmune diseases and/or inflammation associated treating or preventing a metabolic disorder. For example, one with autoimmune diseases such as organ-tissue autoimmune or more sirtuin-modulating compounds that increase the level diseases (e.g., Raynaud's syndrome), Scleroderma, myasthe and/or activity of a sirtuin protein may be administered in nia gravis, transplant rejection, endotoxin shock, sepsis, pso combination with one or more anti-diabetic agents. Exem riasis, eczema, dermatitis, multiple Sclerosis, autoimmune plary anti-diabetic agents include, for example, an aldose thyroiditis, uveitis, Systemic lupus erythematosis. Addison's reductase inhibitor, a glycogen phosphorylase inhibitor, a disease, autoimmune polyglandular disease (also known as Sorbitol dehydrogenase inhibitor, a protein tyrosine phos autoimmune polyglandular syndrome), and Grave's disease. phatase I B inhibitor, a dipeptidyl protease inhibitor, insulin In certain embodiments, one or more sirtuin-modulating (including orally bioavailable insulin preparations), an insu 10 compounds that increase the level and/or activity of a sirtuin lin mimetic, metformin, acarbose, a peroxisome proliferator protein may be taken alone or in combination with other activated receptor-Y (PPAR-Y) ligand such as troglitazone, compounds useful for treating or preventing inflammation. rosaglitaZone, pioglitaZone or GW-1929, a Sulfonylurea, gli Exemplary anti-inflammatory agents include, for example, pazide, glyburide, or chlorpropamide wherein the amounts of steroids (e.g., cortisol, cortisone, fludrocortisone, prednisone, the first and second compounds result in a therapeutic effect. 15 6.C.-methylprednisone, triamcinolone, betamethasone or dex Other anti-diabetic agents include a glucosidase inhibitor, a amethasone), nonsteroidal antiinflammatory drugs (NSAIDS glucagon-like peptide-1 (GLP-1), insulin, a PPAR C/Y dual (e.g., aspirin, acetaminophen, tolmetin, ibuprofen, mefe agonist, a meglitimide and an OP2 inhibitor. In an exemplary namic acid, piroXicam, nabumetone, rofecoxib, celecoxib, embodiment, an anti-diabetic agent may be a dipeptidyl pep etodolac or nimesulide). In another embodiment, the other tidase IV (DP-IV or DPP-IV) inhibitor, such as, for example therapeutic agent is an antibiotic (e.g., Vancomycin, penicil LAF237 from Novartis (NVP DPP728; 1-2-(5-cyanopy lin, amoxicillin, ampicillin, cefotaxime, ceftriaxone, ridin-2-yl)aminoethylaminoacetyl-2-cyano-(S)-pyrroli cefixime, rifampinmetronidazole, doxycycline or Streptomy dine) or MK-04301 from Merck (see e.g., Hughes et al., cin). In another embodiment, the other therapeutic agent is a Biochemistry 38: 11597-603 (1999)). PDE4 inhibitor (e.g., roflumilast or rolipram). In another Inflammatory Diseases 25 embodiment, the other therapeutic agent is an antihistamine In other aspects, sirtuin-modulating compounds that (e.g., cyclizine, hydroxy Zine, promethazine or diphenhy increase the level and/or activity of a sirtuin protein can be dramine). In another embodiment, the other therapeutic agent used to treat or prevent a disease or disorder associated with is an anti-malarial (e.g., artemisinin, artemether, artsunate, inflammation. Sirtuin-modulating compounds that increase chloroquine phosphate, mefloquine hydrochloride, doxycy the level and/or activity of a sirtuin protein may be adminis 30 cline hyclate, proguanil hydrochloride, atovaquone or halo tered prior to the onset of at, or after the initiation of inflam fantrine). In one embodiment, the other therapeutic agent is mation. When used prophylactically, the compounds are pref drotrecogin alfa. erably provided in advance of any inflammatory response or Further examples of anti-inflammatory agents include, for symptom. Administration of the compounds may prevent or example, aceclofenac, acemetacin, e-acetamidocaproic acid, attenuate inflammatory responses or symptoms. 35 acetaminophen, acetaminosalol, acetanilide, acetylsalicylic Exemplary inflammatory conditions include, for example, acid, S-adenosylmethionine, alclofenac, alclometaSone, multiple Sclerosis, rheumatoid arthritis, psoriatic arthritis, alfentanil, algestone, allylprodine, alminoprofen, aloxiprin, degenerative joint disease, spondouloarthropathies, gouty alphaprodine, aluminum bis(acetylsalicylate), amcinonide, arthritis, Systemic lupus erythematosus, juvenile arthritis, amfenac, aminochlorthenoxazin, 3-amino-4-hydroxybutyric rheumatoid arthritis, osteoarthritis, osteoporosis, diabetes 40 acid, 2-amino-4-picoline, aminopropylon, aminopyrine, (e.g., insulin dependent diabetes mellitus or juvenile onset amixetrine, ammonium salicylate, ampiroXicam, amtolimetin diabetes), menstrual cramps, cystic fibrosis, inflammatory guacil, anilleridine, antipyrine, antrafenine, apaZone, beclom bowel disease, irritable bowel syndrome, Crohn's disease, ethasone, bendazac, benorylate, benoxaprofen, benzpipery mucous colitis, ulcerative colitis, gastritis, esophagitis, pan lon, benzydamine, benzylmorphine, bermoprofen, creatitis, peritonitis, Alzheimer's disease, shock, ankylosing 45 betamethasone, betamethasone-17-valerate, bezitramide, spondylitis, gastritis, conjunctivitis, pancreatis (acute or C.-bisabolol, bromfenac, p-bromoacetanilide, 5-bromosali chronic), multiple organ injury syndrome (e.g., secondary to cylic acid acetate, bromosaligenin, bucetin, bucloxic acid, septicemia or trauma), myocardial infarction, atherosclero bucolome, budesonide, bufeXamac, bumadizon, buprenor sis, stroke, reperfusion injury (e.g., due to cardiopulmonary phine, butacetin, butibufen, butorphanol, carbamazepine, car bypass or kidney dialysis), acute glomerulonephritis, Vascu 50 biphene, carprofen, carsalam, chlorobutanol, chloropred litis, thermal injury (i.e., Sunburn), necrotizing enterocolitis, nisone, chlorthenoxazin, choline Salicylate, cinchophen, granulocyte transfusion associated syndrome, and/or cinmetacin, ciramadol, clidanac, clobetasol, clocortolone, Sjogren's syndrome. Exemplary inflammatory conditions of clometacin, clonitaZene, clonixin, clopirac, cloprednol, the skin include, for example, eczema, atopic dermatitis, con clove, codeine, codeine methyl bromide, codeine phosphate, tact dermatitis, urticaria, Schleroderma, psoriasis, and derma 55 codeine Sulfate, cortisone, cortivaZol, cropropamide, tosis with acute inflammatory components. crotethamide, cyclazocine, deflazacort, dehydrotestosterone, In another embodiment, sirtuin-modulating compounds desomorphine, desonide, desoximetaSone, dexamethasone, that increase the level and/or activity of a sirtuin protein may dexamethasone-21-isonicotinate, dexoxadrol, dextromora be used to treat or prevent allergies and respiratory condi mide, dextropropoxyphene, deoxycorticosterone, dezocine, tions, including asthma, bronchitis, pulmonary fibrosis, aller 60 diampromide, diamorphone, diclofenac, difenamizole, difen gic rhinitis, oxygen toxicity, emphysema, chronic bronchitis, piramide, diflorasone, diflucortolone, diflunisal, diflupred acute respiratory distress syndrome, and any chronic obstruc nate, dihydrocodeine, dihydrocodeinone enol acetate, dihy tive pulmonary disease (COPD). The compounds may be dromorphine, dihydroxyaluminum acetylsalicylate, used to treat chronic hepatitis infection, including hepatitis B dimenoxadol, dimepheptanol, dimethylthiambutene, diox and hepatitis C. 65 aphetylbutyrate, diplpanone, diprocetyl, dipyrone, ditazol. Additionally, sirtuin-modulating compounds that increase droxicam, emorfaZone, enfenamic acid, enoXolone, epirizole, the level and/or activity of a sirtuin protein may be used to eptazocine, etersalate, ethenZamide, ethoheptazine, ethoxa US 8,178,536 B2 155 156 Zene, ethylmethylthiambutene, ethylmorphine, etodolac, (methylsulfonyl)phenyl-3-(2H)-pyridazinone, etofenamate, etonitaZene, eugenol, felbinac, fenbufen, fen fluorophenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl) clozic acid, fendosal, fenoprofen, fentanyl, fentiazac, fepra benzenesulfonamide, tert-butyl 1 benzyl-4-(4-oxopiperidin dinol, feprazone, floctafenine, fluazacort, flucloronide, flufe 1-yl)sulfonylpiperidine-4-carboxylate, 4-5-(phenyl)-3- namic acid, flumethasone, flunisolide, flunixin, (trifluoromethyl)-1H-pyrazol-1-ylbenzenesulfonamide, flunoxaprofen, fluocinolone acetonide, fluocinonide, fluoci salts and prodrugs thereof. nolone acetonide, fluocortin butyl, fluocortolone, fluoresone, Flushing fluorometholone, fluperolone, flupirtine, fluprednidene, flu In another aspect, sirtuin-modulating compounds that prednisolone, fluproduaZone, flurandrenolide, flurbiprofen, increase the level and/or activity of a sirtuin protein may be fluticasone, formocortal, fosfosal, gentisic acid, glafenine, 10 used for reducing the incidence or severity of flushing and/or glucametacin, glycol salicylate, guaiaZulene, halcinonide, hot flashes which are symptoms of a disorder. For instance, halobetasol, halometaSone, haloprednone, heroin, hydroc the Subject method includes the use of sirtuin-modulating odone, hydrocortamate, hydrocortisone, hydrocortisone compounds that increase the level and/or activity of a sirtuin acetate, hydrocortisone succinate, hydrocortisone hemisuc protein, alone or in combination with other agents, for reduc cinate, hydrocortisone 21-lysinate, hydrocortisone cypi 15 ing incidence or severity of flushing and/or hot flashes in onate, hydromorphone, hydroxypethidine, ibufenac, ibupro cancer patients. In other embodiments, the method provides fen, ibuproxam, imidazole salicylate, indomethacin, for the use of sirtuin-modulating compounds that increase the indoprofen, isofeZolac, isoflupredone, isoflupredone acetate, level and/or activity of a sirtuin protein to reduce the inci isoladol, isomethadone, isonixin, isoxepac, isoxicam, keto dence or severity of flushing and/or hot flashes in menopausal bemidone, ketoprofen, ketorolac, p-lactophenetide, lefe and post-menopausal woman. tamine, levallorphan, levorphanol, levophenacyl-morphan, In another aspect, sirtuin-modulating compounds that lofentanil, lonazolac, lomoxicam, loxoprofen, lysine acetyl increase the level and/or activity of a sirtuin protein may be salicylate, maZipredone, meclofenamic acid, medrysone, used as a therapy for reducing the incidence or severity of mefenamic acid, meloxicam, meperidine, meprednisone, flushing and/or hot flashes which are side-effects of another meptazinol, mesalamine, metazocine, methadone, methotri 25 drug therapy, e.g., drug-induced flushing. In certain embodi meprazine, methylprednisolone, methylprednisolone ments, a method for treating and/or preventing drug-induced acetate, methylprednisolone sodium Succinate, methylpred flushing comprises administering to a patient in need thereof nisolone Suleptinate, metiazinic acid, metofoline, metopon, a formulation comprising at least one flushing inducing com mofebutaZone, mofeZolac, mometasone, moraZone, mor pound and at least one sirtuin-modulating compound that phine, morphine hydrochloride, morphine Sulfate, morpho 30 increases the level and/or activity of a sirtuin protein. In other line salicylate, myrophine, nabumetone, nalbuphine, nalor embodiments, a method for treating drug induced flushing phine, 1-naphthyl salicylate, naproxen, narceline, nefopam, comprises separately administering one or more compounds nicomorphine, nifenaZone, niflumic acid, nimeSulide, 5'-ni that induce flushing and one or more sirtuin-modulating com tro-2'-propoxyacetanilide, norlevorphanol, normethadone, pounds, e.g., wherein the sirtuin-modulating compound and normorphine, norpipanone, olsalazine, opium, oxaceprol, 35 flushing inducing agent have not been formulated in the same Oxametacine, Oxaprozin, oxycodone, oxymorphone, compositions. When using separate formulations, the sirtuin oxyphenbutaZone, papaveretum, paramethasone, paranyline, modulating compound may be administered (1) at the same as parsalmide, pentazocine, perisoxal, phenacetin, phenadox administration of the flushing inducing agent, (2) intermit one, phenazocine, phenaZopyridine hydrochloride, pheno tently with the flushing inducing agent, (3) staggered relative coll, phenoperidine, phenopyrazone, phenomorphan, phenyl 40 to administration of the flushing inducing agent, (4) prior to acetylsalicylate, phenylbutaZone, phenyl salicylate, phenyra administration of the flushing inducing agent, (5) Subsequent midol, piketoprofen, piminodine, pipebuZone, piperylone, to administration of the flushing inducing agent, and (6) vari piraZolac, piritramide, piroxicam, pirprofen, pranoprofen, ous combination thereof. Exemplary flushing inducing prednicarbate, prednisolone, prednisone, prednival, pred agents include, for example, niacin, faloxifene, antidepres nylidene, proglumetacin, proheptazine, promedol, propac 45 sants, anti-psychotics, chemotherapeutics, calcium channel etamol, properidine, propiram, propoxyphene, propy blockers, and antibiotics. phenaZone, produaZone, protizinic acid, proxazole, In one embodiment, sirtuin-modulating compounds that ramifenaZone, remifentanil, rimazolium metilsulfate, salac increase the level and/or activity of a sirtuin protein may be etamide, Salicin, Salicylamide, salicylamide o-acetic acid, used to reduce flushing side effects of a vasodilator or an salicylic acid, salicylsulfuric acid, Salsalate, salverine, sim 50 antilipemic agent (including anticholesteremic agents and etride, Sufentanil, SulfaSalazine, Sulindac, Superoxide dismu lipotropic agents). In an exemplary embodiment, a sirtuin tase, Suprofen, SuxibuZone, talniflumate, tenidap, tenoxicam, modulating compound that increases the level and/or activity terofenamate, tetrandrine, thiazolinobutaZone, tiaprofenic of a sirtuin protein may be used to reduce flushing associated acid, tiaramide, tilidine, tinoridine, tiXocortol, tolfenamic with the administration of niacin. acid, tolmetin, tramadol, triamcinolone, triamcinolone 55 Nicotinic acid, 3-pyridinecarboxylic acid or niacin, is an acetonide, tropesin, Viminol, Xenbucin, Ximoprofen, Zalto antilipidemic agent that is marketed under, for example, the profen and Zomepirac. trade names Nicolar R, SloNiacin R, NicobidR and Time In an exemplary embodiment, a sirtuin-modulating com Release Niacinr. Nicotinic acid has been used for many pound that increases the level and/or activity of a sirtuin years in the treatment of lipidemic disorders such as hyper protein may be administered with a selective COX-2 inhibitor 60 lipidemia, hypercholesterolemia and atherosclerosis. This for treating or preventing inflammation. Exemplary selective compound has long been known to exhibit the beneficial COX-2 inhibitors include, for example, deracoxib, pare effects of reducing total cholesterol, low density lipoproteins coxib, celecoxib, Valdecoxib, rofecoxib, etoricoxib, lumira or “LDL cholesterol.” triglycerides and apolipoproteina (Lp coxib, 2-(3,5-difluorophenyl)-3-4-(methylsulfonyl)phe (a)) in the human body, while increasing desirable high den nyl-2-cyclopenten-1-one, (S)-6,8-dichloro-2-(triflu 65 sity lipoproteins or “HDL cholesterol. oromethyl)-2H-1-benzopyran-3-carboxylic acid, 2-(3,4- Typical doses range from about 1 gram to about 3 grams difluorophenyl)-4-(3-hydroxy-3-methyl-1-butoxy)-5-4- daily. Nicotinic acid is normally administered two to four US 8,178,536 B2 157 158 times per day after meals, depending upon the dosage form inhibitor (SSRI), such as a fluoxetinoid (fluoxetine, norflu selected. Nicotinic acid is currently commercially available oxetine) or a nefazodonoid (nefazodone, hydroxynefaz in two dosage forms. One dosage form is an immediate or odone, oxonefazodone). Other exemplary SSRIs include rapid release tablet which should be administered three or dulloxetine, Venlafaxine, milnacipran, citalopram, fluvoxam four times per day. Immediate release (“IR”) nicotinic acid ine, paroxetine and Sertraline. The sirtuin-modulating com formulations generally release nearly all of their nicotinic pound that increases the level and/or activity of a sirtuin acid within about 30 to 60 minutes following ingestion. The protein can also be used as part of a treatment with sedative other dosage form is a Sustained release form which is Suit hypnotic drug, Such as selected from the group consisting of able for administration two to four times per day. In contrast a benzodiazepine (such as alprazolam, chlordiazepoxide, to IR formulations, sustained release (“SR) nicotinic acid 10 formulations are designed to release significant quantities of clonazepam, chloraZepate, clobazam, diazepam, halazepam, drug for absorption into the blood stream over specific timed lorazepam, oxazepam and prazepam), Zolpidem, and barbi intervals in order to maintain therapeutic levels of nicotinic turates. In still other embodiments, a sirtuin-modulating com acid over an extended period such as 12 or 24 hours after pound that increases the level and/or activity of a sirtuin ingestion. 15 protein may be used as part of a treatment with a 5-HT1A As used herein, the term "nicotinic acid' is meant to receptor partial agonist, Such as selected from the group con encompass nicotinic acid or a compound other than nicotinic sisting of buspirone, flesinoxan, gepirone and ipsapirone. acid itself which the body metabolizes into nicotinic acid, Sirtuin-modulating compounds that increase the level and/or thus producing essentially the same effect as nicotinic acid. activity of a sirtuin protein can also used as part of a treatment Exemplary compounds that produce an effect similar to that with a norepinephrine reuptake inhibitor, such as selected of nicotinic acid include, for example, nicotinyl alcohol tar from tertiary amine tricyclics and secondary amine tricyclics. trate, d-glucitol hexanicotinate, aluminum nicotinate, nicer Exemplary tertiary amine tricyclic include amitriptyline, clo itrol and d. 1-alpha-tocopheryl nicotinate. Each Such com mipramine, doxepin, imipramine and trimipramine. Exem pound will be collectively referred to herein as “nicotinic plary secondary amine tricyclic include amoxapine, acid.” 25 desipramine, maprotiline, nortriptyline and protriptyline. In In another embodiment, the invention provides a method certain embodiments, sirtuin-modulating compounds that for treating and/or preventing hyperlipidemia with reduced increase the level and/or activity of a sirtuin protein may be flushing side effects. The method comprises the steps of used as part of a treatment with a monoamine oxidase inhibi administering to a Subject in need thereof a therapeutically tor, such as selected from the group consisting of isocarbox effective amount of nicotinic acid and a sirtuin-modulating 30 azid, phenelZine, tranylcypromine, selegiline and moclobe compound that increases the level and/or activity of a sirtuin protein in an amount sufficient to reduce flushing. In an exem mide. plary embodiment, the nicotinic acid and/or sirtuin-modulat In still another representative embodiment, sirtuin-modu ing compound may be administered nocturnally. lating compounds that increase the level and/or activity of a In another representative embodiment, the method 35 sirtuin protein may be used to reduce flushing side effects of involves the use of Sirtuin-modulating compounds that chemotherapeutic agents, such as cyclophosphamide, tamox increase the level and/or activity of a sirtuin protein to reduce ifen. flushing side effects of raloxifene. Raloxifene acts like estro In another embodiment, sirtuin-modulating compounds gen in certain places in the body, but is not a hormone. It helps that increase the level and/or activity of a sirtuin protein may prevent osteoporosis in women who have reached meno 40 be used to reduce flushing side effects of calcium channel pause. Osteoporosis causes bones to gradually grow thin, blockers, such as amlodipine. fragile, and more likely to break. Evista slows down the loss In another embodiment, sirtuin-modulating compounds of bone mass that occurs with menopause, lowering the risk of that increase the level and/or activity of a sirtuin protein may spine fractures due to osteoporosis. A common side effect of be used to reduce flushing side effects of antibiotics. For raloxifene is hot flashes (Sweating and flushing). This can be 45 example, sirtuin-modulating compounds that increase the uncomfortable for women who already have hot flashes due level and/or activity of a sirtuin protein can be used in com to menopause. bination with levofloxacin. Levofloxacin is used to treat In another representative embodiment, the method infections of the sinuses, skin, lungs, ears, airways, bones, involves the use of Sirtuin-modulating compounds that and joints caused by Susceptible bacteria. Levofloxacin also is increase the level and/or activity of a sirtuin protein to reduce 50 frequently used to treat urinary infections, including those flushing side effects of antidepressants or anti-psychotic resistant to other antibiotics, as well as prostatitis. Levofloxa agent. For instance, sirtuin-modulating compounds that cin is effective in treating infectious diarrheas caused by E. increase the level and/or activity of a sirtuin protein can be coli, campylobacter jejuni, and Shigella bacteria. Levofloxa used in conjunction (administered separately or together) cin also can be used to treat various obstetric infections, with a serotonin reuptake inhibitor, a 5HT2 receptor antago 55 including mastitis. nist, an anticonvulsant, a norepinephrine reuptake inhibitor, Ocular Disorders an C.-adrenoreceptor antagonist, an NK-3 antagonist, an One aspect of the present invention is a method for inhib NK-1 receptor antagonist, a PDE4 inhibitor, an Neuropeptide iting, reducing or otherwise treating vision impairment by Y5 Receptor Antagonists, a D4 receptor antagonist, a 5HT1A administering to a patient a therapeutic dosage of sirtuin receptor antagonist, a 5HT1D receptor antagonist, a CRF 60 modulator selected from a compound disclosed herein, or a antagonist, a monoamine oxidase inhibitor, or a sedative pharmaceutically acceptable salt, prodrug or a metabolic hypnotic drug. derivative thereof. In certain embodiments, sirtuin-modulating compounds In certain aspects of the invention, the vision impairment is that increase the level and/or activity of a sirtuin protein may caused by damage to the optic nerve or central nervous sys be used as part of a treatment with a serotonin reuptake 65 tem. In particular embodiments, optic nerve damage is caused inhibitor (SRI) to reduce flushing. In certain preferred by high intraocular pressure, such as that created by glau embodiments, the SRI is a selective serotonin reuptake coma. In other particular embodiments, optic nerve damage is US 8,178,536 B2 159 160 caused by Swelling of the nerve, which is often associated sis, Best Disease, Branch Retinal Artery Occlusion, Branch with an infection or an immune (e.g., autoimmune) response Retinal Vein Occlusion, Cancer Associated and Related Such as in optic neuritis. Autoimmune Retinopathies, Central Retinal Artery Occlu Glaucoma describes a group of disorders which are asso sion, Central Retinal Vein Occlusion, Central Serous Chori ciated with a visual field defect, cupping of the optic disc, and oretinopathy, Eales Disease, Epimacular Membrane, Lattice optic nerve damage. These are commonly referred to as glau Degeneration, Macroaneurysm, Diabetic Macular Edema, comatous optic neuropathies. Most glaucomas are usually, Irvine-Gass Macular Edema, Macular Hole, Subretinal but not always, associated with a rise in intraocular pressure. Neovascular Membranes, Diffuse Unilateral Subacute Neu Exemplary forms of glaucoma include Glaucoma and Pen roretinitis, Nonpseudophakic Cystoid Macular Edema, Pre etrating Keratoplasty, Acute Angle Closure, Chronic Angle 10 Closure, Chronic Open Angle, Angle Recession, Aphakic and sumed Ocular Histoplasmosis Syndrome, Exudative Retinal Pseudophakic, Drug-Induced. Hyphema, Intraocular Detachment, Postoperative Retinal Detachment, Prolifera Tumors, Juvenile, Lens-Particle, Low Tension, Malignant, tive Retinal Detachment, Rhegmatogenous Retinal Detach Neovascular, Phacolytic, Phacomorphic, Pigmentary, Pla ment, Tractional Retinal Detachment, Retinitis Pigmentosa, teau Iris, Primary Congenital, Primary Open Angle, Pseu 15 CMV Retinitis, Retinoblastoma, Retinopathy of Prematurity, doexfoliation, Secondary Congenital, Adult Suspect, Unilat Birdshot Retinopathy, Background Diabetic Retinopathy, eral, Uveitic, Ocular Hypertension, Ocular Hypotony, Proliferative Diabetic Retinopathy, Hemoglobinopathies Posner-Schlossman Syndrome and Scleral Expansion Proce Retinopathy, Purtscher Retinopathy, Valsalva Retinopathy, dure in Ocular Hypertension & Primary Open-angle Glau Juvenile Retinoschisis, Senile Retinoschisis, Terson Syn COa. drome and White Dot Syndromes. Intraocular pressure can also be increased by various Sur Other exemplary diseases include ocular bacterial infec gical procedures, such as phacoemulsification (i.e., cataract tions (e.g. conjunctivitis, keratitis, tuberculosis, syphilis, Surgery) and implanation of structures such as an artificial gonorrhea), viral infections (e.g. Ocular Herpes Simplex lens. In addition, spinal Surgeries in particular, or any Surgery Virus, Varicella Zoster Virus, Cytomegalovirus retinitis, in which the patient is prone for an extended period of time 25 Human Immunodeficiency Virus (HIV)) as well as progres can lead to increased interOccular pressure. sive outer retinal necrosis secondary to HIV or other HIV Optic neuritis (ON) is inflammation of the optic nerve and associated and other immunodeficiency-associated ocular causes acute loss of vision. It is highly associated with mul diseases. In addition, ocular diseases include fungal infec tiple sclerosis (MS) as 15-25% of MS patients initially tions (e.g. Candida choroiditis, histoplasmosis), protozoal present with ON, and 50-75% of ON patients are diagnosed 30 infections (e.g. toxoplasmosis) and others such as ocular with MS. ON is also associated with infection (e.g., viral toxocariasis and sarcoidosis. infection, meningitis, syphilis), inflammation (e.g., from a One aspect of the invention is a method for inhibiting, reduc vaccine), infiltration and ischemia. ing or treating vision impairment in a subject undergoing Another condition leading to optic nerve damage is ante treatment with a chemotherapeutic drug (e.g., a neurotoxic rior ischemic optic neuropathy (AION). There are two types 35 drug, a drug that raises intraocular pressure Such as a steroid), of AION. Arteritic AION is due to giant cell arteritis (vascu by administering to the Subject in need of Such treatment a litis) and leads to acute vision loss. Non-arteritic AION therapeutic dosage of a sirtuin modulator disclosed herein. encompasses all cases of ischemic optic neuropathy other Another aspect of the invention is a method for inhibiting, than those due to giant cell arteritis. The pathophysiology of reducing or treating vision impairment in a Subject undergo AION is unclear although it appears to incorporate both 40 ing Surgery, including ocular or other Surgeries performed in inflammatory and ischemic mechanisms. the prone position Such as spinal cord Surgery, by administer Other damage to the optic nerve is typically associated with ing to the Subject in need of Such treatment a therapeutic demyleination, inflammation, ischemia, toxins, or trauma to dosage of a sirtuin modulator disclosed herein. Ocular Sur the optic nerve. Exemplary conditions where the optic nerve geries include cataract, iridotomy and lens replacements. is damaged include Demyelinating Optic Neuropathy (Optic 45 Another aspect of the invention is the treatment, including Neuritis, Retrobulbar Optic Neuritis), Optic Nerve Sheath inhibition and prophylactic treatment, of age related ocular Meningioma, Adult Optic Neuritis, Childhood Optic Neuri diseases include cataracts, dry eye, retinal damage and the tis, Anterior Ischemic Optic Neuropathy, Posterior Ischemic like, by administering to the Subject in need of such treatment Optic Neuropathy, Compressive Optic Neuropathy, Papille a therapeutic dosage of a sirtuin modulator disclosed herein. dema, Pseudopapilledema and Toxic/Nutritional Optic Neu 50 The formation of cataracts is associated with several bio ropathy. chemical changes in the lens of the eye. Such as decreased Other neurological conditions associated with vision loss, levels of antioxidants ascorbic acid and glutathione, albeit not directly associated with damage to the optic nerve, increased lipid, amino acid and protein oxidation, increased include Amblyopia, Bells Palsy, Chronic Progressive Exter Sodium and calcium, loss of amino acids and decreased lens nal Ophthalmoplegia, Multiple Sclerosis, Pseudotumor Cere 55 metabolism. The lens, which lacks blood vessels, is sus bri and Trigeminal Neuralgia. pended in extracellular fluids in the anterior part of the eye. In certain aspects of the invention, the vision impairment is Nutrients, such as ascorbic acid, glutathione, Vitamin E, sele caused by retinal damage. In particular embodiments, retinal nium, bioflavonoids and carotenoids are required to maintain damage is caused by disturbances in blood flow to the eye the transparency of the lens. Low levels of selenium results in (e.g., arteriosclerosis, vasculitis). In particular embodiments, 60 an increase of free radical-inducing hydrogen peroxide, retinal damage is caused by disrupton of the macula (e.g., which is neutralized by the selenium-dependent antioxidant exudative or non-exudative macular degeneration). enzyme glutathione peroxidase. Lens-protective glutathione Exemplary retinal diseases include Exudative Age Related peroxidase is also dependent on the amino acids methionine, Macular Degeneration, Nonexudative Age Related Macular cysteine, glycine and glutamic acid. Degeneration, Retinal Electronic Prosthesis and RPE Trans 65 Cataracts can also develop due to an inability to properly plantation Age Related Macular Degeneration, Acute Multi metabolize galactose found in dairy products that contain focal Placoid Pigment Epitheliopathy, Acute Retinal Necro lactose, a disaccharide composed of the monosaccharide US 8,178,536 B2 161 162 galactose and glucose. Cataracts can be prevented, delayed, in Bruch's membrane and may grow under the RPE, detach slowed and possibly even reversed if detected early and meta ing it from the choroid, and leaking fluid or bleeding. bolically corrected. Macular pigment, one of the protective factors that prevent Retinal damage is attributed, interalia, to free radical ini Sunlight from damaging the retina, is formed by the accumu tiated reactions in glaucoma, diabetic retinopathy and age- 5 lation of nutritionally derived carotenoids, such as lutein, the related macular degeneration (AMD). The eye is a part of the fatty yellow pigment that serves as a delivery vehicle for other central nervous system and has limited regenerative capabil important nutrients and Zeaxanthin. Antioxidants such as ity. The retina is composed of numerous nerve cells which Vitamins C and E, beta-carotene and lutein, as well as Zinc, contain the highest concentration of polyunsaturated fatty Selenium and copper, are all found in the healthy macula. In acids (PFA) and subject to oxidation. Free radicals are gen- 10 addition to providing nourishment, these antioxidants protect erated by UV light entering the eye and mitochondria in the against free radical damage that initiates macular degenera rods and cones, which generate the energy necessary to trans tion. form light into visual impulses. Free radicals cause peroxi Another aspect of the invention is the prevention or treat dation of the PFA by hydroxyl or superoxide radicals which in ment of damage to the eye caused by stress, chemical insult or turn propagate additional free radicals. The free radicals 15 radiation, by administering to the Subject in need of Such cause temporary or permanent damage to retinal tissue. treatment a therapeutic dosage of a sirtuin modulator dis Glaucoma is usually viewed as a disorder that causes an closed herein. Radiation or electromagnetic damage to the elevated intraocular pressure (IOP) that results in permanent eye can include that caused by CRTs or exposure to sunlight damage to the retinal nerve fibers, but a sixth of all glaucoma or UV. cases do not develop an elevated IOP. This disorder is now 20 In one embodiment, a combination drug regimen may perceived as one of reduced vascular perfusion and an include drugs or compounds for the treatment or prevention increase in neurotoxic factors. Recent studies have implicated of ocular disorders or secondary conditions associated with elevated levels of glutamate, nitric oxide and peroxynitirite in these conditions. Thus, a combination drug regimen may the eye as the causes of the death of retinal ganglion cells. include one or more sirtuin activators and one or more thera Neuroprotective agents may be the future of glaucoma care. 25 peutic agents for the treatment of an ocular disorder. For For example, nitric oxide synthase inhibitors block the for example, one or more sirtuin-activating compounds can be mation of peroxynitrite from nitric oxide and Superoxide. In a combined with an effective amount of one or more of: an recent study, animals treated with aminoguanidine, a nitric agent that reduces intraocular pressure, an agent for treating oxide synthase inhibitor, had a reduction in the loss of retinal glaucoma, an agent for treating optic neuritis, an agent for ganglion cells. It was concluded that nitric oxide in the eye 30 treating CMV Retinopathy, an agent for treating multiple caused cytotoxicity in many tissues and neurotoxicity in the Sclerosis, and/or an antibiotic, etc. central nervous system. In one embodiment, a sirtuin modulator can be adminis Diabetic retinopathy occurs when the underlying blood tered in conjunction with a therapy for reducing intraocular vessels develop microvascular abnormalities consisting pri pressure. One group of therapies involves blocking aqueous marily of microaneurysms and intraretinal hemorrhages. 35 production. For example, topical beta-adrenergic antagonists Oxidative metabolites are directly involved with the patho (timolol and betaxolol) decrease aqueous production. Topical genesis of diabetic retinopathy and free radicals augment the timolol causes IOP to fall in 30 minutes with peak effects in generation of growth factors that lead to enhanced prolifera 1-2 hours. A reasonable regimen is Timoptic 0.5%, one drop tive activity. Nitric oxide produced by endothelial cells of the every 30 minutes for 2 doses. The carbonic anhydrase inhibi vessels may also cause Smooth muscle cells to relax and result 40 tor, acetazolamide, also decreases aqueous production and in vasodilation of segments of the vessel. Ischemia and should be given in conjunction with topical beta-antagonists. hypoxia of the retina occur after thickening of the arterial An initial dose of 500 mg is administered followed by 250 mg basement membrane, endothelial proliferation and loss of every 6 hours. This medication may be given orally, intramus pericytes. The inadequate oxygenation causes capillary oblit cularly, or intravenously. In addition, alpha2-agonists (e.g., eration or nonperfusion, arteriolar-Venular shunts, sluggish 45 Apraclonidine) act by decreasing aqueous production. Their blood flow and an impaired ability of RBCs to release oxygen. effects are additive to topically administered beta-blockers. Lipid peroxidation of the retinal tissues also occurs as a result They have been approved for use in controlling an acute rise of free radical damage. in pressure following anterior chamber laser procedures, but The macula is responsible for our acute central vision and has been reported effective in treating acute closed-angle composed of light-sensing cells (cones) while the underlying 50 glaucoma. A reasonable regimen is 1 drop every 30 minutes retinal pigment epithelium (RPE) and choroid nourish and for 2 doses. help remove waste materials. The RPE nourishes the cones A second group of therapies for reducing intraocular pres with the vitamin A substrate for the photosensitive pigments Sure involve reducing vitreous Volume. Hyperosmotic agents and digests the cones shed outer tips. RPE is exposed to high can be used to treat an acute attack. These agents draw water levels of UV radiation, and secretes factors that inhibitangio- 55 out of the globe by making the blood hyperosmolar. Oral genesis. The choroid contains a dense vascular network that glycerol in a dose of 1 mL/kg in a cold 50% solution (mixed provides nutrients and removes the waste materials. with lemon juice to make it more palatable) often is used. In AMD, the shed cone tips become indigestible by the Glycerol is converted to glucose in the liver; persons with RPE, where the cells swell and die after collecting too much diabetes may need additional insulin if they become hyperg undigested material. Collections of undigested waste mate- 60 lycemic after receiving glycerol. Oral isosorbide is a meta rial, called drusen, form under the RPE. Photoxic damage bolically inert alcohol that also can be used as an osmotic also causes the accumulation of lipofuscin in RPE cells. The agent for patients with acute angle-closure glaucoma. Usual intracellular lipofuscin and accumulation of drusen in dose is 100 g taken p.o. (220 cc of a 45% solution). This inert Bruch's membrane interferes with the transport of oxygen alcohol should not be confused with isosorbide dinitrate, a and nutrients to the retinal tissues, and ultimately leads to 65 nitrate-based cardiac medication used forangina and for con RPE and photoreceptor dysfunction. In exudative AMD, gestive heart failure. Intravenous mannitol in a dose of 1.0-1.5 blood vessels grow from the choriocapillaris through defects mg/kg also is effective and is well tolerated in patients with US 8,178,536 B2 163 164 nausea and vomiting. These hyperosmotic agents should be Mitochondrial-Associated Diseases and Disorders used with caution in any patient with a history of congestive In certain embodiments, the invention provides methods heart failure. for treating diseases or disorders that would benefit from A third group of therapies involve facilitating aqueous increased mitochondrial activity. The methods involve outflow from the eye. Miotic agents pull the iris from the 5 administering to a subject in need thereof a therapeutically iridocorneal angle and may help to relieve the obstruction of effective amount of a sirtuin activating compound. Increased the trabecular meshwork by the peripheral iris. Pilocarpine mitochondrial activity refers to increasing activity of the 2% (blue eyes)-4% (brown eyes) can be administered every mitochondria while maintaining the overall numbers of mito 15 minutes for the first 1-2 hours. More frequent administra chondria (e.g., mitochondrial mass), increasing the numbers tion or higher doses may precipitate a systemic cholinergic 10 of mitochondria thereby increasing mitochondrial activity crisis. NSAIDS are sometimes used to reduce inflammation. (e.g., by Stimulating mitochondrial biogenesis), or combina Exemplary therapeutic agents for reducing intraocular tions thereof. In certain embodiments, diseases and disorders pressure include ALPHAGANR P (Allergan) (brimonidine that would benefit from increased mitochondrial activity tartrate ophthalmic solution), AZOPTR (Alcon) (brinzola include diseases or disorders associated with mitochondrial mide ophthalmic suspension), BETAGANR) (Allergan) 15 dysfunction. (levobunolol hydrochloride ophthalmic solution, USP), In certain embodiments, methods for treating diseases or BETIMOL(R) (Vistakon) (timolol ophthalmic solution), disorders that would benefit from increased mitochondrial BETOPTIC SR (Alcon) (betaxolol HCl), BRIMONIDINE activity may comprise identifying a subject Suffering from a TARTRATE (Bausch & Lomb), CARTEOLOL HYDRO mitochondrial dysfunction. Methods for diagnosing a mito CHLORIDE (Bausch & Lomb), COSOPTR (Merck) (dor chondrial dysfunction may involve molecular genetic, patho Zolamide hydrochloride-timolol maleate ophthalmic solu logic and/or biochemical analysis are Summarized in Cohen tion), LUMIGANR) (Allergan) (bimatoprost ophthalmic and Gold, Cleveland Clinic Journal of Medicine, 68: 625-642 solution), OPTIPRANOLOL(R) (Bausch & Lomb) (metipra (2001). One method for diagnosing a mitochondrial dysfunc nolol ophthalmic solution), TIMOLOL GFS (Falcon) tion is the Thor-Byrne-ier scale (see e.g., Cohen and Gold, (timolol maleate ophthalmic gel forming solution), TIMOP 25 supra: Collin S. et al., Eur Neurol. 36:260-267 (1996)). Other TICR (Merck) (timolol maleate ophthalmic solution), TRA methods for determining mitochondrial number and function VATAN(R) (Alcon) (travoprost ophthalmic solution), TRU include, for example, enzymatic assays (e.g., a mitochondrial SOPTR (Merck) (dorzolamide hydrochloride ophthalmic enzyme oran ATPbiosynthesis factor such as an ETC enzyme solution) and XALATANR) (Pharmacia & Upjohn) (latano or a Krebs cycle enzyme), determination or mitochondrial prost ophthalmic solution). 30 mass, mitochondrial Volume, and/or mitochondrial number, In one embodiment, a sirtuin modulator can be adminis quantification of mitochondrial DNA, monitoring intracellu tered in conjunction with a therapy for treating and/or pre lar calcium homeostasis and/or cellular responses to pertur venting glaucoma. An example of a glaucoma drug is bations of this homeostasis, evaluation of response to an DARANIDE(R) Tablets (Merck) (Dichlorphenamide). apoptogenic stimulus, determination of free radical produc In one embodiment, a sirtuin modulator can be adminis 35 tion. Such methods are known in the art and are described, for tered in conjunction with a therapy for treating and/or pre example, in U.S. Patent Publication No. 2002/0049176 and venting optic neuritis. Examples of drugs for optic neuritis references cited therein. include DECADRONR Phosphate Injection (Merck) (Dex Mitochondria are critical for the survival and proper func amethasone Sodium Phosphate), DEPO-MEDROL(R) (Phar tion of almost all types of eukaryotic cells. Mitochondria in macia & Upjohn)(methylprednisolone acetate), HYDRO 40 virtually any cell type can have congenital or acquired defects CORTONER) Tablets (Merck) (Hydrocortisone), that affect their function. Thus, the clinically significant signs ORAPREDR) (Biomarin) (prednisolone sodium phosphate and symptoms of mitochondrial defects affecting respiratory oral solution) and PEDIAPREDR (Celltech) (prednisolone chain function are heterogeneous and variable depending on sodium phosphate, USP). the distribution of defective mitochondria among cells and In one embodiment, a sirtuin modulator can be adminis 45 the severity of their deficits, and upon physiological demands tered in conjunction with a therapy for treating and/or pre upon the affected cells. Nondividing tissues with high energy venting CMV Retinopathy. Treatments for CMV retinopathy requirements, e.g. nervous tissue, skeletal muscle and cardiac include CYTOVENER (ganciclovir capsules) and VAL muscle are particularly susceptible to mitochondrial respira CYTER (Roche Laboratories) (valganciclovir hydrochloride tory chain dysfunction, but any organ system can be affected. tablets). 50 Diseases and disorders associated with mitochondrial dys In one embodiment, a sirtuin modulator can be adminis function include diseases and disorders in which deficits in tered in conjunction with a therapy for treating and/or pre mitochondrial respiratory chain activity contribute to the venting multiple Sclerosis. Examples of Such drugs include development of pathophysiology of Such diseases or disor DANTRIUM(R) (Procter & Gamble Pharmaceuticals) (dant ders in a mammal. This includes 1) congenital genetic defi rolene sodium), NOVANTRONE(R) (Serono) (mitoxantrone), 55 ciencies in activity of one or more components of the mito AVONEX(R) (Biogen Idec) (Interferon beta-1a), BETASE chondrial respiratory chain; and 2) acquired deficiencies in RONR) (Berlex) (Interferon beta-1b), COPAXONER) (Teva the activity of one or more components of the mitochondrial Neuroscience) (glatiramer acetate injection) and REBIF(R) respiratory chain, wherein Such deficiencies are caused by a) (Pfizer) (interferon beta-1a). oxidative damage during aging; b) elevated intracellular cal In addition, macrollide and/or mycophenolic acid, which 60 cium; c) exposure of affected cells to nitric oxide; d) hypoxia has multiple activities, can be co-administered with a sirtuin or ischemia; e) microtubule-associated deficits in axonal modulator. Macrollide antibiotics include tacrolimus, transport of mitochondria, or f) expression of mitochondrial cyclosporine, Sirolimus, everolimus, ascomycin, erythromy uncoupling proteins. cin, azithromycin, clarithromycin, clindamycin, lincomycin, Diseases or disorders that would-benefit from increased dirithromycin, josamycin, spiramycin, diacetyl-midecamy 65 mitochondrial activity generally include for example, dis cin, tylosin, roxithromycin, ABT-773, tellithromycin, leuco eases in which free radical mediated oxidative injury leads to mycins, and lincosamide. tissue degeneration, diseases in which cells inappropriately US 8,178,536 B2 165 166 undergo apoptosis, and diseases in which cells fail to undergo topenia and leukemia syndrome, MARIAHS syndrome apoptosis. Exemplary diseases or disorders that would benefit (Mitrochondrial ataxia, recurrent infections, aphasia, hypou from increased mitochondrial activity include, for example, ricemia/hypomyelination, seizures, and dicarboxylic aci AD (Alzheimer's Disease), ADPD (Alzheimer's Disease and duria), ND6 dystonia, Cyclic vomiting syndrome with Parkinsons’s Disease). AMDF (Ataxia, Myoclonus and Deaf declines during infection, 3-Hydroxyisobutryic aciduria with ness), auto-immune disease, cancer, CIPO (Chronic Intesti lactic acidemia, Diabetes mellitus with lactic acidemia, Uri nal Pseudoobstruction with myopathy and Ophthalmople dine responsive neurologic syndrome (URNS), Dilated car gia), congenital muscular dystrophy, CPEO (Chronic diomyopathy, Splenic Lymphoma, and Renal Tubular Acido Progressive External Ophthalmoplegia), DEAF (Maternally sis/Diabetes/Ataxis syndrome. inherited DEAFness or aminoglycoside-induced DEAF 10 In other embodiments, the invention provides methods for ness), DEMCHO (Dementia and Chorea), diabetes mellitus treating a subject suffering from mitochondrial disorders aris (Type I or Type II), DIDMOAD (Diabetes Insipidus, Diabetes ing from, but not limited to, post-traumatic head injury and Mellitus, Optic Atrophy, Deafness), DMDF (Diabetes Melli cerebral edema, stroke (invention methods useful for prevent tus and Deafness), dystonia, Exercise Intolerance, ESOC ing or preventing reperfusion injury), Lewy body dementia, (Epilepsy, Strokes, Optic atrophy, and Cognitive decline), 15 hepatorenal syndrome, acute liver failure, NASH (non-alco FBSN (Familial Bilateral Striatal Necrosis), FICP (Fatal holic Steatohepatitis), Anti-metastasis/prodifferentiation Infantile Cardiomyopathy Plus, a MELAS-associated cardi therapy of cancer, idiopathic congestive heart failure, atrial omyopathy), GER (Gastrointestinal Reflux), HD (Hunting fibrilation (non-valvular), Wolff-Parkinson-White Syn ton's Disease), KSS (Kearns Sayre Syndrome), “later-onset drome, idiopathic heart block, prevention of reperfusion myopathy, LDYT (Leber's hereditary optic neuropathy and injury in acute myocardial infarctions, familial migraines, DYsTonia), Leigh's Syndrome, LHON (Leber Hereditary irritable bowel syndrome, secondary prevention of non-Q Optic Neuropathy), LIMM (Lethal Infantile Mitochondrial wave myocardial infarctions, Premenstrual syndrome, Pre Myopathy), MDM (Myopathy and Diabetes Mellitus), vention of renal failure in hepatorenal syndrome, anti-phos MELAS (Mitochondrial Encephalomyopathy, Lactic Acido pholipid antibody syndrome, eclampsia/pre-eclampsia, sis, and Stroke-like episodes), MEPR (Myoclonic Epilepsy 25 oopause infertility, ischemic heart disease/angina, and Shy and Psychomotor Regression), MERME (MERRF/MELAS Drager and unclassified dysautonomia syndromes. overlap disease), MERRF (Myoclonic Epilepsy and Ragged In still another embodiment, there are provided methods Red Muscle Fibers), MHCM (Maternally Inherited Hyper for the treatment of mitochondrial disorders associated with trophic CardioMyopathy), MICM (Maternally Inherited Car pharmacological drug-related side effects. Types of pharma diomyopathy), MILS (Maternally Inherited Leigh Syn 30 ceutical agents that are associated with mitochondrial disor drome), Mitochondrial Encephalocardiomyopathy, ders include reverse transcriptase inhibitors, protease inhibi Mitochondrial Encephalomyopathy, MM (Mitochondrial tors, inhibitors of DHOD, and the like. Examples of reverse Myopathy), MMC (Maternal Myopathy and Cardiomyopa transcriptase inhibitors include, for example, AZidothymi thy), MNGIE (Myopathy and external ophthalmoplegia, dine (AZT), Stavudine (D4T), Zalcitabine (ddC), Didanosine Neuropathy, Gastro-Intestinal, Encephalopathy), Multisys 35 (DDI), Fluoroiodoarauracil (FIAU), Lamivudine (3TC), tem. Mitochondrial Disorder (myopathy, encephalopathy, Abacavir and the like. Examples of protease inhibitors blindness, hearing loss, peripheral neuropathy), NARP (Neu include, for example, Ritonavir, Indinavir, Saquinavir, Nelfi rogenic muscle weakness, Ataxia, and Retinitis Pigmentosa; navir and the like. Examples of inhibitors of dihydroorotate alternate phenotype at this locus is reported as Leigh Dis dehydrogenase (DHOD) include, for example, Leflunomide, ease), PD (Parkinson's Disease), Pearson’s Syndrome. PEM 40 Brequinar, and the like. (Progressive Encephalopathy), PEO (Progressive External Reverse transcriptase inhibitors not only inhibit reverse Ophthalmoplegia), PME (Progressive Myoclonus Epilepsy), transcriptase but also polymerase gamma which is required PMPS (Pearson Marrow-Pancreas Syndrome), psoriasis, for mitochondrial function. Inhibition of polymerase gamma RTT (Rett Syndrome), schizophrenia, SIDS (Sudden Infant activity (e.g., with a reverse transcriptase inhibitor) therefore Death Syndrome), SNHL (Sensorineural Hearing Loss), Var 45 leads to mitochondrial dysfunction and/or a reduced mito ied Familial Presentation (clinical manifestations range from chondrial mass which manifests itselfin patients as hyperlac spastic paraparesis to multisystem progressive disorder & tatemia. This type of condition may benefit from an increase fatal cardiomyopathy to truncal ataxia, dysarthria, severe in the number of mitochondria and/or an improvement in hearing loss, mental regression, ptosis, ophthalmoparesis, mitochondrial function, e.g., by administration of a sirtuin distal cyclones, and diabetes mellitus), or Wolfram syndrome. 50 activating compound. Other diseases and disorders that would benefit from Common symptoms of mitochondrial diseases include car increased mitochondrial activity include, for example, Frie diomyopathy, muscle weakness and atrophy, developmental dreich's ataxia and other ataxias, amyotrophic lateral Sclero delays (involving motor, language, cognitive or executive sis (ALS) and other motor neuron diseases, macular degen function), ataxia, epilepsy, renal tubular acidosis, peripheral eration, epilepsy, Alpers syndrome, Multiple mitochondrial 55 neuropathy, optic neuropathy, autonomic neuropathy, neuro DNA deletion syndrome, MtDNA depletion syndrome, Com genic bowel dysfunction, sensorineural deafness, neurogenic plex I deficiency, Complex II (SDH) deficiency, Complex III bladder dysfunction, dilating cardiomyopathy, migraine, deficiency, Cytochrome c oxidase (COX, Complex IV) defi hepatic failure, lactic acidemia, and diabetes mellitus. ciency, Complex V deficiency, Adenine Nucleotide Translo In certain embodiments, the invention provides methods cator (ANT) deficiency, Pyruvate dehydrogenase (PDH) defi 60 for treating a disease or disorder that would benefit from ciency, Ethylmalonic aciduria with lactic acidemia, 3-Methyl increased mitochondrial activity that involves administering glutaconic aciduria with lactic acidemia, Refractory epilepsy to a subject in need thereof one or more sirtuin activating with declines during infection, Asperger syndrome with compounds in combination with another therapeutic agent declines during infection, Autism with declines during infec Such as, for example, an agent useful for treating mitochon tion, Attention deficit hyperactivity disorder (ADHD), Cere 65 drial dysfunction (such as antioxidants, vitamins, or respira bral palsy with declines during infection, Dyslexia with tory chain cofactors), an agent useful for reducing a symptom declines during infection, materially inherited thrombocy associated with a disease or disorder involving mitochondrial US 8,178,536 B2 167 168 dysfunction (such as, an anti-seizure agent, an agent useful impairment of mitochondrial function. In certain embodi for alleviating neuropathic pain, an agent for treating cardiac ments, sirtuin activating compounds may be used for reduc dysfunction), a cardiovascular agent (as described further ing the rate of decline in muscular functional capacities and below), a chemotherapeutic agent (as described further for improving muscular functional status in patients with below), or an anti-neurodegeneration agent (as described fur- 5 muscular dystrophy. ther below). In an exemplary embodiment, the invention pro Multiple sclerosis (MS) is a neuromuscular disease char vides methods for treating a disease or disorder that would acterized by focal inflammatory and autoimmune degenera benefit from increased mitochondrial activity that involves tion of cerebral white matter. Periodic exacerbations or administering to a Subject in need thereof one or more sirtuin attacks are significantly correlated with upper respiratory activating compounds in combination with one or more of the 10 tract and other infections, both bacterial and viral, indicating following: coenzyme Qo, L-carnitine, thiamine, riboflavin, that mitochondrial dysfunction plays a role in MS. Depres niacinamide, folate, Vitamin E, selenium, lipoic acid, or pred sion of neuronal mitochondrial respiratory chain activity nisone. Compositions comprising Such combinations are also caused by Nitric Oxide (produced by astrocytes and other provided herein. cells involved in inflammation) is implicated as a molecular In exemplary embodiments, the invention provides meth- 15 mechanism contributing to MS. In certain embodiments, sir ods for treating diseases or disorders that would benefit from tuin activating compounds may be used for treatment of increased mitochondrial acitivty by administering to a subject patients with multiple Sclerosis, both prophylactically and a therapeutically effective amount of a sirtuin activating dis during episodes of disease exacerbation. orders (e.g., Friedreich's Ataxia, muscular dystrophy, mul Epilepsy is often present in patients with mitochondrial tiple Sclerosis, etc.), disorders of neuronal instability (e.g., 20 cytopathies, involving a range of seizure severity and fre seizure disorders, migrane, etc.), developmental delay, neu quency, e.g. absence, tonic, atonic, myoclonic, and status rodegenerative disorders (e.g., Alzheimer's Disease, Parkin epilepticus, occurring in isolated episodes or many times son’s Disease, amyotrophic lateral Sclerosis, etc.), ischemia, daily. In certain embodiments, sirtuin activating compounds renal tubular acidosis, age-related neurodegeneration and may be used for treating patients with seizures secondary to cognitive decline, chemotherapy fatigue, age-related or che- 25 mitochondrial dysfunction, including reducing frequency motherapy-induced menopause or irregularities of menstrual and severity of seizure activity. cycling or ovulation, mitochondrial myopathies, mitochon Metabolic studies on patients with recurrent migraine drial damage (e.g., calcium accumulation, excitotoxicity, headaches indicate that deficits in mitochondrial activity are nitric oxide exposure, hypoxia, etc.), and mitochondrial commonly associated with this disorder, manifesting as deregulation. 30 impaired-oxidative phosphorylation and excess lactate pro A gene defect underlying Friedreich's Ataxia (FA), the duction. Such deficits are not necessarily due to genetic most common hereditary ataxia, was recently identified and is defects in mitochondrial DNA. Migraineurs are hypersensi designated “frataxin'. In FA, after a period of normal devel tive to nitric oxide, an endogenous inhibitor of Cytochrome c opment, deficits in coordination develop which progress to Oxidase. In addition, patients with mitochondrial cytopa paralysis and death, typically between the ages of 30 and 40. 35 thies, e.g. MELAS, often have recurrent migraines. In certain The tissues affected most severely are the spinal cord, periph embodiments, sirtuin activating compounds may be used for eral nerves, myocardium, and pancreas. Patients typically treating patients with recurrent migraine headaches, includ lose motor control and are confined to wheel chairs, and are ing headaches refractory to ergot compounds or serotonin commonly afflicted with heart failure and diabetes. The receptor antagonists. genetic basis for FA involves GAA trinucleotide repeats in an 40 Delays in neurological or neuropsychological develop intron region of the gene encoding frataxin. The presence of ment are often found in children with mitochondrial diseases. these repeats results in reduced transcription and expression Development and remodeling of neural connections requires of the gene. Frataxin is involved in regulation of mitochon intensive biosynthetic activity, particularly involving synthe drial iron content. When cellular frataxin content is subnor sis of neuronal membranes and myelin, both of which require mal, excess iron accumulates in mitochondria, promoting 45 pyrimidine nucleotides as cofactors. Uridine nucleotides are oxidative damage and consequent mitochondrial degenera involved inactivation and transfer of Sugars to glycolipids and tion and dysfunction. When intermediate numbers of GAA glycoproteins. Cytidine nucleotides are derived from uridine repeats are present in the frataxin gene intron, the severe nucleotides, and are crucial for synthesis of major membrane clinical phenotype of ataxia may not develop. However, these phospholipid constituents like phosphatidylcholine, which intermediate-length trinucleotide extensions are found in 25 50 receives its choline moiety from cytidine diphosphocholine. to 30% of patients with non-insulin dependent diabetes mel In the case of mitochondrial dysfunction (due to either mito litus, compared to about 5% of the nondiabetic population. In chondrial DNA defects or any of the acquired or conditional certain embodiments, sirtuin activating compounds may be deficits like exicitoxic or nitric oxide-mediated mitochondrial used for treating patients with disorders related to deficien dysfunction) or other conditions resulting in impaired pyri cies or defects in frataxin, including Friedreich's Ataxia, 55 midine synthesis, cell proliferation and axonal extension is myocardial dysfunction, diabetes mellitus and complications impaired at crucial stages in development of neuronal inter of diabetes like peripheral neuropathy. connections and circuits, resulting in delayed or arrested Muscular dystrophy refers to a family of diseases involving development of neuropsychological functions like language, deterioration of neuromuscular structure and function, often motor, social, executive function, and cognitive skills. In resulting in atrophy of skeletal muscle and myocardial dys- 60 autism for example, magnetic resonance spectroscopy mea function. In the case of Duchenne muscular dystrophy, muta Surements of cerebral phosphate compounds indicates that tions or deficits in a specific protein, dystrophin, are impli there is global underSynthesis of membranes and membrane cated in its etiology. Mice with their dystrophin genes precursors indicated by reduced levels of uridine diphospho inactivated display some characteristics of muscular dystro Sugars, and cytidine nucleotide derivatives involved in mem phy, and have an approximately 50% deficit in mitochondrial 65 brane synthesis. Disorders characterized by developmental respiratory chain activity. A final common pathway for neu delay include Rett's Syndrome, pervasive developmental romuscular degeneration in most cases is calcium-mediated delay (or PDD-NOS“pervasive developmental delay not oth US 8,178,536 B2 169 170 erwise specified to distinguish it from specific Subcategories embodiments, sirtuin activating compounds may be useful like autism), autism, Asperger's Syndrome, and Attention for preventing delayed cell death (apoptosis in regions like the Deficit/Hyperactivity Disorder (ADHD), which is becoming hippocampus or cortex occurring about 2 to 5 days after an recognized as a delay or lag in development of neural cir episode of cerebral ischemia) after ischemic or hypoxic insult cuitry underlying executive functions. In certain embodi 5 to the brain. ments, sirtuin activating compounds may be useful for treat Acidosis due to renal dysfunction is often observed in ing treating patients with neurodevelopmental delays (e.g., patients with mitochondrial disease, whether the underlying involving motor, language, executive function, and cognitive respiratory chain dysfunction is congenital or induced by skills), or other delays or arrests of neurological and neurop ischemia or cytotoxic agents like cisplatin. Renal tubular sychological development in the nervous system and Somatic 10 acidosis often requires administration of exogenous sodium development in non-neural tissues like muscle and endocrine bicarbonate to maintain blood and tissue pH. In certain glands. embodiments, sirtuin activating compounds may be useful The two most significant severe neurodegenerative dis for treating renal tubular acidosis and other forms of renal eases associated with aging, Alzheimer's Disease (AD) and dysfunction caused by mitochondrial respiratory chain defi Parkinson's Disease (PD), both involve mitochondrial dys 15 cits. function in their pathogenesis. Complex I deficiencies in par During normal aging, there is a progressive decline in ticular are frequently found not only in the nigrostriatal neu mitochondrial respiratory chain function. Beginning about rons that degenerate in Parkinson's disease, but also in age 40, there is an exponential rise in accumulation of mito peripheral tissues and cells like muscle and platelets of Par chondrial DNA defects in humans, and a concurrent decline kinson's Disease patients. In Alzheimer's Disease, mitochon in nuclear-regulated elements of mitochondrial respiratory drial respiratory chain activity is often depressed, especially activity. Many mitochondrial DNA lesions have a selection Complex IV (Cytochrome c Oxidase). Moreover, mitochon advantage during mitochondrial turnover, especially in post drial respiratory function altogether is depressed as a conse mitotic cells. The proposed mechanism is that mitochondria quence of aging, further amplifying the deleterious sequelae with a defective respiratory chain produce less oxidative dam of additional molecular lesions affecting respiratory chain 25 age to themselves than do mitochondria with intact functional function. Other factors in addition to primary mitochondrial respiratory chains (mitochondrial respiration is the primary dysfunction underlie neurodegeneration in AD, PD, and source of free radicals in the body). Therefore, normally related disorders. Excitotoxic stimulation and nitric oxide are functioning mitochondria accumulate oxidative damage to implicated in both diseases, factors which both exacerbate membrane lipids more rapidly than do defective mitochon mitochondrial respiratory chain deficits and whose deleteri 30 dria, and are therefore "tagged' for degradation by lysos ous actions are exaggerated on a background of respiratory omes. Since mitochondria within cells have a half life of chain dysfunction. Huntington’s Disease also involves mito about 10 days, a selection advantage can result in rapid chondrial dysfunction in affected brain regions, with coop replacement of functional mitochondria with those with erative interactions of excitotoxic stimulation and mitochon diminished respiratory activity, especially in slowly dividing drial dysfunction contributing to neuronal degeneration. In 35 cells. The net result is that once a mutation in a gene for a certain embodiments, sirtuin activating compounds may be mitochondrial protein that reduces oxidative damage to mito useful for treating and attenuating progression of age-related chondria occurs, such defective mitochondria will rapidly neurodegenerative diseases including AD and PD. populate the cell, diminishing or eliminating its respiratory One of the major genetic defects in patients with Amyo capabilities. The accumulation of Such cells results in aging trophic Lateral Sclerosis (ALS or Lou Gehrig's Disease) is 40 or degenerative disease at the organismal level. This is con mutation or deficiency in Copper-Zinc Superoxide Dismu sistent with the progressive mosaic appearance of cells with tase (SOD 1), an antioxidant enzyme. Mitochondria both defective electron transport activity in muscle, with cells produce and are primary targets for reactive oxygen species. almost devoid of Cytochromec Oxidase (COX) activity inter Inefficient transfer of electrons to oxygen in mitochondria is spersed randomly amidst cells with normal activity, and a the most significant physiological source of free radicals in 45 higher incidence of COX-negative cells in biopsies from mammalian systems. Deficiencies in antioxidants or antioxi older Subjects. The organism, during aging, or in a variety of dant enzymes can result in or exacerbate mitochondrial mitochondrial diseases, is thus faced with a situation in which degeneration. Mice transgenic for mutated SOD1 develop irreplaceable postmitotic cells (e.g. neurons, skeletal and car symptoms and pathology similar to those in human ALS. The diac muscle) must be preserved and their function maintained development of the disease in these animals has been shown 50 to a significant degree, in the face of an inexorable progressive to involve oxidative destruction of mitochondria followed by decline in mitochondrial respiratory chain function. Neurons functional decline of motor neurons and onset of clinical with dysfunctional mitochondria become progressively more symptoms. Skeletal muscle from ALS patients has low mito sensitive to insults like excitotoxic injury. Mitochondrial fail chondrial Complex I activity. In certain embodiments, sirtuin ure contributes to most degenerative diseases (especially neu activating compounds may be useful for treating ALS, for 55 rodegeneration) that accompany aging. Congenital mito reversing or slowing the progression of clinical symptoms. chondrial diseases often involve early-onset Oxygen deficiency results in both direct inhibition of mito neurodegeneration similar in fundamental mechanism to dis chondrial respiratory chain activity by depriving cells of a orders that occur during aging of people born with normal terminal electron acceptor for Cytochrome c reoxidation at mitochondria. In certain embodiments, sirtuin activating Complex IV, and indirectly, especially in the nervous system, 60 compounds may be useful for treating or attenuating cogni via secondary post-anoxic excitotoxicity and nitric oxide for tive decline and other degenerative consequences of aging. mation. In conditions like cerebral anoxia, angina or sickle Mitochondrial DNA damage is more extensive and persists cell anemia crises, tissues are relatively hypoxic. In Such longer than nuclear DNA damage in cells subjected to oxida cases, compounds that increase mitochondrial activity pro tive stress or cancer chemotherapy agents like cisplatin due to vide protection of affected tissues from deleterious effects of 65 both greater Vulnerability and less efficient repair of mito hypoxia, attenuate secondary delayed cell death, and accel chondrial DNA. Although mitochondrial DNA may be more erate recovery from hypoxic tissue stress and injury. In certain sensitive to damage than nuclear DNA, it is relatively resis US 8,178,536 B2 171 172 tant, in some situations, to mutagenesis by chemical carcino MERFF syndrome (myoclonic epilepsy and ragged red gens. This is because mitochondria respond to Some types of fibers), limb-girdle distribution weakness, and infantile mitochondrial DNA damage by destroying their defective myopathy (benign or severe and fatal). Muscle biopsy speci genomes rather than attempting to repair them. This results in mens stained with modified Gomori's trichrome stain show global mitochondrial dysfunction for a period after cytotoxic ragged red fibers due to excessive accumulation of mitochon chemotherapy. Clinical use of chemotherapy agents like cis dria. Biochemical defects in substrate transport and utiliza platin, mitomycin, and cytoxan is often accompanied by tion, the Krebs cycle, oxidative phosphorylation, or the res debilitating “chemotherapy fatigue’, prolonged periods of piratory chain are detectable. Numerous mitochondrial DNA weakness and exercise intolerance which may persist even point mutations and deletions have been described, transmit after recovery from hematologic and gastrointestinal toxici 10 ties of such agents. In certain embodiments, sirtuin activating ted in a maternal, nonmendelian inheritance pattern. Muta compounds may be useful for treatment and prevention of tions in nuclear-encoded mitochondrial enzymes occur. side effects of cancer chemotherapy related to mitochondrial In certain embodiments, sirtuin activating compounds may dysfunction. be useful for treating patients Suffering from toxic damage to A crucial function of the ovary is to maintain integrity of 15 mitochondria, such as, toxic damage due to calcium accumu the mitochondrial genome in oocytes, since mitochondria lation, excitotoxicity, nitric oxide exposure, drug induced passed onto a fetus are all derived from those present in toxic damage, or hypoxia. oocytes at the time of conception. Deletions in mitochondrial A fundamental mechanism of cell injury, especially in DNA become detectable around the age of menopause, and excitable tissues, involves excessive calcium entry into cells, are also associated with abnormal menstrual cycles. Since as a result of either leakage through the plasma membrane or cells cannot directly detect and respond to defects in mito defects in intracellular calcium handling mechanisms. Mito chondrial DNA, but can only detect secondary effects that chondria are major sites of calcium sequestration, and pref affect the cytoplasm, like impaired respiration, redox status, erentially utilize energy from the respiratory chain for taking or deficits in pyrimidine synthesis, such products of mito up calcium rather than for ATP synthesis, which results in a chondrial function participate as a signal for oocyte selection 25 downward spiral of mitochondrial failure, since calcium and follicular atresia, ultimately triggering menopause when uptake into mitochondria results in diminished capabilities maintenance of mitochondrial genomic fidelity and func for energy transduction. tional activity can no longer be guaranteed. This is analogous Excessive stimulation of neurons with excitatory amino to apoptosis in cells with DNA damage, which undergo an acids is a common mechanism of cell death or injury in the active process of cellular Suicide when genomic fidelity can 30 no longer beachieved by repair processes. Women with mito central nervous system. Activation of glutamate receptors, chondrial cytopathies affecting the gonads often undergo pre especially of the subtype designated NMDA receptors, mature menopause or display primary cycling abnormalities. results in mitochondrial dysfunction, in part through eleva Cytotoxic cancer chemotherapy often induces premature tion of intracellular calcium during excitotoxic stimulation. menopause, with a consequent increased risk of osteoporosis. 35 Conversely, deficits in mitochondrial respiration and oxida Chemotherapy-induced amenorrhea is generally due to pri tive phosphorylation sensitizes cells to excitotoxic stimuli, mary ovarian failure. The incidence of chemotherapy-in resulting in cell death or injury during exposure to levels of duced amenorrhea increases as a function of age in premeno excitotoxic neurotransmitters or toxins that would be innocu pausal women receiving chemotherapy, pointing toward ous to normal cells. mitochondrial involvement. Inhibitors of mitochondrial res 40 Nitric oxide (about 1 micromolar) inhibits cytochrome piration or protein synthesis inhibit hormone-induced ovula oxidase (Complex IV) and thereby inhibits mitochondrial tion, and furthermore inhibit production of ovarian steroid respiration; moreover, prolonged exposure to nitric oxide hormones in response to pituitary gonadotropins. Women (NO) irreversibly reduces Complex I activity. Physiological with Down's syndrome typically undergo menopause prema or pathophysiological concentrations of NO thereby inhibit turely, and also are subject to early onset of Alzheimer-like 45 pyrimidine biosynthesis. Nitric oxide is implicated in a vari dementia. Low activity of cytochrome oxidase is consistently ety of neurodegenerative disorders including inflammatory found in tissues of Down's patients and in late-onset Alzhe and autoimmune diseases of the central nervous system, and imer's Disease. Appropriate Support of mitochondrial func is involved in mediation of excitotoxic and post-hypoxic tion or compensation for mitochondrial dysfunction therefore damage to neurons. is useful for protecting againstage-related or chemotherapy 50 Oxygen is the terminal electron acceptor in the respiratory induced menopause or irregularities of menstrual cycling or chain. Oxygen deficiency impairs electron transport chain ovulation. In certain embodiments, sirtuin activating com activity, resulting in diminished pyrimidine synthesis as well pounds may be useful for treating and preventing amenor as diminished ATP synthesis via oxidative phosphorylation. rhea, irregular ovulation, menopause, or secondary conse Human cells proliferate and retain viability under virtually quences of menopause. 55 anaerobic conditions if provided with uridine and pyruvate In certain embodiments, sirtuin modulating compounds (or a similarly effective agent for oxidizing NADH to opti may be useful for treatment mitochondrial myopathies. Mito mize glycolytic ATP production). chondrial myopathies range from mild, slowly progressive In certain embodiments, sirtuin activating compounds may weakness of the extraocular muscles to severe, fatal infantile be useful for treating diseases or disorders associated with myopathies and multisystem encephalomyopathies. Some 60 mitochondrial deregulation. syndromes have been defined, with some overlap between Transcription of mitochondrial DNA encoding respiratory them. Established syndromes affecting muscle include pro chain components requires nuclear factors. In neuronal gressive external ophthalmoplegia, the Kearns-Sayre Syn axons, mitochondria must shuttle back and forth to the drome (with ophthalmoplegia, pigmentary retinopathy, car nucleus in order to maintain respiratory chain activity. If diac conduction defects, cerebellar ataxia, and sensorineural 65 axonal transport is impaired by hypoxia or by drugs like taxol deafness), the MELAS syndrome (mitochondrial encepha which affect microtubule stability, mitochondria distant from lomyopathy, lactic acidosis, and stroke-like episodes), the the nucleus undergo loss of cytochrome oxidase activity. US 8,178,536 B2 173 174 Accordingly, treatment with a sirtuin activating compound For example, disruption of skeletal muscle nitrogen metabo may be useful for promoting nuclear-mitochondrial interac lism as well as depletion of sources of metabolic energy occur tions. during extensive muscle activity. Amino acids, including Mitochondria are the primary source of free radicals and branched-chain amino acids, are released from muscles fol reactive oxygen species, due to spillover from the mitochon lowed by their deamination to elevate serum ammonia and drial respiratory chain, especially when defects in one or local oxidation as muscle fuel Sources, which augments meta more respiratory chain components impairs orderly transfer bolic acidosis. In addition, there is a decline in catalytic of electrons from metabolic intermediates to molecular oxy efficiency of muscle contraction events, as well as an alter gen. To reduce oxidative damage, cells can compensate by ation of enzymatic activities of nitrogen and energy metabo expressing mitochondrial uncoupling proteins (UCP), of 10 lism. Further, protein catabolism is initiated where rate of which several have been identified. UCP-2 is transcribed in protein synthesis is decreased coupled with an increase in the response to oxidative damage, inflammatory cytokines, or degradation of non-contractible protein. These metabolic excess lipid loads, e.g. fatty liver and steatohepatitis. UCPs processes are also accompanied by free radical generation reduce spillover of reactive oxygen species from mitochon which further damages muscle cells. dria by discharging proton gradients across the mitochondrial 15 Recovery from fatigue during acute and extended exercise inner membrane, in effect wasting energy produced by requires reversal of metabolic and non-metabolic fatiguing metabolism and rendering cells Vulnerable to energy stress as factors. Known factors that participate in human muscle a trade-off for reduced oxidative injury. fatigue, Such as lactate, ammonia, hydrogen ion, etc., provide Muscle Performance an incomplete and unsatisfactory explanation of the fatigue/ In other embodiments, the invention provides methods for recovery process, and it is likely that additional unknown enhancing muscle performance by administering a therapeu agents participate (Baker et al., J. Appl. Physiol. 74:2294 tically effective amount of a sirtuin activating compound. For 2300, 1993; Bazzarre et al., J. Am. Coll. Nutr. 11:505-511, example, sirtuin activating compounds may be useful for 1992; Dohm et al., Fed. Proc. 44:348-352, 1985; Edwards In: improving physical endurance (e.g., ability to perform a Biochemistry of Exercise, Proceedings of the Fifth Interna physical task Such as exercise, physical labor, sports activi 25 tional Symposium on the Biochemistry of Exercise (Kutrgen, ties, etc.), inhibiting or retarding physical fatigues, enhancing Vogel, Poormans, eds.), 1983; MacDougall et al., Acta blood oxygen levels, enhancing energy in healthy individu Physiol. Scand. 146:403-404, 1992; Walseret al., Kidney Int. als, enhance working capacity and endurance, reducing 32: 123-128, 1987). Several studies have also analyzed the muscle fatigue, reducing stress, enhancing cardiac and car effects of nutritional Supplements and herbal Supplements in diovascular function, improving sexual ability, increasing 30 enhancing muscle performance. muscle ATP levels, and/or reducing lactic acid in blood. In Aside from muscle performance during endurance exer certain embodiments, the methods involve administering an cise, free radicals and oxidative stress parameters are affected amount of a sirtuin activating compound that increase mito in pathophysiological states. A Substantial body of data now chondrial activity, increase mitochondrial biogenesis, and/or Suggests that oxidative stress contributes to muscle wasting or increase mitochondrial mass. 35 atrophy in pathophysiological states (reviewed in Clarkson, P. Sports performance refers to the ability of the athlete's M. Antioxidants and physical performance. Crit. Rev. Food muscles to perform when participating in sports activities. Sci. Nutr. 35: 31-41; 1995; Powers, S. K. Lennon, S. L. Enhanced sports performance, strength, speed and endurance Analysis of cellular responses to free radicals: Focus on exer are measured by an increase in muscular contraction strength, cise and skeletal muscle. Proc. Nutr. Soc. 58: 1025-1033; increase in amplitude of muscle contraction, shortening of 40 1999). For example, with respect to muscular disorders where muscle reaction time between stimulation and contraction. both muscle endurance and function are compensated, the Athlete refers to an individual who participates in sports at role of nitric oxide (NO), has been implicated. In muscular any level and who seeks to achieve an improved level of dystrophies, especially those due to defects in proteins that strength, speed and endurance in their performance, such as, make up the dystrophin-glycoprotein complex (DGC), the for example, body builders, bicyclists, long distance runners, 45 enzyme that synthesizes NO, nitric oxide synthase (NOS), short distance runners, etc. An athlete may be hard training, has been associated. Recent studies of dystrophies related to that is, performs sports activities intensely more than three DGC defects suggest that one mechanism of cellular injury is days a week or for competition. An athlete may also be a functional ischemia related to alterations in cellular NOS and fitness enthusiast who seeks to improve general health and disruption of a normal protective action of NO. This protec well-being, improve energy levels, who works out for about 50 tive action is the prevention of localischemia during contrac 1-2 hours about 3 times a week. Enhanced sports performance tion-induced increases in sympathetic vasoconstriction. in manifested by the ability to overcome muscle fatigue, Rando (Microsc Res Tech 55(4):223-35, 2001), has shown ability to maintain activity for longer periods of time, and that oxidative injury precedes pathologic changes and that have a more effective workout. muscle cells with defects in the DGC have an increased In the arena of athlete muscle performance, it is desirable to 55 Susceptibility to oxidant challenges. Excessive lipid peroxi create conditions that permit competition or training at higher dation due to free radicals has also been shown to be a factor levels of resistance for a prolonged period of time. However, in myopathic diseases Such as McArdle's disease (Russo et acute and intense anaerobic use of skeletal muscles often al., Med Hypotheses. 39(2): 147-51, 1992). Furthermore, results in impaired athletic performance, with losses in force mitochondrial dysfunction is a well-known correlate of age and work output, and increased onset of muscle fatigue, Sore 60 related muscle wasting (sarcopenia) and free radical damage ness, and dysfunction. It is now recognized that even a single has been Suggested, though poorly investigated, as a contrib exhaustive exercise session, or for that matter any acute uting factor (reviewed in Navarro, A.; Lopez-Cepero, J. M.: trauma to the body Such as muscle injury, resistance or Sanchez del Pino, M. L. Front. Biosci. 6: D26-44; 2001). exhaustive muscle exercise, or elective Surgery, is character Other indications include acute sarcopenia, for example ized by perturbed metabolism that affects muscle perfor 65 muscle atrophy and/or cachexia associated with burns, bed mance in both short and long term phases. Both muscle meta rest, limb immobilization, or major thoracic, abdominal, and/ bolic/enzymatic activity and gene expression are affected. or orthopedic Surgery. It is contemplated that the methods of US 8,178,536 B2 175 176 the present invention will also be effective in the treatment of would be applied to bees involved in the production of honey. muscle related pathological conditions. Generally, the methods described herein may be applied to In certain embodiments, the invention provides novel any organism, e.g., eukaryote, that may have commercial dietary compositions comprising sirtuin modulators, a importance. For example, they can be applied to fish (aquac method for their preparation, and a method of using the com ulture) and birds (e.g., chicken and fowl). positions for improvement of sports performance. Accord Higher doses of sirtuin-modulating compounds that ingly, provided are therapeutic compositions, foods and bev increase the level and/or activity of a sirtuin protein may also erages that have actions of improving physical endurance be used as a pesticide by interfering with the regulation of and/or inhibiting physical fatigues for those people involved silenced genes and the regulation of apoptosis during devel in broadly-defined exercises including sports requiring 10 endurance and labors requiring repeated muscle exertions. opment. In this embodiment, a compound may be applied to Such dietary compositions may additional comprise electro plants using a method known in the art that ensures the com lytes, caffeine, Vitamins, carbohydrates, etc. pound is bio-available to insect larvae, and not to plants. Other Uses At least in view of the link between reproduction and Sirtuin-modulating compounds that increase the level and/ 15 longevity (Longo and Finch, Science, 2002), sirtuin-modu or activity of a sirtuin protein may be used for treating or lating compounds that increase the level and/or activity of a preventing viral infections (such as infections by influenza, sirtuin protein can be applied to affect the reproduction of herpes or papilloma virus) or as antifungal agents. In certain organisms such as insects, animals and microorganisms. embodiments, sirtuin-modulating compounds that increase 4. Assays the level and/or activity of a sirtuin protein may be adminis Yet other methods contemplated herein include screening tered as part of a combination drug therapy with another methods for identifying compounds or agents that modulate therapeutic agent for the treatment of viral diseases, includ sirtuins. An agent may be a nucleic acid, such as an aptamer. ing, for example, acyclovir, ganciclovir and Zidovudine. In Assays may be conducted in a cell based or cell free format. another embodiment, sirtuin-modulating compounds that For example, an assay may comprise incubating (or contact increase the level and/or activity of a sirtuin protein may be 25 ing) a sirtuin with a test agent under conditions in which a administered as part of a combination drug therapy with sirtuin can be modulated by an agent known to modulate the anotheranti-fungal agent including, for example, topical anti sirtuin, and monitoring or determining the level of modula fungals such as ciclopiroX, clotrimazole, econazole, micona tion of the sirtuin in the presence of the test agent relative to Zole, nystatin, oxiconazole, terconazole, and tolnaftate, or the absence of the test agent. The level of modulation of a systemic anti-fungal Such as fluconazole (Diflucan), itracona 30 sirtuin can be determined by determining its ability to Zole (Sporanox), ketoconazole (NiZoral), and miconazole deacetylate a Substrate. Exemplary Substrates are acetylated (Monistat I.V.). peptides which can be obtained from BIOMOL (Plymouth Subjects that may be treated as described herein include Meeting, Pa.). Preferred substrates include peptides of p53, eukaryotes, such as mammals, e.g., humans, Ovines, bovines, Such as those comprising an acetylated K382. A particularly equines, porcines, canines, felines, non-human primate, 35 preferred substrate is the Fluor de Lys-SIRT1 (BIOMOL), mice, and rats. Cells that may be treated include eukaryotic i.e., the acetylated peptide Arg-His-Lys-Lys. Other Substrates cells, e.g., from a Subject described above, or plant cells, yeast are peptides from human histones H3 and H4 or an acetylated cells and prokaryotic cells, e.g., bacterial cells. For example, amino acid. Substrates may be fluorogenic. The sirtuin may modulating compounds may be administered to farm animals be SIRT1, Sir2, SIRT3, or a portion thereof. For example, to improve their ability to withstand farming conditions 40 recombinant SIRT1 can be obtained from BIOMOL. The longer. reaction may be conducted for about 30 minutes and stopped, Sirtuin-modulating compounds that increase the level and/ e.g., with nicotinamide. The HDAC fluorescent activity or activity of a sirtuin protein may also be used to increase assay/drug discovery kit (AK-500, BIOMOL Research Labo lifespan, stress resistance, and resistance to apoptosis in ratories) may be used to determine the level of acetylation. plants. In one embodiment, a compound is applied to plants, 45 Similar assays are described in Bitterman et al. (2002).J. Biol. e.g., on a periodic basis, or to fungi. In another embodiment, Chem. 277:45099. The level of modulation of the sirtuin in an plants are genetically modified to produce a compound. In assay may be compared to the level of modulation of the another embodiment, plants and fruits are treated with a com sirtuin in the presence of one or more (separately or simulta pound prior to picking and shipping to increase resistance to neously) compounds described herein, which may serve as damage during shipping. Plant seeds may also be contacted 50 positive or negative controls. Sirtuins for use in the assays with compounds described herein, e.g., to preserve them. may be full length sirtuin proteins orportions thereof. Since it In other embodiments, sirtuin-modulating compounds that has been shown herein that activating compounds appear to increase the level and/or activity of a sirtuin protein may be interact with the N-terminus of SIRT1, proteins for use in the used for modulating lifespan in yeast cells. Situations in assays include N-terminal portions of Sirtuins, e.g., about which it may be desirable to extend the lifespan of yeast cells 55 amino acids 1-176 or 1-255 of SIRT1; about amino acids include any process in which yeast is used, e.g., the making of 1-174 or 1-252 of Sir2. beer, yogurt, and bakery items, e.g., bread. Use of yeast In one embodiment, a screening assay comprises (i) con having an extended lifespan can result in usingless yeast or in tacting a sirtuin with a test agent and an acetylated Substrate having the yeast be active for longer periods of time. Yeast or under conditions appropriate for the sirtuin to deacetylate the other mammalian cells used for recombinantly producing 60 Substrate in the absence of the testagent; and (ii) determining proteins may also be treated as described herein. the level of acetylation of the substrate, wherein a lower level Sirtuin-modulating compounds that increase the level and/ of acetylation of the substrate in the presence of the test agent or activity of a sirtuin protein may also be used to increase relative to the absence of the test agent indicates that the test lifespan, stress resistance and resistance to apoptosis in agent stimulates deacetylation by the sirtuin, whereas a insects. In this embodiment, compounds would be applied to 65 higher level of acetylation of the substrate in the presence of useful insects, e.g., bees and other insects that are involved in the test agent relative to the absence of the test agent indicates pollination of plants. In a specific embodiment, a compound that the test agent inhibits deacetylation by the sirtuin. US 8,178,536 B2 177 178 Methods for identifying an agent that modulates, e.g., sodium lauryl sulphate). The tablets may be coated by meth stimulates or inhibits, sirtuins in vivo may comprise (i) con ods well known in the art. Liquid preparations for oral admin tacting a cell with a test agent and a Substrate that is capable istration may take the form of for example, solutions, syrups of entering a cell in the presence of an inhibitor of class I and or Suspensions, or they may be presented as a dry product for class II HDACs under conditions appropriate for the sirtuinto constitution with water or other suitable vehicle before use. deacetylate the Substrate in the absence of the test agent; and Such liquid preparations may be prepared by conventional (ii) determining the level of acetylation of the substrate, means with pharmaceutically acceptable additives Such as wherein a lower level of acetylation of the substrate in the Suspending agents (e.g., Sorbitol syrup, cellulose derivatives presence of the test agent relative to the absence of the test or hydrogenated edible fats); emulsifying agents (e.g., leci agent indicates that the test agent stimulates deacetylation by 10 thin or acacia); non-aqueous vehicles (e.g., ationd oil, oily the sirtuin, whereas a higher level of acetylation of the sub esters, ethyl alcohol or fractionated vegetable oils); and pre strate in the presence of the test agent relative to the absence servatives (e.g., methyl or propyl-p-hydroxybenzoates or Sor of the test agent indicates that the test agent inhibits deacety bic acid). The preparations may also contain buffer salts, lation by the sirtuin. A preferred substrate is an acetylated flavoring, coloring and Sweetening agents as appropriate. peptide, which is also preferably fluorogenic, as further 15 Preparations for oral administration may be suitably formu described herein. The method may further comprise lysing lated to give controlled release of the active compound. the cells to determine the level of acetylation of the substrate. For administration by inhalation (e.g., pulmonary deliv Substrates may be added to cells at a concentration ranging ery), sirtuin-modulating compounds may be conveniently from about 1 uM to about 10 mM, preferably from about 10 delivered in the form of an aerosol spray presentation from uM to 1 mM, even more preferably from about 100 uM to 1 pressurized packs or a nebuliser, with the use of a suitable mM, such as about 200 uM. A preferred substrate is an acety propellant, e.g., dichlorodifluoromethane, trichlorofluo lated lysine, e.g., e-acetyl lysine (Fluor de Lys, FdL) or Fluor romethane, dichlorotetrafluoroethane, carbon dioxide or de Lys-SIRT1. A preferred inhibitor of class I and class II other suitable gas. In the case of a pressurized aerosol the HDACs is trichostatin A (TSA), which may be used at con dosage unit may be determined by providing a valve to deliver centrations ranging from about 0.01 to 100 uM, preferably 25 a metered amount. Capsules and cartridges of e.g., gelatin, for from about 0.1 to 10 M, such as 1 uM. Incubation of cells use in an inhaler or insufflator may be formulated containing with the test compound and the substrate may be conducted a powder mix of the compound and a suitable powder base for about 10 minutes to 5 hours, preferably for about 1-3 Such as lactose or starch. hours. Since TSA inhibits all class I and class II HDACs, and Sirtuin-modulating compounds may be formulated for that certain Substrates, e.g., Fluor de LyS, is a poor Substrate 30 parenteral administration by injection, e.g., by bolus injection for SIRT2 and even less a substrate for SIRT3-7, such an or continuous infusion. Formulations for injection may be assay may be used to identify modulators of SIRT1 in vivo. presented in unit dosage form, e.g., in ampoules or in multi 5. Pharmaceutical Compositions dose containers, with an added preservative. The composi The sirtuin-modulating compounds described herein may tions may take Such forms as Suspensions, solutions or emul be formulated in a conventional manner using one or more 35 sions in oily or aqueous vehicles, and may contain physiologically acceptable carriers or excipients. For formulatory agents such as Suspending, stabilizing and/or example, sirtuin-modulating compounds and their physi dispersing agents. Alternatively, the active ingredient may be ologically acceptable salts and Solvates may be formulated in powder form for constitution with a Suitable vehicle, e.g., for administration by, for example, injection (e.g. SubO, IM, sterile pyrogen-free water, before use. IP), inhalation or insufflation (either through the mouth or the 40 Sirtuin-modulating compounds may also be formulated in nose) or oral, buccal, Sublingual, transdermal, nasal, rectal compositions such as Suppositories or retention parenteral or rectal administration. In one embodiment, a enemas, e.g., containing conventional Suppository bases Such sirtuin-modulating compound may be administered locally, as cocoa butter or other glycerides. at the site where the target cells are present, i.e., in a specific In addition to the formulations described previously, sir tissue, organ, or fluid (e.g., blood, cerebrospinal fluid, etc.). 45 tuin-modulating compounds may also be formulated as a Sirtuin-modulating compounds can be formulated for a depot preparation. Such long acting formulations may be variety of modes of administration, including systemic and administered by implantation (for example Subcutaneously or topical or localized administration. Techniques and formula intramuscularly) or by intramuscular injection. Thus, for tions generally may be found in Remington's Pharmaceutical example, sirtuin-modulating compounds may be formulated Sciences. Meade Publishing Co., Easton, Pa. For parenteral 50 with suitable polymeric or hydrophobic materials (for administration, injection is preferred, including intramuscu example as an emulsion in an acceptable oil) or ion exchange lar, intravenous, intraperitoneal, and Subcutaneous. For injec resins, or as sparingly soluble derivatives, for example, as a tion, the compounds can be formulated in liquid Solutions, sparingly soluble salt. Controlled release formula also preferably in physiologically compatible buffers such as includes patches. Hank's Solution or Ringer's Solution. In addition, the com 55 In certain embodiments, the compounds described herein pounds may be formulated in solid form and redissolved or can be formulated for delivery to the central nervous system Suspended immediately prior to use. Lyophilized forms are (CNS) (reviewed in Begley, Pharmacology & Therapeutics also included. 104: 29-45 (2004)). Conventional approaches for drug deliv For oral administration, the pharmaceutical compositions ery to the CNS include: neuroSurgical strategies (e.g., intrac may take the form of for example, tablets, loZanges, or cap 60 erebral injection or intracerebroventricular infusion); Sules prepared by conventional means with pharmaceutically molecular manipulation of the agent (e.g., production of a acceptable excipients such as binding agents (e.g., pregelati chimeric fusion protein that comprises a transport peptide nised maize starch, polyvinylpyrrolidone or hydroxypropyl that has an affinity for an endothelial cell surface molecule in methylcellulose); fillers (e.g., lactose, microcrystalline cellu combination with an agent that is itself incapable of crossing lose or calcium hydrogen phosphate); lubricants (e.g., mag 65 the BBB) in an attempt to exploit one of the endogenous nesium Stearate, talc or silica); disintegrants (e.g., potato transport pathways of the BBB. pharmacological strategies starch or sodium starch glycolate); or wetting agents (e.g., designed to increase the lipid solubility of an agent (e.g., US 8,178,536 B2 179 180 conjugation of water-soluble agents to lipid or cholesterol propylcellulose, hydroxypropylmethylcellulose phthalate, carriers); and the transitory disruption of the integrity of the noncrystalline cellulose, magnesium aluminum silicate, tri BBB by hyperosmotic disruption (resulting from the infusion ethanolamine, polyvinyl alcohol, and polyvinylpyrrolidone of a mannitol solution into the carotid artery or the use of a (PVP). Most of these surface modifiers are known pharma biologically active agent Such as an angiotensin peptide). ceutical excipients and are described in detail in the Hand One possibility to achieve sustained release kinetics is book of Pharmaceutical Excipients, published jointly by the embedding or encapsulating the active compound into nano American Pharmaceutical Association and The Pharmaceu particles. Nanoparticles can be administrated as powder, as a tical Society of Great Britain, the Pharmaceutical Press, 1986. powder mixture with added excipients or as Suspensions. Further description on preparing nanoparticles can be Colloidal suspensions of nanoparticles can easily be admin 10 found, for example, in U.S. Pat. No. 6,264,922, the contents istrated through a cannula with Small diameter. of which are incorporated herein by reference. Nanoparticles are particles with a diameter from about 5 Liposomes are a further drug delivery system which is nm to up to about 1000 nm. The term “nanoparticles” as it is easily injectable. Accordingly, in the method of invention the used hereinafter refers to particles formed by a polymeric active compounds can also be administered in the form of a matrix in which the active compound is dispersed, also known 15 liposome delivery system. Liposomes are well-known by a as "nanospheres', and also refers to nanoparticles which are person skilled in the art. Liposomes can be formed from a composed of a core containing the active compound which is variety of phospholipids, such as cholesterol, Stearylamine of Surrounded by a polymeric membrane, also known as "nano phosphatidylcholines. Liposomes being usable for the capsules”. In certain embodiments, nanoparticles are pre method of invention encompass all types of liposomes includ ferred having a diameter from about 50 nm to about 500 nm, ing, but not limited to, Small unilamellar vesicles, large unila in particular from about 100 nm to about 200 nm. mellar vesicles and multilamellar vesicles. Nanoparticles can be prepared by in situ polymerization of Liposomes are used for a variety of therapeutic purposes, dispersed monomers or by using preformed polymers. Since and in particular, for carrying therapeutic agents to target polymers prepared in situ are often not biodegradable and/or cells. Advantageously, liposome-drug formulations offer the contain toxicological serious byproducts, nanoparticles from 25 potential of improved drug-delivery properties, which preformed polymers are preferred. Nanoparticles from pre include, for example, controlled drug release. An extended formed polymers can be prepared by different techniques, circulation time is often needed for liposomes to reacha target e.g., by emulsion evaporation, solvent displacement, salting region, cell or site. In particular, this is necessary where the out, mechanical grinding, microprecipitation, and by emulsi target region, cell or site is not located near the site of admin fication diffusion. 30 istration. For example, when liposomes are administered sys With the methods described above, nanoparticles can be temically, it is desirable to coat the liposomes with a hydro formed with various types of polymers. For use in the method philic agent, for example, a coating of hydrophilic polymer of the present invention, nanoparticles made from biocom chains such as polyethylene glycol (PEG) to extend the blood patible polymers are preferred. The term “biocompatible” circulation lifetime of the liposomes. Such surface-modified refers to material that after introduction into a biological 35 liposomes are commonly referred to as “long circulating” or environment has no serious effects to the biological environ “sterically stabilized' liposomes. ment. From biocompatible polymers those polymers are One Surface modification to a liposome is the attachment of especially preferred which are also biodegradable. The term PEG chains, typically having a molecular weight from about “biodegradable' refers to material that after introduction into 1000 daltons (Da) to about 5000 Da, and to about 5 mole a biological environment is enzymatically or chemically 40 percent (%) of the lipids making up the liposomes (see, for degraded into Smaller molecules, which can be eliminated example, Stealth Liposomes, CRC Press, Lasic, D. and Mar Subsequently. Examples are polyesters from hydroxycar tin, F., eds., Boca Raton, Fla., (1995)), and the cited refer boxylic acids such as poly(lactic acid) (PLA), poly(glycolic ences therein. The pharmacokinetics exhibited by such lipo acid) (PGA), polycaprolactone (PCL), copolymers of lactic Somes are characterized by a dose-independent reduction in acid and glycolic acid (PLGA), copolymers of lactic acid and 45 uptake of liposomes by the liver and spleen via the mono caprolactone, polyepsilon caprolactone, polyhyroxybutyric nuclear phagocyte system (MPS), and significantly pro acid and poly(ortho)esters, polyurethanes, polyanhydrides, longed blood circulation time, as compared to non-surface polyacetals, polydihydropyrians, polycyanoacrylates, natural modified liposomes, which tend to be rapidly removed from polymers such as alginate and other polysaccharides includ the blood and accumulated in the liver and spleen. ing dextran and cellulose, collagen and albumin. 50 In certain embodiments, the complex is shielded to Suitable surface modifiers can preferably be selected from increase the circulatory half-life of the complex or shielded to known organic and inorganic pharmaceutical excipients. increase the resistance of nucleic acid to degradation, for Such excipients include various polymers, low molecular example degradation by nucleases. weight oligomers, natural products and Surfactants. Preferred As used herein, the term 'shielding, and its cognates Such Surface modifiers include nonionic and ionic Surfactants. 55 as "shielded, refers to the ability of "shielding moieties” to Representative examples of surface modifiers include gelatin, reduce the non-specific interaction of the complexes casein, lecithin (phosphatides), gum acacia, cholesterol, described herein with serum complement or with other spe tragacanth, Stearic acid, benzalkonium chloride, calcium cies present in serum in vitro or in vivo. Shielding moieties Stearate, glycerol monostearate, cetostearyl alcohol, may decrease the complex interaction with orbinding to these cetomacrogol emulsifying wax, Sorbitan esters, polyoxyeth 60 species through one or more mechanisms, including, for ylenealkyl ethers, e.g., macrogolethers such as cetomacrogol example, non-specific steric or non-specific electronic inter 1000, polyoxyethylene castor oil derivatives, polyoxyethyl actions. Examples of Such interactions include non-specific ene Sorbitan fatty acid esters, e.g., the commercially available electrostatic interactions, charge interactions, Van der Waals TweensTM, polyethylene glycols, polyoxyethylene stearates, interactions, steric-hindrance and the like. For a moiety to act colloidal silicon dioxide, phosphates, Sodium dodecylsulfate, 65 as a shielding moiety, the mechanism or mechanisms by carboxymethylcellulose calcium, carboxymethylcellulose which it may reduce interaction with, association with or sodium, methylcellulose, hydroxyethylcellulose, hydroxy binding to the serum complement or other species does not US 8,178,536 B2 181 182 have to be identified. One can determine whethera moiety can As mentioned above, the compositions of matter of the act as a shielding moiety by determining whether or to what invention comprise an aqueous preparation of preferably Sub extent a complex binds serum species. stituted amorphous cyclodextrin and one or more sirtuin It should be noted that "shielding moieties’ can be multi modulators. The relative amounts of sirtuin modulators and functional. For example, a shielding moiety may also func cyclodextrin will vary depending upon the relative amount of tion as, for example, a targeting factor. A shielding moiety each of the sirtuin modulators and the effect of the cyclodex may also be referred to as multifunctional with respect to the trin on the compound. In general, the ratio of the weight of mechanism(s) by which it shields the complex. While not compound of the sirtuin modulators to the weight of cyclo wishing to be limited by proposed mechanism or theory, dextrin compound will be in a range between 1:1 and 1:100. examples of Such a multifunctional shielding moiety are pH 10 A weight to weight ratio in a range of 1:5 to 1:50 and more sensitive endosomal membrane-disruptive synthetic poly preferably in a range of 1:10 to 1:20 of the compound selected mers, such as PPAA or PEAA. Certain poly(alkylacrylic from sirtuin modulators to cyclodextrin are believed to be the acids) have been shown to disrupt endosomal membranes most effective for increased circulating availability of the while leaving the outer cell surface membrane intact (Stayton sirtuin modulator. et al. (2000) J. Controll. Release 65:203-220; Murthy et al. 15 Importantly, if the aqueous solution comprising the sirtuin (1999) J. Controll. Release 61:137-143: WO 99/34831), modulators and a cyclodextrin is to be administered parenter thereby increasing cellular bioavailability and functioning as ally, especially via the intravenous route, a cyclodextrin will a targeting factor. However, PPAA reduces binding of serum be substantially free of pyrogenic contaminants. Various complement to complexes in which it is incorporated, thus forms of cyclodextrin, Such as forms of amorphous cyclodex functioning as a shielding moiety. trin, may be purchased from a number of Vendors including Another way to produce a formulation, particularly a solu Sigma-Aldrich, Inc. (St. Louis, Mo., USA). A method for the tion, of a sirtuin modulator Such as resveratrol or a derivative production of hydroxypropyl-3-cyclodextrin is disclosed in thereof, is through the use of cyclodextrin. By cyclodextrin is Pitha et al., U.S. Pat. No. 4,727,064 which is incorporated meant C.-, 3-, or Y-cyclodextrin. Cyclodextrins are described herein by reference. in detail in Pitha et al., U.S. Pat. No. 4,727,064, which is 25 Additional description of the use of cyclodextrin for solu incorporated herein by reference. Cyclodextrins are cyclic bilizing compounds can be found in US 2005/0026849, the oligomers of glucose; these compounds form inclusion com contents of which are incorporated herein by reference. plexes with any drug whose molecule can fit into the lipo Rapidly disintegrating or dissolving dosage forms are use phile-seeking cavities of the cyclodextrin molecule. ful for the rapid absorption, particularly buccal and sublin The cyclodextrin of the compositions according to the 30 gual absorption, of pharmaceutically active agents. Fast melt invention may be Cl-, 3-, or Y-cyclodextrin, C.-cyclodextrin dosage forms are beneficial to patients, such as aged and contains six glucopyranose units: B-cyclodextrin contains pediatric patients, who have difficulty in Swallowing typical seven glucopyranose units; and Y-cyclodextrin contains eight Solid dosage forms, such as caplets and tablets. Additionally, glucopyranose units. The molecule is believed to form a trun fast melt dosage forms circumvent drawbacks associated cated cone having a core opening of 4.7-5.3 angstroms, 6.0- 35 with, for example, chewable dosage forms, wherein the 6.5 angstroms, and 7.5-8.3 angstroms in C-, 3-, or Y-cyclo length of time an active agent remains in a patient’s mouth dextrin respectively. The composition according to the plays an important role in determining the amount of taste invention may comprise a mixture of two or more of the Cl-, masking and the extent to which a patient may object to throat B-, or Y-cyclodextrins. Typically, however, the composition grittiness of the active agent. according to the invention will comprise only one of the Cl-, 40 To overcome such problems manufacturers have devel B-, or Y-cyclodextrins. oped a number of fast melt solid dose oral formulations. Most preferred cyclodextrins in the compositions accord These are available from manufacturers including Cima ing to the invention are amorphous cyclodextrin compounds. Labs, FuisZ Technologies Ltd., Prographarm, R. P. Scherer, By amorphous cyclodextrin is meant non-crystalline mix Yamanouchi-Shaklee, and McNeil-PPC, Inc. All of these tures of cyclodextrins wherein the mixture is prepared from 45 manufacturers market different types of rapidly dissolving Cl-, 3-, or Y-cyclodextrin. In general, the amorphous cyclo Solid oral dosage forms. See e.g., patents and publications by dextrin is prepared by non-selective alkylation of the desired Cima Labs such as U.S. Pat. Nos. 5,607,697, 5,503,846, cyclodextrin species. Suitable alkylation agents for this pur 5,223,264, 5,401,513, 5,219,574, and 5,178,878, WO pose include but are not limited to propylene oxide, glycidol. 98/46215, WO 98/14179; patents to Fuisz Technologies, now iodoacetamide, chloroacetate, and 2-diethylaminoethlychlo 50 part of BioVail, such as U.S. Pat. Nos. 5,871,781, 5,869,098, ride. Reactions are carried out to yield mixtures containing a 5,866,163, 5,851,553, 5,622,719, 5,567,439, and 5,587,172: plurality of components thereby preventing crystallization of 5,464.632 to Prographarm; patents to R. P. Scherer such as the cyclodextrin. Various alkylated cyclodextrins can be made U.S. Pat. Nos. 4,642,903, 5,188,825, 5,631,023 and 5,827, and of course will vary, depending upon the starting species of 541; patents to Yamanouchi-Shaklee such as U.S. Pat. Nos. cyclodextrin and the alkylating agent used. Among the amor 55 5,576,014 and 5,446.464; patents to Janssen such as U.S. Pat. phous cyclodextrins suitable for compositions according to Nos. 5,807,576, 5,635,210, 5,595,761, 5,587,180 and 5,776, the invention are hydroxypropyl, hydroxyethyl, glucosyl, 491; 5,639,475 and 5,709,886 to Eurand America, Inc.; U.S. maltosyl and maltotriosyl derivatives of B-cyclodextrin, car Pat. Nos. 5,807,578 and 5,807,577 to L.A.B. Pharmaceutical boxyamidomethyl-3-cyclodextrin, carboxymethyl-3-cyclo Research; patents to Schering Corporation such as U.S. Pat. dextrin, hydroxypropyl-3-cyclodextrin and diethylamino-B- 60 Nos. 5,112,616 and 5,073,374; 4,616,047 to Laboratoire L. cyclodextrin. LaFon; U.S. Pat. No. 5,501,861 to Takeda Chemicals Inc., One example of resveratrol dissolved in the presence of a Ltd.; and U.S. Pat. No. 6,316,029 to Elan. cyclodextrin is provided in Marier et al., J. Pharmacol. Exp. In one example of fast melt tablet preparation, granules for Therap. 302:369-373 (2002), the contents of which are incor fast melt tablets made by either the spray drying or pre porated herein by reference, where a 6 mg/mL solution of 65 compacting processes are mixed with excipients and com resveratrol was prepared using 0.9% saline containing 20% pressed into tablets using conventional tablet making machin hydroxylpropyl-3-cyclodextrin. ery. The granules can be combined with a variety of carriers US 8,178,536 B2 183 184 including low density, high moldability Saccharides, low In one embodiment, a sirtuin-modulating compound moldability saccharides, polyol combinations, and then described herein, is incorporated into a topical formulation directly compressed into a tablet that exhibits an improved containing a topical carrier that is generally Suited to topical dissolution and disintegration profile. drug administration and comprising any such material known The tablets according to the present invention typically in the art. The topical carrier may be selected so as to provide have a hardness of about 2 to about 6 Strong-Cobb units (scu). the composition in the desired form, e.g., as an ointment, Tablets within this hardness range disintegrate or dissolve lotion, cream, microemulsion, gel, oil, Solution, or the like, rapidly when chewed. Additionally, the tablets rapidly disen and may be comprised of a material of either naturally occur tegrate in water. On average, a typical 1.1 to 1.5 gram tablet ring or synthetic origin. It is preferable that the selected disintegrates in 1-3 minutes without stirring. This rapid dis 10 carrier not adversely affect the active agent or other compo integration facilitates delivery of the active material. nents of the topical formulation. Examples of suitable topical The granules used to make the tablets can be, for example, carriers for use herein include water, alcohols and other non mixtures of low density alkali earth metal salts or carbohy toxic organic solvents, glycerin, mineral oil, silicone, petro drates. For example, a mixture of alkali earth metal salts leum jelly, lanolin, fatty acids, vegetable oils, parabens, includes a combination of calcium carbonate and magnesium 15 waxes, and the like. hydroxide. Similarly, a fast melt tablet can be prepared Formulations may be colorless, odorless ointments, according to the methods of the present invention that incor lotions, creams, microemulsions and gels. porates the use of A) spray dried extra light calcium carbon Sirtuin-modulating compounds may be incorporated into ate/maltodextrin, B) magnesium hydroxide and C) a eutectic ointments, which generally are semisolid preparations which polyol combination including Sorbitol Instant, Xylitol and are typically based on petrolatum or other petroleum deriva mannitol. These materials have been combined to produce a tives. The specific ointment base to be used, as will be appre low density tablet that dissolves very readily and promotes ciated by those skilled in the art, is one that will provide for the fast disintegration of the active ingredient. Additionally, optimum drug delivery, and, preferably, will provide for other the pre-compacted and spray dried granules can be combined desired characteristics as well, e.g., emolliency or the like. As in the same tablet. 25 with other carriers or vehicles, an ointment base should be For fast melt tablet preparation, a sirtuin modulator useful inert, stable, nonirritating and nonsensitizing. As explained in in the present invention can be in a form Such as Solid, par Remington's (Supra) ointment bases may be grouped in four ticulate, granular, crystalline, oily or solution. The sirtuin classes: oleaginous bases; emulsifiable bases; emulsion modulator for use in the present invention may be a spray bases; and water-soluble bases. Oleaginous ointment bases dried product or an adsorbate that has been pre-compacted to 30 include, for example, vegetable oils, fats obtained from ani a harder granular form that reduces the medicament taste. A mals, and semisolid hydrocarbons obtained from petroleum. pharmaceutical active ingredient for use in the present inven Emulsifiable ointment bases, also known as absorbent oint tion may be spray dried with a carrier that prevents the active ment bases, contain little or no water and include, for ingredient from being easily extracted from the tablet when example, hydroxyStearin Sulfate, anhydrous lanolin and chewed. 35 hydrophilic petrolatum. Emulsion ointment bases are either In addition to being directly added to the tablets of the water-in-oil (W/O) emulsions or oil-in-water (O/W) emul present invention, the medicament drug itself can be pro sions, and include, for example, cetyl alcohol, glyceryl cessed by the pre-compaction process to achieve an increased monostearate, lanolin and Stearic acid. Exemplary water density prior to being incorporated into the formulation. soluble ointment bases are prepared from polyethylene gly The pre-compaction process used in the present invention 40 cols (PEGs) of varying molecular weight; again, reference can be used to deliver poorly soluble pharmaceutical materi may be had to Remington's, Supra, for further information. als so as to improve the release of Such pharmaceutical mate Sirtuin-modulating compounds may be incorporated into rials over traditional dosage forms. This could allow for the lotions, which generally are preparations to be applied to the use of lower dosage levels to deliver equivalent bioavailable skin Surface without friction, and are typically liquid or semi levels of drug and thereby lower toxicity levels of both cur 45 liquid preparations in which solid particles, including the rently marketed drug and new chemical entities. Poorly active agent, are present in a water or alcohol base. Lotions soluble pharmaceutical materials can be used in the form of are usually suspensions of solids, and may comprise a liquid nanoparticles, which are nanometer-sized particles. oily emulsion of the oil-in-water type. Lotions are preferred In addition to the active ingredient and the granules pre formulations for treating large body areas, because of the ease pared from low density alkali earth metal salts and/or water 50 of applying a more fluid composition. It is generally neces soluble carbohydrates, the fast melt tablets can beformulated sary that the insoluble matter in a lotion be finely divided. using conventional carriers or excipients and well established Lotions will typically contain Suspending agents to produce pharmaceutical techniques. Conventional carriers or excipi better dispersions as well as compounds useful for localizing ents include, but are not limited to, diluents, binders, adhe and holding the active agent in contact with the skin, e.g., sives (i.e., cellulose derivatives and acrylic derivatives), lubri 55 methylcellulose, sodium carboxymethylcellulose, or the like. cants (i.e., magnesium or calcium Stearate, vegetable oils, An exemplary lotion formulation for use in conjunction with polyethylene glycols, talc, sodium lauryl Sulphate, polyoxy the present method contains propylene glycol mixed with a ethylene monoStearate), disintegrants, colorants, flavorings, hydrophilic petrolatum such as that which may be obtained preservatives, Sweeteners and miscellaneous materials such under the trademark Aquaphor(R) from Beiersdorf, Inc. (Nor as buffers and adsorbents. 60 walk, Conn.). Additional description of the preparation of fast melt tablets Sirtuin-modulating compounds may be incorporated into can be found, for example, in U.S. Pat. No. 5,939,091, the creams, which generally are viscous liquid or semisolid emul contents of which are incorporated herein by reference. sions, either oil-in-water or water-in-oil. Cream bases are Pharmaceutical compositions (including cosmetic prepa water-washable, and contain an oil phase, an emulsifier and rations) may comprise from about 0.00001 to 100% such as 65 an aqueous phase. The oil phase is generally comprised of from 0.001 to 10% or from 0.1% to 5% by weight of one or petrolatum and a fatty alcohol Such as cetyl or Stearyl alcohol; more sirtuin-modulating compounds described herein. the aqueous phase usually, although not necessarily, exceeds US 8,178,536 B2 185 186 the oil phase in Volume, and generally contains a humectant. %, preferably 2 wt.% to 20 wt.%, emulsifiers; 2 wt.% to 20 The emulsifier in a cream formulation, as explained in Rem wt.% emollient; and 0.01 to 0.2 wt.% preservative, with the ington's, Supra, is generally a nonionic, anionic, cationic or active agent and carrier (e.g., water) making of the remainder amphoteric Surfactant. of the formulation. Sirtuin-modulating compounds may be incorporated into A skin permeation enhancer serves to facilitate passage of microemulsions, which generally are thermodynamically therapeutic levels of active agent to pass through a reasonably stable, isotropically clear dispersions of two immiscible liq sized area of unbroken skin. Suitable enhancers are well uids, such as oil and water, stabilized by an interfacial film of known in the art and include, for example: lower alkanols surfactant molecules (Encyclopedia of Pharmaceutical Tech 10 Such as methanol ethanol and 2-propanol; alkyl methyl Sul nology (New York: Marcel Dekker, 1992), volume 9). For the foxides such as dimethylsulfoxide (DMSO), decylmethylsul preparation of microemulsions, Surfactant (emulsifier), co foxide (CoMSO) and tetradecylmethyl sulfboxide: pyrroli Surfactant (co-emulsifier), an oil phase and a water phase are dones such as 2-pyrrolidone, N-methyl-2-pyrrolidone and necessary. Suitable Surfactants include any surfactants that N-(-hydroxyethyl)pyrrolidone; urea; N,N-diethyl-m-tolua are useful in the preparation of emulsions, e.g., emulsifiers 15 mide: C-C alkanediols; miscellaneous solvents such as that are typically used in the preparation of creams. The dimethyl formamide (DMF), N,N-dimethylacetamide co-surfactant (or “co-emulsifer') is generally selected from (DMA) and tetrahydrofurfuryl alcohol; and the 1-substituted the group of polyglycerol derivatives, glycerol derivatives azacycloheptan-2-ones, particularly 1-n-dodecylcyclazacy and fatty alcohols. Preferred emulsifier/co-emulsifier combi cloheptan-2-one (laurocapram; available under the trademark nations are generally although not necessarily selected from AZone(R) from Whitby Research Incorporated, Richmond, the group consisting of glyceryl monostearate and polyoxy Va.). ethylene Stearate; polyethylene glycol and ethylene glycol Examples of solubilizers include, but are not limited to, the palmitostearate; and caprilic and capric triglycerides and ole following: hydrophilic ethers such as diethylene glycol oyl macrogolglycerides. The water phase includes not only 25 monoethyl ether (ethoxydiglycol, available commercially as water but also, typically, buffers, glucose, propylene glycol, Transcutol R) and diethylene glycol monoethyl ether oleate polyethylene glycols, preferably lower molecular weight (available commercially as SoftcutolR); polyethylene castor polyethylene glycols (e.g., PEG 300 and PEG 400), and/or oil derivatives such as polyoxy 35 castor oil, polyoxy 40 glycerol, and the like, while the oil phase will generally 30 hydrogenated castor oil, etc.; polyethylene glycol, particu comprise, for example, fatty acid esters, modified vegetable larly lower molecular weight polyethylene glycols such as oils, silicone oils, mixtures of mono- di- and triglycerides, PEG 300 and PEG 400, and polyethylene glycol derivatives mono- and di-esters of PEG (e.g., oleoyl macrogol glycer such as PEG-8 caprylic/capric glycerides (available commer ides), etc. cially as Labrasol(R); alkyl methylsulfoxides such as DMSO; Sirtuin-modulating compounds may be incorporated into 35 pyrrolidones Such as 2-pyrrolidone and N-methyl-2-pyrroli gel formulations, which generally are semisolid systems con done; and DMA. Many solubilizers can also act as absorption sisting of either Suspensions made up of Small inorganic enhancers. A single solubilizer may be incorporated into the particles (two-phase systems) or large organic molecules dis formulation, or a mixture of solubilizers may be incorporated tributed substantially uniformly throughout a carrier liquid therein. 40 (single phase gels). Single phase gels can be made, for Suitable emulsifiers and co-emulsifiers include, without example, by combining the active agent, a carrier liquid and a limitation, those emulsifiers and co-emulsifiers described Suitable gelling agent such as tragacanth (at 2 to 5%), sodium with respect to microemulsion formulations. Emollients alginate (at 2-10%), gelatin (at 2-15%), methylcellulose (at include, for example, propylene glycol, glycerol, isopropyl 3-5%), sodium carboxymethylcellulose (at 2-5%), carbomer 45 myristate, polypropylene glycol-2 (PPG-2) myristyl ether (at 0.3-5%) or polyvinyl alcohol (at 10-20%) together and propionate, and the like. mixing until a characteristic semisolid product is produced. Other active agents may also be included in formulations, Other Suitable gelling agents include methylhydroxycellu e.g., other anti-inflammatory agents, analgesics, antimicro lose, polyoxyethylene-polyoxypropylene, hydroxyethylcel bial agents, antifungal agents, antibiotics, vitamins, antioxi lulose and gelatin. Although gels commonly employ aqueous 50 dants, and Sunblock agents commonly found in Sunscreen carrier liquid, alcohols and oils can be used as the carrier formulations including, but not limited to, anthranilates, ben liquid as well. Zophenones (particularly benzophenone-3), camphor deriva Various additives, known to those skilled in the art, may be tives, cinnamates (e.g., octyl methoxycinnamate), dibenzoyl included in formulations, e.g., topical formulations. 55 methanes (e.g., butyl methoxydibenzoyl methane), p-ami Examples of additives include, but are not limited to, solubi nobenzoic acid (PABA) and derivatives thereof, and salicy lizers, skin permeation enhancers, opacifiers, preservatives lates (e.g., octyl salicylate). (e.g., anti-oxidants), gelling agents, buffering agents, Surfac In certain topical formulations, the active agent is present tants (particularly nonionic and amphoteric Surfactants), in an amount in the range of approximately 0.25 wt.% to 75 emulsifiers, emollients, thickening agents, stabilizers, 60 wt.% of the formulation, preferably in the range of approxi humectants, colorants, fragrance, and the like. Inclusion of mately 0.25 wt.% to 30 wt.% of the formulation, more solubilizers and/or skin permeation enhancers is particularly preferably in the range of approximately 0.5 wt.% to 15 wt. preferred, along with emulsifiers, emollients and preserva % of the formulation, and most preferably in the range of tives. An optimum topical formulation comprises approxi 65 approximately 1.0 wt.% to 10 wt.% of the formulation. mately: 2 wt.% to 60 wt.%, preferably 2 wt.% to 50 wt.%, Topical skin treatment compositions can be packaged in a solubilizer and/or skin permeation enhancer; 2 wt.% to 50 wt. suitable container to suit its viscosity and intended use by the US 8,178,536 B2 187 188 consumer. For example, a lotion or cream can be packaged in 1996; and Hematopoietic Stem Cell Therapy, E. D. Ball, J. a bottle or a roll-ball applicator, or a propellant-driven aerosol Lister & P. Law, Churchill Livingstone, 2000. device or a container fitted with a pump suitable for finger Toxicity and therapeutic efficacy of sirtuin-modulating operation. When the composition is a cream, it can simply be compounds can be determined by standard pharmaceutical stored in a non-deformable bottle or squeeze container, Such 5 procedures in cell cultures or experimental animals. The as a tube or a lidded jar. The composition may also be LDs is the dose lethal to 50% of the population. The EDs is included in capsules such as those described in U.S. Pat. No. the dose therapeutically effective in 50% of the population. 5,063,507. Accordingly, also provided are closed containers The dose ratio between toxic and therapeutic effects (LDso/ containing a cosmetically acceptable composition as herein 10 EDso) is the therapeutic index. Sirtuin-modulating com defined. pounds that exhibit large therapeutic indexes are preferred. While sirtuin-modulating compounds that exhibit toxic side In an alternative embodiment, a pharmaceutical formula effects may be used, care should be taken to design a delivery tion is provided for oral or parenteral administration, in which system that targets such compounds to the site of affected case the formulation may comprises a modulating com 15 tissue in order to minimize potential damage to uninfected pound-containing microemulsion as described above, but cells and, thereby, reduce side effects. may contain alternative pharmaceutically acceptable carriers, The data obtained from the cell culture assays and animal vehicles, additives, etc. particularly Suited to oral or studies can be used informulating a range of dosage for use in parenteral drug administration. Alternatively, a modulating humans. The dosage of Such compounds may lie within a compound-containing microemulsion may be administered range of circulating concentrations that include the EDs with orally or parenterally substantially as described above, with little or no toxicity. The dosage may vary within this range out modification. depending upon the dosage form employed and the route of Phospholipids complexes, e.g., resveratrol-phospholipid administration utilized. For any compound, the therapeuti complexes, and their preparation are described in U.S. Patent 25 cally effective dose can be estimated initially from cell culture Application Publication No. 2004/116386. Methods for sta assays. A dose may be formulated in animal models to bilizing active components using polyol/polymer microcap achieve a circulating plasma concentration range that sules, and their preparation are described in US20040 108608. includes the ICs (i.e., the concentration of the test compound Processes for dissolving lipophilic compounds in aqueous 30 that achieves a half-maximal inhibition of symptoms) as solution with amphiphilic block copolymers are described in determined in cell culture. Such information can be used to WO 04/035O13. more accurately determine useful doses in humans. Levels in Conditions of the eye can be treated or prevented by, e.g., plasma may be measured, for example, by high performance systemic, topical, intraocular injection of a sirtuin-modulat liquid chromatography. ing compound, or by insertion of a Sustained release device 35 6. Kits that releases a sirtuin-modulating compound. A sirtuin Also provided herein are kits, e.g., kits for therapeutic modulating compound that increases or decreases the level purposes or kits for modulating the lifespan of cells or modu and/or activity of a sirtuin protein may be delivered in a lating apoptosis. A kit may comprise one or more sirtuin pharmaceutically acceptable ophthalmic vehicle, such that modulating compounds, e.g., in premeasured doses. A kit the compound is maintained in contact with the ocular Surface 40 may optionally comprise devices for contacting cells with the for a sufficient time period to allow the compound to penetrate compounds and instructions for use. Devices include the corneal and internal regions of the eye, as for example the Syringes, stents and other devices for introducing a sirtuin anterior chamber, posterior chamber, vitreous body, aqueous modulating compound into a Subject (e.g., the blood vessel of humor, vitreous humor, cornea, iris/ciliary, lens, choroid/ 45 a Subject) or applying it to the skin of a Subject. retina and Sclera. The pharmaceutically-acceptable oph Another type of kit contemplated by the invention are kits thalmic vehicle may, for example, be an ointment, vegetable for identifying sirtuin-modulating compounds. Such kits con oil or an encapsulating material. Alternatively, the com tain (1) a sirtuin or sirtuin-containing material and (2) a sir pounds of the invention may be injected directly into the tuin-modulating compound of the invention, which are in vitreous and aqueous humour. In a further alternative, the 50 separate vessels. Such kits can be used, for example, to per compounds may be administered systemically, such as by form a competition-type assay to test other compounds (typi intravenous infusion or injection, for treatment of the eye. cally provided by the user) for sirtuin-modulating activity. In Sirtuin-modulating compounds described herein may be certain embodiments, these kits further comprise means for stored in oxygen free environment according to methods in 55 determining sirtuin activity (e.g., a peptide with an appropri the art. For example, resveratrol or analog thereof can be ate indicator, Such as those disclosed in the Exemplification). prepared in an airtight capsule for oral administration, such as In yet another embodiment, the invention provides a com Capsugel from Pfizer, Inc. position of matter comprising a sirtruin modulator of this Cells, e.g., treated ex vivo with a sirtuin-modulating com invention and another therapeutic agent the same ones used pound, can be administered according to methods for admin 60 in combination therapies and combination compositions in istering a graft to a subject, which may be accompanied, e.g., separate dosage forms, but associated with one another. The by administration of an immunosuppressant drug, e.g., term “associated with one another as used herein means that cyclosporin A. For general principles in medicinal formula the separate dosage forms are packaged together or otherwise tion, the reader is referred to Cell Therapy: StemCell Trans 65 attached to one another Such that it is readily apparent that the plantation, Gene Therapy, and Cellular Immunotherapy, by separate dosage forms are intended to be sold and adminis G. Morstyn & W. Sheridan eds, Cambridge University Press, tered as part of the same regimen. The agent and the sirtruin US 8,178,536 B2 189 190 modulator are preferably packaged together in a blister pack (R. I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells or other multi-chamber package, or as connected, separately And Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide sealed containers (such as foil pouches or the like) that can be To Molecular Cloning (1984); the treatise, Methods In Enzy separated by the user (e.g., by tearing on score lines between mology (Academic Press, Inc., N.Y.); Gene Transfer Vectors the two containers). 5 For Mammalian Cells (J. H. Miller and M. P. Calos eds., 1987, In still another embodiment, the invention provides a kit Cold Spring Harbor Laboratory); Methods In Enzymology, comprising in separate vessels, a) a sirtruin modulator of this Vols. 154 and 155 (Wu et al. eds.), Immunochemical Methods invention; and b) another another therapeutic agent such as In Cell And Molecular Biology (Mayer and Walker, eds., those described elsewhere in the specification. 10 Academic Press, London, 1987); Handbook Of Experimental The practice of the present methods will employ, unless Immunology, Volumes I-IV (D. M. Weir and C. C. Blackwell, otherwise indicated, conventional techniques of cell biology, eds., 1986); Manipulating the Mouse Embryo, (Cold Spring cell culture, molecular biology, transgenic biology, microbi Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986). ology, recombinant DNA, and immunology, which are within Exemplification the skill of the art. Such techniques are explained fully in the 15 The invention now being generally described, it will be literature. See, for example, Molecular Cloning A Laboratory more readily understood by reference to the following Manual, 2" Ed., ed. by Sambrook, Fritsch and Maniatis examples which are included merely for purposes of illustra (Cold Spring Harbor Laboratory Press: 1989); DNA Cloning, tion of certain aspects and embodiments of the present inven Volumes I and II (D. N. Glover ed., 1985); Oligonucleotide tion, and are not intended to limit the invention in any way. Synthesis (M. J. Gait ed., 1984); Mullis et al. U.S. Pat. No: EXAMPLE 1. 4,683, 195; Nucleic Acid Hybridization (B. D. Hames & S.J. Higgins eds. 1984); Transcription And Translation (B. D. Synthesis and Characterization of Sirtuin Modulators Hames & S. J. Higgins eds. 1984); Culture Of Animal Cells General Schemes
Scheme 1: )- s.S r
He Her He C 4N HC O2 NH2 - C2 NH2 CC reflux NO O Smith et al, Sulfur Lett. 1994 vol 17, p. 197 O NO and E. Ma, Molecules 2003, vol. 8, p. 678-686
Et3N, CHCl2
NO NH2 S Sodium S A / 2N N)-() hydrosulfide N2 N -()
lsO pen- R C R
H R N-R N-KH HN-K N1,N-SO) () O N1S-SO) () O