USOO854.6432B2

(12) United States Patent (10) Patent No.: US 8,546.432 B2 Bahadoor et al. (45) Date of Patent: Oct. 1, 2013

(54) TETRAZOLONES AS INHIBITORS OF FATTY 5,339,163 A 8, 1994 Homma et al. ACID SYNTHASE 5,347,010 A 9, 1994 Goto et al. 5,362,704 A 11, 1994 Goto et al. (75) Inventors: Adilah Bahadoor, Cambridge, MA 3. A E. Mingal (US); Alfredo C. Castro, Winchester, 5,466,220 A 1 1/1995 Brenneman et al. MA (US); Lawrence K. Chan, 5,466,708 A 1 1/1995 Derungs et al. Brookline, MA (US); Gregg F. Keaney, S! A A. 3. NEN et al al Lexington, MA (US); Marta 5,520,639- W - A 5/1996 PetersonCSO et C.al. a. Nevalainen, Weymouth, MA (US); Vesa 5,527.288 A 6/1996 Gross et al. Nevalainen, Weymouth, MA (US); 5,569,189 A 10/1996 Parsons et al. Stephane Peluso, Brookline, MA (US); 5,589,439 A 12/1996 Goto et al. Daniel A. Snyder, Somerville, MA 5,599,302 A 3. 1997 Lilley et al (US); Thomas T. Tibbitts, Westford, 3229 A 32 sea MA (US) 5,649912 A 7/1997 Peterson et al. 5,650,374. A 7/1997 Goto et al. (73) Assignee: Infinity Pharmaceuticals, Inc., 5,652,198 A 7/1997 Goto et al. Cambridge, MA (US) 5,654,257 A 8/1997 Goto et al. 5,668,087 A 9, 1997 Goto et al. (*) Notice: Subject to any disclaimer, the term of this S. A 2. SE patent is extended or adjusted under 35 5,747,420 A 5/1998 Goto et al. U.S.C. 154(b) by 103 days. (Continued) (21) Appl. No.: 13/101.978 FOREIGN PATENT DOCUMENTS DE 4437197 4f1996 (22) Filed: May 5, 2011 EP O712850 5, 1996 (65) Prior Publication Data (Continued) OTHER PUBLICATIONS US 2011 FO274655A1 Nov. 10, 2011 Patani et al., Chem Rev, 1996, vol. 96 (8), especially p. 3149.* O O Adams et al., “Rabbitpox virus and vaccinia virus infection of rabbits Related U.S. Application Data as a model for human Smallpox.” JVirol. 81 (20): 11084-11095, Epub (60) Provisional application No. 61/331.575, filed on May Aug. 8, 2007. 5, 2010, provisional application No. 61/331,644, filed Agostini et al., “Fatty acid synthase is required for the proliferation of on May 5, 2010, provisional application No. human(2004) oral squamous carcinoma cells.” Oral Oncol. 40(7):728-735 61/419.174, filed on Dec. 2, 2010, provisional Allen et al., “Identification and characterization of mutations in hepa application No. 61/437.5 64, filed on Jan. 28, 2011, titis B virus resistant to lamivudine. Lamivudine Clinical Investiga provisional application No. 61/472,566, filed on Apr. tion Group.” Hepatology 27(6):1670-1677 (1998). 6, 2011. (Continued) (51) Int. Cl. in a A6CO7D IK3I/4I 249/00 (2006.01) (74)Ely Attorney, Euli Agent, f orsh Firm — cus Jones DayD (52) U.S. Cl. (57) ABSTRACT USPC ...... 514/381: 548/250 Provided herein are tetrazolone FASN inhibitors of the for (58) Field of Classification Search mula (I): USPC ...... 514/381: 548/250 See application file for complete search history. (I) (56) References Cited O O

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U.S. Patent Oct. 1, 2013 Sheet 2 of 2 US 8,546.432 B2

US 8,546,432 B2 1. 2 TETRAZOLONES AS INHIBITORS OF EATTY (See, e.g., Hepatology, 48: 1396 (2008); Trends Endocrine ACID SYNTHASE Metabol., 21:33 (2010); and Virology, 394: 130 (2009)). With regard to other various viruses, it was reported that the This application claims priority to U.S. Provisional Appli FASN expression is increased in the cells infected by cox cation Nos. 61/331,575, filed May 5, 2010: 61/331,644, filed 5 sackievirus B3(CVB3), a picornavirus, and the replication of May 5, 2010: 61/419,174, filed Dec. 2, 2010: 61/437,564, CVB3 is blocked by FASN inhibitors. (See Rassmann et al., filed Jan. 28, 2011; and 61/472,566, filed Apr. 6, 2011. The Antiviral Research, 76: 150-158 (2007)). FASN was reported contents of U.S. Provisional Application Nos. 61/331.575, to be important in lytic viral replication of Epstein-Barr virus filed May 5, 2010: 61/331,644, filed May 5, 2010: 61/437, (EBV), and it was suggested that FASN inhibition may be a 564, filed Jan. 28, 2011; and 61/472,566, filed Apr. 6, 2011 are 10 novel approach for blocking the EBV replication. (Li et al., incorporated herein in their entireties. Journal of Virology, 78(8): 4197-4206 (2004)). The role of A text file of sequence listing (SEQLIST 12928-033 FASN in the replication of dengue virus has also been impli 999.TXT, created May 4, 2011; size 20,000 bytes), filed cated. (See, e.g., Heaton et al., Proc. Natl. Acad. Sci., 107(40): electronically, is incorporated herein by reference. 17345-17350 (2010); and Samsa et al., PLoS Pathegens, 15 5(10): e1000632 (2009)). Moreover, aside from being a BACKGROUND potential target for anti-viral therapy, the role of FASN has also been implicated in diabetes or regulation of the general Fatty acid synthase (FASN) is a key enzyme for the syn wellness of the liver. (See, e.g., Wu et al., PNAS Early Edition, thesis of long-chain fatty acids from acetyl-coenzyme A www.pnas.org/cgi/doi/10.1073/pnas.1002588108 (2011)). (CoA) and malonyl-CoA that uses reduced nicotinamide Thus, there is a need for effective inhibitors of FASN, which adenine dinucleotidephosphate as a cofactor. FASN is mini can be potentially used as therapies for microbial infections, mally expressed in most normal human tissues except the including, but not limited to viral infections, or other diseases liver and adipose tissue, where it is expressed at high levels. and disorders. Since FASN expression is markedly increased in several 25 human cancers compared with the corresponding normal tis SUMMARY sue, and FASN overexpression in tumors has been associated with a poor prognosis, FASN inhibitors have long been Provided herein are tetraZolone FASN inhibitors of the viewed as potential therapeutics for the treatment of cancer. formula (I): FASN inhibitors have also shown promise in the treatment of 30 other FASN-mediated diseases, disorders or conditions, such (I) as obesity, lack of appetite control and inflammatory condi O O tions. Furthermore, FASN has been identified as a target for R NN -l - RB treatment of microbial infections. In particular, it was 35 V f N reported that fatty acid synthesis or the level of fatty acid is NRN I critical in viral pathogenesis. For example, it was reported that the formation of a novel vesicular compartment (i.e., remodelled golgi apparatus), on the Surface of which viral or a pharmaceutically acceptable form thereof; wherein the RNA replication takes place, requires fatty acid biosynthesis. 40 variables R, R and R are defined below and herein. (See Cherry et al., PLoS Pathogens, 2010): e102 (2006)). In Also provided herein are pharmaceutical compositions addition, fatty acid biosynthesis has been indentified as a comprising at least one compound of formula (I) or a phar target for anti-viral therapy using a metabolic profiling of the maceutically acceptable form thereof. Also provided herein hosts upon viral infection. (See Munger et al., Nature Bio are methods of treating cancer comprising administering at technology, 26: 1179-1186 (2008). It was also reported that 45 least one compound of formula (I) or a pharmaceutically inhibition of fatty acid biosynthesis (e.g., inhibition of fatty acceptable form thereof, or a pharmaceutical composition acid synthase) results in reduced replication of human thereof, to a subject in need thereof. Also provided herein are cytomegalomous virus (HCMV) and influenza A viruses. methods of treating microbial infections comprising admin (Id.). istering at least one compound of formula (I) or a pharma Reports establishing FASN as a valid target for the treat 50 ceutically acceptable form thereof, or a pharmaceutical com ment of viral infections are available for various viruses. For position thereof, to a subject in need thereof. example, the role of FASN has been implicated in the patho The details of additional or alternative embodiments are set genesis of an enveloped virus Such as human cytomegalo forth in the accompanying Detailed Description and Exem mous virus (HCMV), influenza A and Hepatitis C (HCV). plification as described below. Other features, objects, and (See Munger et al., Nature Biotechnology, 26: 1179-1186 55 advantages will be apparent from this description and from (2008); Syed et al., Trends in Endocrinology and Metabolism, the claims. 21: 33-40 (2009); Sakamoto et al., Nature Chemical Biology, 1: 333-337 (2005): Yang et al., Hepatology, 48: 1396-1403 SEQUENCE IDENTIFICATION NUMBERS (2008)). With regard to HCV, it was reported that an elevated level of fatty acid biosynthesis enzymes, including FASN, 60 contributes to liver Steatosis, leading to cirrhosis and hepato cellular carcinoma, upon HCV infection. (Fukusawa et al., SEQ ID NO. 1: Biol. Pharm. Bull., 29(9): 1958-1961 (2006)). HCV replica Homo sapiens FASN amino acid sequence: tion was reported to be regulated by, among others, fatty acid MEEWVIAGMSGKLPESENLOEFWDNLIGGVDMVTDDDRRWKAGLYGLPRR biosynthesis. (Kapadia et al., Proc. Natl. Acad. Sci., 102(7): 65 SGKLKDLSRFDASFFGVHPKOAHTMDPOLRLLLEVTYEAIVDGGINPDSL 2561-2566 (2005)). Other reports establishing FASN as a potential host-target against HCV have also been published. US 8,546,432 B2 3 4 - Continued - Continued RGTHTGVWVGVSGSETSEALSRDPETLVGYSMVGCORAMMANRLSFFFDF DLGLDSLMSVEVROTLERELNLVLSWREVROLTLRKLQELSSKADEASEL RGPSIALDTACSSSLMALONAYOAIHSGQCPAAIWGGINWLLKPNTSVOF ACPTPKEDGLAQQQTOLNLRSLLVNPEGPTLMRLNSWOSSERPLFLVHPI LRLGMLSPEGTCKAFDTAGNGYCRSEGWWAWLLTKKSLARRWYATILNAG EGSTTVFHSLASRLSIPTYGLOCTRAAPLDSIHSLAAYYIDCIROVOPEG TNTDGFKEOGWTFPSGDIOEOLIRSLYOSAGVAPESFEYIEAHGTGTKVG PYRVAGYSYGACVAFEMCSOLOAOOSPAPTHNSLFLFDGSPTYWLAYTOS DPOELNGITRALCATROEPLLIGSTKSNMGHPEPASGLAALAKVLLSLEH YRAKLTPGCEAEAETEAICFFVOOFTDMEHNRVLEALLPLKGLEERVAAA GLWAPNLHFHSPNPEIPALLDGRLOVVDOPLPVRGGNVGINSFGFGGSNW 10 VDLIIKSHOGLDROELSFAARSFYYKLRAAEOYTPKAKYHGNWMLLRAKT HIILRPNTOPPPAPAPHATLPRLLRASGRTPEAVOKLLEOGLRHSODLAF GGAYGEDLGADYNLSQVCDGKVSWHVIEGDHRTLLEGSGLESIISIIHSS LSMLNDIAAVPATAMPFRGYAVLGGERGGPEVOOWPAGERPLWFICSGMG LAEPRWSWREG TOWRGMGLSLMRLDRFRDSILRSDEAVKPFGLKVSOLLLSTDESTFDDIV 15 HSFWSLTAIQIGLIDLLSCMGLRPDGIWGHSLGEVACGYADGCLSOEEAV DEFINITIONS LAAYWRGOCIKEAHLPPGAMAAVGLSWEECKORCPPGVWPACHNSKDTVT Definitions of specific functional groups and chemical ISGPOAPWFEFVEOLRKEGVFAKEVRTGGMAFHSYFMEAIAPPLLOELKK terms are described in more detail below. The chemical ele ments are identified in accordance with the Periodic Table of VIREPKPRSARWLSTSIPEAOWHSSLARTSSAEYNVNNLVSPWLFOEALW the Elements, CAS version, Handbook of Chemistry and HVPEHAVVLEIAPHALLOAVLKRGLKPSCTIIPLMKKDHRDNLEFFLAGI Physics, 75" Ed., inside cover, and specific functional groups GRLHLSGIDANPNALFPPWEFPAPRGTPLISPLIKWDHSLAWDWPAAEDF are generally defined as described therein. Additionally, gen 25 eral principles of organic chemistry, as well as specific func PNGSGSPSAAIYNIDTSSESPDHYLWDHTLDGRWLFPATGYLSIWWKTLA tional moieties and reactivity, are described in Organic Chemistry, Thomas Sorrell, University Science Books, Sau RALGLGVEOLPVVFEDWWLHOATILPKTGTVSLEVRLLEASRAFEVSENG salito, 1999; Smith and March March's Advanced Organic NLVVSGKVYOWDDPDPRLFDHPESPTPNPTEPLFLAOAEWYKELRLRGYD Chemistry, 5' Edition, John Wiley & Sons, Inc., New York, 30 2001: Larock, Comprehensive Organic Transformations, YGPHFOGILEASLEGDSGRLLWKDNWWSFMDTMLOMSILGSAKHGLYLPT VCHPublishers, Inc., New York, 1989; and Carruthers, Some Modern Methods of Organic Synthesis, 3" Edition, Cam RVTAIHIDPATHROKLYTLODKAOVADVVVSRWLRVTVAGGVHISGLHTE bridge University Press, Cambridge, 1987. SAPRRQQEQOWPILEKFCFTPHTEEGCLSERAALQEELOLCKGLVOALOT Certain compounds provided herein can comprise one or 35 more asymmetric centers, and thus can exist in various iso KVTOOGLKMVWPGLDGAQIPRDPSQQELPRLLSAACRLOLNGNLOLELAQ meric forms, e.g., enantiomers and/or diastereomers and/or WLAQERPKLPEDPLLSGLLDSPALKACLDTAVENMPSLKMKWWEWLAGHG Stereoisomers. The compounds provided herein can be in the form of an individual enantiomer, diastereomer or geometric HLYSRIPGLLSPHPLLOLSYTATDRHPOALEAAQAELOOHDVAOGOWDPA isomer, or can be in the form of a mixture of Stereoisomers, DPAPSALGSADLLWCNCAWAALGDPASALSNMWAALREGGFL.LL.HTLLRG 40 including racemic mixtures and mixtures enriched in one or more Stereoisomer. In certain embodiments, the compounds HPLGDIVAFLTSTEPOYGOGILSODAWESLFSRVSLRLVGLKKSFYGSTL provided herein are enantiopure compounds. In certain other embodiments, mixtures of Stereoisomers are provided. FLCRRPTPODSPIFLPWDDTSFRWVESLKGILADEDSSRPVWLKAINCAT Furthermore, certain compounds, as described herein can SGVWGLVNCLRREPGGNRLRCWLLSNLSSTSHVPEVDPGSAELOKVLOGD 45 have one or more double bonds that can exist as either the cis or trans, or the E or Z isomer, unless otherwise indicated. Also WMNWYRDGAWGAFRHFLLEEDKPEEPTAHAFWSTLTRGDLSSIRWWCSS encompassed are the compounds as individual isomers Sub LRHAOPTCPGAOLCTVYYASLNFRDIMLATGKLSPDAIPGKWTSODSLLG stantially free of other isomers, and alternatively, as mixtures of various isomers, e.g., racemic mixtures of E/Z isomers or MEFSGRDASGKRWMGLWPAKGLATSWLLSPDFLWDWPSNWTLEEAASWPW 50 mixtures enriched in one E/Z isomer. The terms “optically enriched”, “enantiomerically WYSTAYYALVWRGRVRPGETLLIHSGSGGVGOAAIAIALSLGCRVFTTVG enriched,” “enantiomerically pure' and “non-racemic, as SAEKRAYLOARFPOLDSTSFANSRDTSFEOHVLWHTGGKGVDLVLNSLAE used interchangeably herein, refer to compositions in which the percent by weight of one enantiomer is greater than the EKLOASVRCLATHGRFLEIGKFDLSONHPLGMAIFLKNVTFHGVLLDAFF 55 amount of that one enantiomer in a control mixture of the racemic composition (e.g., greater than 1:1 by weight). In NESSADWREVWALVOAGIRDGVWRPLKCTVFHGAOVEDAFRYMAOGKHIG addition, the term “non-racemic can apply more broadly to KVVVOVLAEEPEAVLKGAKPKLMSAISKTFCPAHKSYIIAGGLGGFGLEL mixtures of stereoisomers, diastereomers or olefin E/Z iso mers. For example, an enantiomerically enriched preparation AOWLIORGWOKLVLTSRSGIRTGYOAKOVRRWRROGWOWOWSTSNISSLE 60 of the (S)-enantiomer, means a preparation of the compound GARGLIAEAAQLGPWGGWFNLAWWLRDGLLENOTPEFFODWCKPKYSGTL having greater than 50% by weight of the (S)-enantiomer relative to the (R)-enantiomer, such as at least 75% by weight, NLDRVTREACPELDYFWVFSSWSCGRGNAGOSNYGFANSAMERICEKRRH and even such as at least 80% by weight. In some embodi EGLPGLAVOWGAIGDVGILVETMSTNDTIVSGTLPORMASCLEVLDLFLN ments, the enrichment can be much greater than 80% by 65 weight, providing a “substantially optically enriched.” “sub QPHMVLSSFWLAEKAAAYRDRDSORDLVEAVAHILGIRDLAAVNLDSSLA stantially enantiomerically enriched.” “substantially enantio merically pure' or a 'substantially non-racemic' preparation, US 8,546,432 B2 5 6 which refers to preparations of compositions which have at loalkyl). In some embodiments, the alkyl moiety has 1 to 3 least 85% by weight of one enantiomer relative to other enan carbon atoms (“Cs perhaloalkyl). In some embodiments, tiomer. Such as at least 90% by weight, and Such as at least the alkyl moiety has 1 to 2 carbon atoms ("C. perha 95% by weight. In some embodiments, the enantiomerically loalkyl). In some embodiments, all of the hydrogen atoms enriched composition has a higher potency with respect to are each replaced with fluoro. In some embodiments, all of the therapeutic utility per unit mass than does the racemic mix hydrogen atoms are each replaced with chloro. Examples of ture of that composition. Enantiomers can be isolated from perhaloalkyl groups include —CF, —CFCF mixtures by methods known to those skilled in the art, includ —CFCFCF —CC1, CFC1, —CF2Cl and the like. ing chiral high pressure liquid chromatography (HPLC) and As used herein, alone or as part of another group, "alkenyl the formation and crystallization of chiral salts; or enanti 10 refers to a monoradical of a straight-chain or branched hydro omers can be prepared by asymmetric syntheses. See, for carbon group having from 2 to 10 carbon atoms and one or example, Jacques, et al., Enantiomers, Racemates and Reso more carbon-carbon double bonds (“Coalkenyl). In some lutions (Wiley Interscience, New York, 1981); Wilen, S.H., et embodiments, an alkenyl group has 2 to 9 carbon atoms al., Tetrahedron 33:2725 (1977); Eliel, E. L. Stereochemistry (“Coalkenyl). In some embodiments, an alkenyl group has of Carbon Compounds (McGraw-Hill, N.Y., 1962); and 15 2 to 8 carbon atoms (“Cs alkenyl). In some embodiments, Wilen, S. H. Tables of Resolving Agents and Optical Resolu an alkenyl group has 2 to 7 carbon atoms (“C, alkenyl). In tions p. 268 (E. L. Eliel, Ed., Univ. of Notre Dame Press, Some embodiments, an alkenyl group has 2 to 6 carbonatoms Notre Dame, Ind. 1972). (“Coalkenyl). In some embodiments, an alkenyl group has As used herein, alone or as part of another group, "halo' 2 to 5 carbon atoms (“Cs alkenyl). In some embodiments, and “halogen refer to fluorine (fluoro, —F), chlorine (chloro, an alkenyl group has 2 to 4 carbonatoms ("Calkenyl). In —Cl), bromine (bromo. —Br), or iodine (iodo. —I). Some embodiments, an alkenyl group has 2 to 3 carbonatoms As used herein, alone or as part of another group, "alkyl (“C- alkenyl). In some embodiments, an alkenyl group has refers to a monoradical of a straight-chain or branched Satu 2 carbon atoms (“C, alkenyl). The one or more carbon rated hydrocarbon group having from 1 to 10 carbon atoms carbon double bonds can be internal (such as in 2-butenyl) or (“Coalkyl). In some embodiments, an alkyl group has 1 to 25 terminal (Such as in 1-butenyl). Examples of C alkenyl 9 carbonatoms (“Coalkyl). In some embodiments, an alkyl groups include ethenyl (C), 1-propenyl (C), 2-propenyl group has 1 to 8 carbonatoms ("Cs alkyl). In some embodi (C), 1-butenyl (C), 2-butenyl (C), butadienyl (C) and the ments, an alkyl group has 1 to 7 carbon atoms (“C, alkyl). like. Examples of Calkenyl groups include the aforemen In Some embodiments, an alkyl group has 1 to 6 carbonatoms tioned Calkenyl groups as well as pentenyl (Cs), pentadi (“C. alkyl). In some embodiments, an alkyl group has 1 to 30 enyl (Cs), hexenyl (C) and the like. Additional examples of 5 carbonatoms ("Cs alkyl). In some embodiments, an alkyl alkenyl include heptenyl (C7), octenyl (Cs), octatrienyl (Cs) group has 1 to 4 carbonatoms ("Calkyl). In some embodi and the like. Unless otherwise specified, each instance of an ments, an alkyl group has 1 to 3 carbon atoms (“C- alkyl). alkenyl group is independently unsubstituted (an "unsubsti In Some embodiments, an alkyl group has 1 to 2 carbonatoms tuted alkenyl) or substituted (a “substituted alkenyl) with 1, (“C, alkyl). In some embodiments, an alkyl group has 1 35 2, 3, 4, or 5 substituents as described herein. In certain carbon atom (“C alkyl). In some embodiments, an alkyl embodiments, the alkenyl group is an unsubstituted Co group has 2 to 6 carbon atoms ("Ce alkyl). Examples of alkenyl. In certain embodiments, the alkenyl group is a Sub C. alkyl groups include methyl (C), ethyl (C), n-propyl stituted Coalkenyl. (C), isopropyl (C), n-butyl (C), tert-butyl (C), sec-butyl As used herein, alone or as part of another group, "alkynyl (C), iso-butyl (C), n-pentyl (Cs), 3-pentanyl (Cs), amyl 40 refers to a monoradical of a straight-chain or branched hydro (Cs), neopentyl (Cs), 3-methyl-2-butanyl (Cs), tertiary amyl carbon group having from 2 to 10 carbon atoms and one or (Cs), and n-hexyl (C). Additional examples of alkyl groups more carbon-carbon triple bonds (“Clio alkynyl). In some include n-heptyl (C7), n-octyl (Cs) and the like. Unless oth embodiments, an alkynyl group has 2 to 9 carbon atoms erwise specified, each instance of an alkyl group is indepen (“Coalkynyl). In some embodiments, an alkynyl group has dently unsubstituted (an "unsubstituted alky1') or substituted 45 2 to 8 carbon atoms ("Cs alkynyl). In some embodiments, (a "substituted alkyl) with 1, 2, 3, 4, or 5 substituents as an alkynyl group has 2 to 7 carbonatoms (“C-7 alkynyl). In described herein. In certain embodiments, the alkyl group is Some embodiments, an alkynyl group has 2 to 6 carbonatoms an unsubstituted Coalkyl (e.g., —CH). In certain embodi ("Ce alkynyl). In some embodiments, an alkynyl group has ments, the alkyl group is a substituted Co alkyl. 2 to 5 carbon atoms ("Cs alkynyl). In some embodiments, When a range of values is listed, it is intended to encompass 50 an alkynyl group has 2 to 4 carbonatoms ("Calkynyl). In each value and Sub-range within the range. For example “C. Some embodiments, an alkynyl group has 2 to 3 carbonatoms alkyl is intended to encompass, C1, C2, Cs, Ca, Cs, C. C. (“C- alkynyl). In some embodiments, an alkynyl group has C1-5. C-4 C-3; C-2, C2-6, C2-s: C2-4, C2-3, C3-6. C3-s: C3-4. 2 carbon atoms (“C, alkynyl). The one or more carbon C4-6. Cas, and CS-6 alkyl. carbon triple bonds can be internal (such as in 2-butynyl) or “Perhaloalkyl as defined herein refers to an alkyl group 55 terminal (such as in 1-butynyl). Examples of C alkynyl having from 1 to 10 carbonatoms wherein all of the hydrogen groups include, without limitation, ethynyl (C), 1-propynyl atoms are each independently replaced by a halogen, e.g., (C), 2-propynyl (C), 1-butynyl (C), 2-butynyl (C) and the selected from fluoro, bromo, chloro or iodo (“Co perha like. Examples of Calkenyl groups include the aforemen loalkyl). In some embodiments, the alkyl moiety has 1 to 9 tioned C. alkynyl groups as well as pentynyl (Cs), hexynyl carbon atoms (“Co perhaloalkyl). In some embodiments, 60 (Ce) and the like. Additional examples of alkynyl include the alkyl moiety has 1 to 8 carbon atoms ("Cs perha heptynyl (C7), octynyl (Cs) and the like. Unless otherwise loalkyl). In some embodiments, the alkyl moiety has 1 to 7 specified, each instance of an alkynyl group is independently carbon atoms (“C, perhaloalkyl). In some embodiments, unsubstituted (an "unsubstituted alkynyl) or substituted (a the alkyl moiety has 1 to 6 carbon atoms ("Ce perha “substituted alkynyl) with 1, 2, 3, 4, or 5 substituents as loalkyl). In some embodiments, the alkyl moiety has 1 to 5 65 described herein. In certain embodiments, the alkynyl group carbon atoms (“Cs perhaloalkyl). In some embodiments, is an unsubstituted Coalkynyl. In certain embodiments, the the alkyl moiety has 1 to 4 carbon atoms ("Ca perha alkynyl group is a Substituted Co alkynyl. US 8,546,432 B2 7 8 As used herein, alone or as part of another group, "het embodiments, the carbocyclyl group is an unsubstituted Co eroaliphatic' refers to a monoradical of an acyclic 3- to carbocyclyl. In certain embodiments, the carbocyclyl group 14-membered straight-chain or branched-chain having from is a Substituted Co carbocyclyl. 2 to 13 carbon atoms and 1 to 4 heteroatoms selected from In some embodiments, “carbocyclyl is a monocyclic, oxygen, Sulfur, phosphorous, and nitrogen, and wherein the saturated carbocyclyl group having from 3 to 10 ring carbon point of attachment is a carbon atom (“3-14 membered het atoms (“Clio cycloalkyl). In some embodiments, a eroaliphatic'). In some embodiments, "heteroaliphatic' is a cycloalkyl group has 3 to 8 ring carbon atoms ("Cs saturated group ("heteroalkyl). In some embodiments, “het cycloalkyl). In some embodiments, a cycloalkyl group has 3 eroaliphatic' is a group containing one or more double bonds to 6 ring carbon atoms (“C cycloalkyl). In some embodi ("heteroalkenyl). In some embodiments, "heteroaliphatic' 10 ments, a cycloalkyl group has 5 to 6 ring carbonatoms ("Cs. is a group containing one or more triple bonds ("heteroalky cycloalkyl). In some embodiments, a cycloalkyl group has 5 nyl'). Exemplary heteroaliphatic groups include, without to 10 ring carbon atoms ("Cso cycloalkyl). Examples of limitation, ethers Such aS methoxyethanyl Cse cycloalkyl groups include cyclopentyl (Cs) and cyclo ( CHCHOCH), ethoxymethanyl ( CHOCHCH), hexyl (Cs). Examples of C. cycloalkyl groups include the (methoxymethoxy)ethanyl ( CHCHOCHOCH), 15 aforementioned Cse cycloalkyl groups as well as cyclopropyl (methoxymethoxy)methanyl ( CHOCHOCH) and (C) and cyclobutyl (C). Examples of Css cycloalkyl groups (methoxyethoxy)methanyl ( CHOCH2CHOCH) and the include the aforementioned C. cycloalkyl groups as well as like: amines such as —CHCH-NHCH, —CH2CHN cycloheptyl (C7) and cyclooctyl (Cs). Unless otherwise speci (CH), —CH-NHCHCH. —CHN(CH2CH)(CH) and fied, each instance of a cycloalkyl group is independently the like. Unless otherwise specified, each instance of a het unsubstituted (an "unsubstituted cycloalkyl) or substituted eroaliphatic group is independently unsubstituted (an (a "substituted cycloalkyl) with 1, 2, 3, 4, or 5 substituents as “unsubstituted heteroaliphatic') or substituted (a "substituted described herein. In certain embodiments, the cycloalkyl heteroaliphatic') with 1-5 substituents as described herein. In group is an unsubstituted Co cycloalkyl. In certain embodi certain embodiments, the heteroaliphatic group is an unsub ments, the cycloalkyl group is a Substituted Co cycloalkyl. stituted 3-14 membered heteroaliphatic. In certain embodi 25 As used herein, alone or as part of another group, "hetero ments, the heteroaliphatic group is a Substituted 3-14 mem cyclyl refers to a radical of a 3- to 14-membered non-aro bered heteroaliphatic. matic ring system having ring carbon atoms and 1 to 4 ring As used herein, alone or as part of another group, 'carbocy heteroatoms, wherein each heteroatom is independently clyl refers to a radical of a non-aromatic cyclic hydrocarbon selected from nitrogen, oxygen, phosphorous, and Sulfur (3- group having from 3 to 10 ring carbonatoms ("Cocarbocy 30 14 membered heterocyclyl). In heterocyclyl groups that con clyl) and Zero heteroatoms in the non-aromatic ring system. tain one or more nitrogen orphosphorous atoms, the point of In some embodiments, a carbocyclyl group has 3 to 9 ring attachment can be a carbon, nitrogen, or phosphorous atom, carbon atoms (“Co carbocyclyl). In some embodiments, a as Valency permits. A heterocyclyl group can either be mono carbocyclyl group has 3 to 8 ring carbon atoms ("Cs car cyclic ("monocyclic heterocyclyl) or polycyclic (e.g., a bocyclyl). In some embodiments, a carbocyclyl group has 3 35 fused, bridged or spiro ring system Such as a bicyclic system to 7 ring carbonatoms ("C-7 carbocyclyl). In some embodi (“bicyclic heterocyclyl) or tricyclic system (“tricyclic het ments, a carbocyclyl group has 3 to 6 ring carbon atoms erocyclyl)), and can be saturated or can contain one or more ("Ce carbocyclyl). In some embodiments, a carbocyclyl carbon-carbon double or triple bonds. Heterocyclyl polycy group has 3 to 5 ring carbon atoms ("Cs carbocyclyl). In clic ring systems can include one or more heteroatoms in one Some embodiments, a carbocyclyl group has 3 to 4 ring car 40 or both rings. “Heterocyclyl also includes ring systems bon atoms (“Cs carbocyclyl). In some embodiments, a wherein the heterocycyl ring, as defined above, is fused with carbocyclyl group has 5 to 10 ring carbon atoms (“Cso one or more carbocycyl groups wherein the point of attach carbocyclyl). Examples of C. carbocyclyl groups include, ment is either on the carbocycyl or heterocyclyl ring, or ring without limitation, cyclopropyl (C), cyclobutyl (C), cyclo systems wherein the heterocyclyl ring, as defined above, is pentyl (Cs), cyclopentenyl (Cs), cyclohexyl (C), cyclohex 45 fused with one or more aryl or heteroaryl groups, wherein the enyl (C), cyclohexadienyl (C) and the like. Examples of point of attachment is on the heterocyclyl ring. In some Cs carbocyclyl groups include the aforementioned C. car embodiments, a heterocyclyl group is a 5-10 membered non bocyclyl groups as well as cycloheptyl (C7), cycloheptadi aromatic ring system having ring carbon atoms and 1-4 ring enyl (C7), cycloheptatrienyl (C7), cyclooctyl (Cs), bicyclo heteroatoms, wherein each heteroatom is independently 2.2.1]heptanyl, bicyclo[2.2.2]octanyl, and the like. 50 selected from nitrogen, oxygen, phosphorous, and Sulfur ("5- Examples of Cocarbocyclyl groups include the aforemen 10 membered heterocyclyl). In some embodiments, a het tioned Cls carbocyclyl groups as well as octahydro-1H-in erocyclyl group is a 5-8 membered non-aromatic ring system denyl, decahydronaphthalenyl, Spiro4.5 decanyl and the having ring carbon atoms and 1-4 ring heteroatoms, wherein like. As the foregoing examples illustrate, in certain embodi each heteroatom is independently selected from nitrogen, ments, the carbocyclyl group is either monocyclic ("mono 55 oxygen, phosphorous, and Sulfur ("5-8 membered heterocy cyclic carbocyclyl) or polycyclic (e.g., containing a fused, clyl). In some embodiments, a heterocyclyl group is a 5-6 bridged or spiro ring system Such as a bicyclic system (“bicy membered non-aromatic ring system having ring carbon clic carbocyclyl) or tricyclic system (“tricyclic carbocy atoms and 1-4 ring heteroatoms, wherein each heteroatom is clyl)) and can be saturated or can contain one or more car independently selected from nitrogen, oxygen, phosphorous, bon-carbon double or triple bonds. “Carbocyclyl also 60 and sulfur (“5-6 membered heterocyclyl). In some embodi includes ring systems wherein the carbocyclyl ring, as ments, the 5-6 membered heterocyclyl has 1-3 ring heteroa defined above, is fused with one or more aryl or heteroaryl toms selected from nitrogen, oxygen, phosphorous, and Sul groups wherein the point of attachment is on the carbocyclyl fur. In some embodiments, the 5-6 membered heterocyclyl ring. Unless otherwise specified, each instance of a carbocy has 1-2 ring heteroatoms selected from nitrogen, oxygen, clyl group is independently unsubstituted (an "unsubstituted 65 phosphorous, and Sulfur. In some embodiments, the 5-6 mem carbocyclyl) or substituted (a “substituted carbocyclyl) bered heterocyclyl has 1 ring heteroatom selected from nitro with 1, 2, 3, 4, or 5 substituents as described herein. In certain gen, oxygen, phosphorous, and Sulfur. Exemplary 3-mem US 8,546,432 B2 9 10 bered heterocyclyls containing 1 heteroatom include, without herein. In certain embodiments, the aryl group is an unsub limitation, azirdinyl, oxiranyl, and thiorenyl. Exemplary stituted Caryl. In certain embodiments, the aryl group is 4-membered heterocyclyls containing 1 heteroatom include, a Substituted Caryl. without limitation, aZetidinyl, oxetanyl and thietanyl. Exem As used herein, alone or part of another group, “aralkyl plary 5-membered heterocyclyls containing heteroatom refers to a Co alkyl group as defined herein Substituted by a include, without limitation, tetrahydrofuranyl, dihydrofura C. aryl group as defined herein, wherein the point of nyl, tetrahydrothiophenyl, dihydrothiophenyl, pyrrolidinyl, attachment is on the alkyl group ("Coaralkyl). dihydropyrrolyl and pyrrolyl-2,5-dione. Exemplary 5-mem As used herein, alone or as part of another group, "het bered heterocyclyls containing 2 heteroatoms include, with eroaryl refers to a radical of a 5-14 membered monocyclic or 10 polycyclic (e.g., bicyclic or tricyclic) aromatic ring system out limitation, dioxolanyl, Oxathiolanyl and dithiolanyl. (e.g., having 6, 10 or 14 telectrons shared in a cyclic array) Exemplary 5-membered heterocyclyls containing 3 heteroa having ring carbon atoms and 1-4 ring heteroatoms provided toms include, without limitation, triazolinyl, oxadiazolinyl, in the aromatic ring system, wherein each heteroatom is inde and thiadiazolinyl. Exemplary 6-membered heterocyclyl pendently selected from nitrogen, oxygen, phosphorous, and groups containing 1 heteroatom include, without limitation, 15 sulfur (“5-14 membered heteroaryl). In heteroaryl groups piperidinyl, tetrahydropyranyl, dihydropyridinyl, and that contain one or more nitrogen orphosphorous atoms, the thianyl. Exemplary 6-membered heterocyclyl groups con point of attachment can be a carbon, phosphorous or nitrogen taining 2 heteroatoms include, without limitation, piperazi atom, as Valency permits. Heteroaryl polycyclic ring systems nyl, morpholinyl, dithianyl, and dioxanyl. Exemplary can include one or more heteroatoms in one or both rings. 6-membered heterocyclyl groups containing 2 heteroatoms “Heteroaryl also includes ring systems wherein the het include, without limitation, triaZinanyl. Exemplary 7-mem eroaryl ring, as defined above, is fused with one or more aryl bered heterocyclyl groups containing 1 heteroatom include, groups wherein the point of attachment is either on the aryl or without limitation, azepanyl, oxepanyl and thiepanyl. Exem on the heteroaryl ring, or wherein the heteroaryl ring, as plary 8-membered heterocyclyl groups containing 1 heteroa defined above, is fused with one or more carbocycyl or het tom include, without limitation, azocanyl, oxecanyl and thio 25 erocycyl groups wherein the point of attachment is on the canyl. Exemplary bicyclic heterocyclyl groups include, heteroaryl ring. For polycyclic heteroaryl groups wherein one without limitation, indolinyl, isoindolinyl, dihydrobenzo ring does not contain a heteroatom (e.g., indolyl, quinolinyl, furanyl, dihydrobenzothienyl, tetrahydrobenzothienyl, tet carbazolyl and the like) the point of attachment can be on rahydrobenzofuranyl, tetrahydroindolyl, tetrahydroquinoli either ring, i.e., either the ring bearing a heteroatom (e.g., nyl, tetrahydroisoquinolinyl, decahydroquinolinyl, 30 2-indolyl) or the ring that does not contain a heteroatom (e.g., decahydroisoquinolinyl, octahydrochromenyl, octahydroiso 5-indolyl). In some embodiments, a heteroaryl group is a 5-10 chromenyl, decahydronaphthyridinyl, decahydro-1.8-naph membered aromatic ring system having ring carbon atoms thyridinyl, octahydropyrrolo3.2-bipyrrole, indolinyl, and 1-4 ring heteroatoms provided in the aromatic ring sys phthalimidyl, naphthalimidyl, chromanyl, chromenyl, tem, wherein each heteroatom is independently selected from 35 nitrogen, oxygen, phosphorous, and Sulfur ('5-10 membered 1H-benzoe 1.4 diazepinyl, 1,4,5,7-tetrahydropyrano3,4- heteroaryl'). In some embodiments, a heteroaryl group is a bipyrrolyl, 5,6-dihydro-4H-furo3.2-bipyrrolyl, 6,7-dihy 5-8 membered aromatic ring system having ring carbon dro-5H-furo3.2-bipyranyl, 5,7-dihydro-4H-thieno2.3-c. atoms and 1-4 ring heteroatoms provided in the aromatic ring pyranyl, 2,3-dihydro-1H-pyrrolo2,3-bipyridinyl, 2.3- system, wherein each heteroatom is independently selected dihydrofuro2,3-bipyridinyl. 4,5,6,7-tetrahydro-1H-pyrrolo 40 from nitrogen, oxygen, phosphorous, and Sulfur ("5-8 mem 2,3-bipyridinyl, 4,5,6,7-tetrahydro furo3.2-cpyridinyl, bered heteroaryl'). In some embodiments, a heteroaryl group 4,5,6,7-tetrahydrothieno 3.2-bipyridinyl, 1,2,3,4-tetrahydro is a 5-6 membered aromatic ring system having ring carbon 1,6-naphthyridinyl, and the like. Unless otherwise specified, atoms and 1-4 ring heteroatoms provided in the aromatic ring each instance of heterocyclyl is independently unsubstituted system, wherein each heteroatom is independently selected (an “unsubstituted heterocyclyl) or substituted (a “substi 45 from nitrogen, oxygen, phosphorous, and Sulfur ("5-6 mem tuted heterocyclyl) with 1, 2, 3, 4, or 5 substituents as bered heteroaryl'). In some embodiments, the 5-6 membered described herein. In certain embodiments, the heterocyclyl heteroaryl has 1-3 ring heteroatoms selected from nitrogen, group is an unsubstituted 3-14 membered heterocyclyl. In oxygen, phosphorous, and Sulfur. In some embodiments, the certain embodiments, the heterocyclyl group is a Substituted 5-6 membered heteroaryl has 1-2 ring heteroatoms selected 3-14 membered heterocyclyl. 50 from nitrogen, oxygen, phosphorous, and Sulfur. In some As used herein, alone or as part of another group, 'aryl embodiments, the 5-6 membered heteroaryl has 1 ring het refers to a radical of a monocyclic or polycyclic (e.g., bicyclic eroatom selected from nitrogen, oxygen, phosphorous, and or tricyclic) aromatic ring system (e.g., having 6, 10 or 14 Ju sulfur. Exemplary 5-membered heteroaryls containing 1 het electrons shared in a cyclic array) having 6-14 ring carbon eroatom include, without limitation, pyrrolyl, furanyl and atoms and Zero heteroatoms provided in the aromatic ring 55 thiophenyl. Exemplary 5-membered heteroaryls containing 2 system ("Caryl). In some embodiments, an aryl group heteroatoms include, without limitation, imidazolyl pyra has 6 ring carbon atoms ("Caryl', e.g., phenyl). In some Zolyl, oxazolyl, isoxazolyl, thiazolyl, and isothiazolyl. Exem embodiments, an aryl group has 10 ring carbon atoms (“Co plary 5-membered heteroaryls containing 3 heteroatoms aryl'; e.g., naphthyl, Such as 1-naphthyl and 2-maphthyl). In include, without limitation, triazolyl, oxadiazolyl, and thia Some embodiments, an aryl group has 14 ring carbon atoms 60 diazolyl. Exemplary 5-membered heteroaryls containing 4 ("Caryl”; e.g., anthracyl). 'Aryl also includes ring sys heteroatoms include, without limitation, tetrazolyl. Exem tems wherein the aryl ring, as defined above, is fused with one plary 6-membered heteroaryls containing 1 heteroatom or more carbocyclyl or heterocyclyl groups wherein the radi include, without limitation, pyridinyl. Exemplary 6-mem cal or point of attachment is on the aryl ring. Unless otherwise bered heteroaryls containing 2 heteroatoms include, without specified, each instance of an aryl group is independently 65 limitation, pyridazinyl, pyrimidinyl and pyrazinyl. Exem unsubstituted (an "unsubstituted aryl') or substituted (a “sub plary 6-membered heteroaryls containing 3 or 4 heteroatoms stituted aryl') with 1, 2, 3, 4, or 5 substituents as described include, without limitation, triazinyl and tetrazinyl, respec US 8,546,432 B2 11 12 tively. Exemplary 7 membered heteroaryls containing 1 het OR, OC( NR)R*, OC( NR)OR*, eroatom include, without limitation, azepinyl, oxepinyl and C(—NR)N(R), OC(—NR)N(R), NRC thiepinyl. Exemplary 5,6-bicyclic heteroaryls include, with (—NR)N(R), C(=O)NR'SOR, NRSOR", out limitation, indolyl, isoindolyl, indazolyl, benzotriazolyl, - SON(R) - SOR", SOOR", OSOR", benzothiophenyl, isobenzothiophenyl, benzofuranyl, ben S(=O)R, OS(=O)R', Si(R), OSi(R), Zoisofuranyl, benzimidazolyl, benzoxazolyl, benzisoxazolyl, C(—S)N(R), C(=O)SR", C(-S)SR', SC(S) benzoxadiazolyl, benzthiazolyl, benzisothiazolyl, benzthia SR', P(=O).R., OP(=O).R., P(=O)(R), diazolyl, indolizinyl, and purinyl. Exemplary 6,6-bicyclic – OP(=O)(R), OP(=O)(OR), P(=O)N(R), heteroaryls include, without limitation, naphthyridinyl, pte – OP(=O)N(R), P(=O)(NR), OP(=O) ridinyl, quinolinyl, isoquinolinyl, cinnolinyl, quinoxalinyl, 10 (NR), NRP(=O)(OR), NRP(=O)(NR), phthalazinyl and quinazolinyl. Exemplary tricyclic heteroar - P(R) P(R), OP(R), OP(R), B(OR), yls include, without limitation, phenanthridinyl, dibenzofura or BR(OR), —O, —S, —NN(R) =NNRC(O) nyl, carbazolyl, acridinyl, phenothiazinyl, phenoxazinyl and R*, NNRCOR, NNRS(O).R., NR, phenazinyl. Unless otherwise specified, each instance of a —NOR, Co alkyl, Co perhaloalkyl, Co alkenyl, heteroaryl group is independently unsubstituted (an "unsub 15 Coalkynyl, 3-14 membered heteroaliphatic, Cocarbocy stituted heteroaryl') or substituted (a "substituted het clyl. 3-14 membered heterocyclyl, C-aryl, and 5-14 mem eroaryl') with 1, 2, 3, 4, or 5 substituents as described herein. bered heteroaryl, wherein each alkyl, alkenyl, alkynyl, car In certain embodiments, the heteroaryl group is an unsubsti bocyclyl, heterocyclyl, aryl, and heteroaryl is independently tuted 5-14 membered heteroaryl. In certain embodiments, the unsubstituted or substituted with 1-5 R“groups; heteroaryl group is a substituted 5-14 membered heteroaryl. wherein: As used herein, alone or part of another group, "het each instance of R' is, independently, selected from Co. eroaralkyl refers to a Co alkyl group as defined herein alkyl, Co perhaloalkyl, Coalkenyl, Co alkynyl, 3-14 substituted by a 5-14 membered heteroaryl group as defined membered heteroaliphatic, Co carbocyclyl, 3-14 mem herein, wherein the point of attachment is on the alkyl group bered heterocyclyl, C. aryl, and 5-14 membered het (“Coheteroaralkyl). 25 eroaryl, wherein eachalkyl, alkenyl, alkynyl, heteroaliphatic, As used herein, a “covalent bond' or “direct bond refers to carbocyclyl, heterocyclyl, aryl, and heteroaryl is indepen a single bond joining two groups. dently unsubstituted or substituted with 1-5 R“groups; As used herein, the term “partially unsaturated refers to a each instance of R” is, independently, selected from ring moiety that includes at least one double or triple bond. hydrogen, OH, -OR", N(R) - CN, C(=O)R“, The term “partially unsaturated' is intended to encompass 30 C(=O)N(R), COR', -SOR", C(=NR) rings having multiple sites of unsaturation, but is not intended OR", C(=NR)N(R), SON(R), SOR, to include aryl or heteroaryl moieties, as herein defined. SOOR*, SOR", C(-S)N(R), C(=O)SR*, As used herein a "divalent group, such as a divalent alkyl, C(=S)SR°, -P(=O).R.", -P(=O)(R) - P(=O), divalent alkenyl, divalent alkynyl, divalent heteroaliphatic, N(R) - P(=O)CNR), Co alkyl, Co perhaloalkyl, divalent carbocyclyl, divalent heterocyclyl divalent aryl or 35 Coalkenyl, Co alkynyl, 3-14 membered heteroaliphatic, divalent heteroaryl group, refers to a bis-radical of the group, Clo carbocyclyl, 3-14 membered heterocyclyl, Caryl, as defined herein. and 5-14 membered heteroaryl, or two R' groups are joined Monovalent or divalent alkyl, alkenyl, alkynyl, het to form a 3-14 membered heterocyclyl or 5-14 membered eroaliphatic, carbocyclyl, heterocyclyl, aryl and heteroaryl heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, het groups, as defined herein, are either “substituted” or “unsub 40 eroaliphatic, carbocyclyl, heterocyclyl, aryl, and heteroaryl is stituted alkyl, “substituted” or “unsubstituted alkenyl, independently unsubstituted or substituted with 1-5 R' “substituted' or “unsubstituted alkynyl, “substituted” or groups: “unsubstituted heteroaliphatic, “substituted” or “unsubsti each instance of R is, independently, selected from tuted carbocyclyl, “substituted' or “unsubstituted hetero hydrogen, Coalkyl, C-operhaloalkyl, Coalkenyl, Co cyclyl, “substituted” or “unsubstituted aryl or “substituted 45 alkynyl, 3-14 membered heteroaliphatic, Co carbocyclyl, or “unsubstituted heteroaryl groups. In general, the term 3-14 membered heterocyclyl, C-aryl, and 5-14 membered “Substituted” means that at least one hydrogen present on a heteroaryl, or two R* groups are joined to form a 3-14 mem group (e.g., a carbon or nitrogen atom, etc.) is replaced with bered heterocyclyl or 5-14 membered heteroaryl ring, a permissible Substituent, e.g., a Substituent which upon Sub wherein eachalkyl, alkenyl, alkynyl, heteroaliphatic carbocy stitution results in a stable compound, e.g., a compound 50 clyl, heterocyclyl, aryl, and heteroaryl is independently which does not spontaneously undergo transformation Such unsubstituted or substituted with 1-5 R“groups; as by rearrangement, cyclization, elimination, or other reac each instance of R" is, independently, selected from halo tion. Unless otherwise indicated, a “substituted’ group can gen, —CN, NO, N, SOH, -SOH, -OH, have a substituent at one or more substitutable positions of the OR, ON(R), N(R), N(OR)R, SH, group, and when more than one position in any given struc 55 - SR, SSR, C(O)R, COH, COR, OC ture is substituted, the substituent is either the same or differ (O)R, OCOR, C(O)N(R), OC(O)N(R), ent at each position. A group referred to as “not hydrogen indicates that the group is an exemplary and permissible substituent as described herein. Exemplary substituents include, but are not limited to, 60 (RV), NRSO.R., SON(RV), SOR, SOOR, halogen (i.e., fluoro ( F), bromo (—Br), chloro (-Cl), and OSOR, SOR, Si(R), OSi(R), C(S)N iodo ( I)), —CN, NO. —N. —SOH, -SOH, -OH, (R), C(O)SR, C(S)SR, SC(S)SR, P(O),R, OR, ON(R), N(R), N(OR)R, SH, -P(O)(R), OP(O)(R), OP(O)(OR), =O, =S, SR', SSR, C(=O)R, COH, CHO, C. alkyl, Ce perhaloalkyl, C- alkenyl, C-alkynyl, 3-14 C(OR), COR", OC(=O)R", OCOR', 65 membered heteroaliphatic, Co carbocyclyl, 3-10 mem bered heterocyclyl, Co aryl, 5-10 membered heteroaryl, wherein each alkyl, alkenyl, alkynyl, heteroaliphatic, car US 8,546,432 B2 13 14 bocyclyl, heterocyclyl, aryl, and heteroaryl is independently acid addition salts are salts of an amino group formed with unsubstituted or substituted with 1-5 R38 groups: inorganic acids such as hydrochloric acid, hydrobromic acid, each instance of R is, independently, selected from C. phosphoric acid, Sulfuric acid and perchloric acid or with alkyl, Ce perhaloalkyl, C- alkenyl, C- alkynyl, 3-14 organic acids such as acetic acid, oxalic acid, maleic acid, membered heteroaliphatic, Cocarbocyclyl, Co aryl, 3-10 tartaric acid, citric acid, Succinic acid or malonic acid or by membered heterocyclyl, and 3-10 membered heteroaryl, using other methods used in the art Such as ion exchange. wherein each alkyl, alkenyl, alkynyl, heteroaliphatic, car Other pharmaceutically acceptable salts include adipate, algi bocyclyl, heterocyclyl, aryl, and heteroaryl is independently nate, ascorbate, aspartate, benzenesulfonate, benzoate, bisul fate, borate, butyrate, camphorate, camphorsulfonate, citrate, unsubstituted or substituted with 1-5 R38 groups: cyclopentanepropionate, digluconate, dodecylsulfate, each instance of Ris, independently, selected from hydro 10 ethanesulfonate, formate, fumarate, glucoheptonate, glycero gen, Calkyl, C. perhaloalkyl, C2-alkenyl, C2-alkynyl, phosphate, gluconate, hemisulfate, heptanoate, hexanoate, 3-14 membered heteroaliphatic, Co carbocyclyl, 3-10 hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lac membered heterocyclyl, C-aryl and 5-10 membered het tate, laurate, lauryl Sulfate, malate, maleate, malonate, meth eroaryl, or two R' groups are joined to forma3-14 membered anesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, heterocyclyl or 5-14 membered heteroaryl ring, wherein each 15 oleate, oxalate, palmitate, pamoate, pectinate, persulfate, alkyl, alkenyl, alkynyl, heteroaliphatic, carbocyclyl, hetero 3-phenylpropionate, phosphate, picrate, pivalate, propionate, cyclyl, aryl, and heteroaryl is independently unsubstituted or Stearate. Succinate, Sulfate, tartrate, thiocyanate, p-toluene substituted with 1-5 R groups; and Sulfonate, undecanoate, Valerate salts, and the like. Salts each instance of R$8 is, independently, halogen, —CN, derived from appropriate bases include alkali metal, alkaline NO, N, —SOH, -SOH, -OH, —OC alkyl, earth metal, ammonium and —N(Calkyl) salts. Repre —ON(C. alkyl). —N(C. alkyl). —N(OC alkyl)(C- sentative alkali or alkaline earth metal salts include Sodium, alkyl). —N(OH)(C. alkyl). —NH(OH), —SH, S(C. lithium, potassium, calcium, magnesium, and the like. Fur alkyl). —SS(C. alkyl). —C(O)(C. alkyl). —COH, ther pharmaceutically acceptable salts include, when appro —CO(C- alkyl). —OC(O)(C. alkyl). —OCO(C- priate, nontoxic ammonium, quaternary ammonium, and alkyl), —C(O)NH2. —C(O)N(C. alkyl). —OC(O)NH 25 amine cations formed using counterions such as halide, (C. alkyl). - NHC(O)(C. alkyl). —N(C. alkyl)C(O) hydroxide, carboxylate, Sulfate, phosphate, nitrate, lower (C. alkyl). —NHCO(C- alkyl). —NHC(O)N alkyl Sulfonate and aryl Sulfonate. (C. alkyl). —NHC(O)NH(C. alkyl). - NHC(O)NH, In certain embodiments, the pharmaceutically acceptable —C(NH)O(C- alkyl), OC(NH)(C. alkyl), OC(NH) form thereof is a hydrate or solvate. As used herein, the term OC alkyl, —C(NH)N(C. alkyl). —C(NH)NH(C. 30 "hydrate” refers to a compound non-covalently associated alkyl), C(NH)NH OC(NH)N(C. alkyl). OC(NH) with one or more molecules of water, which in some embodi NH(C. alkyl), OC(NH)NH, NHC(NH)N ments can be crystalline. Likewise, “solvate” refers to a com (C. alkyl). - NHC(NH)NH, NHSO(C- alkyl). pound non-covalently associated with one or more molecules —SON(C. alkyl). —SONHC alkyl). —SONH2, of an organic solvent, which in Some embodiments can be —SOC alkyl, -SOOC alkyl, —OSOC alkyl, 35 crystalline. —SOC alkyl, - Si(C. alkyl). —OSi(C. alkyl). In certain embodiments, the pharmaceutically acceptable —C(S)N(C. alkyl), —C(S)NH(C. alkyl), —C(S)NH2, form thereof is a prodrug. As used herein, the term “prodrug —C(O)S(C. alkyl). —C(S)SC alkyl, - SC(S)SC refers to a derivative of a parent compound that requires alkyl, -P(O)(C. alkyl). —P(O)(C. alkyl). —OP(O) transformation within the body in order to release the parent (C. alkyl). —OP(O)(OC alkyl), C alkyl, C. perha 40 compound. loalkyl, Ce alkenyl, C- alkynyl, 3-14 membered het The term “prodrug” refers to compounds that are trans eroaliphatic, Co carbocyclyl, Co aryl, 3-10 membered formed in vivo to yield a disclosed compound or a pharma heterocyclyl, 5-10 membered heteroaryl, =O or =S. ceutically acceptable form of the compound. The transforma These and other exemplary substituents are described in tion can occur by various mechanisms, such as, but not more detail in the Detailed Description, the Exemplification 45 limited to, through hydrolysis in blood. In certain cases, a and in the claims. The term “substituents’ is not intended to prodrug has improved physical and/or delivery properties be limited in any manner by the above exemplary listing of over the parent compound. Prodrugs are typically designed to Substituents. enhance pharmaceutically and/or pharmacokinetically based As used herein, a “pharmaceutically acceptable form properties associated with the parent compound. Exemplary thereof includes pharmaceutically acceptable salts, 50 advantages of a prodrug can include, but are not limited to, its hydrates, Solvates, prodrugs, tautomers, isomers, and/or poly physical properties, such as enhanced water solubility for morphs of a compound provided herein, as defined below and parenteral administration at physiological pH compared to herein. the parent compound, or it enhances absorption from the In certain embodiments, the pharmaceutically acceptable digestive tract, or it can enhance drug stability for long-term form thereof is a pharmaceutically acceptable salt. As used 55 Storage. herein, the term “pharmaceutically acceptable salt” refers to For example, if a disclosed compound or a pharmaceuti those salts which are, within the scope of Sound medical cally acceptable form of the compound contains a carboxylic judgment, Suitable for use in contact with the tissues of acid functional group, a prodrug can comprise an ester humans and lower animals without undue toxicity, irritation, formed by the replacement of the hydrogenatom of the acid allergic response and the like, and are commensurate with a 60 group with a group Such as (C-Cs)alkyl, (C-C)alkanoy reasonable benefit/risk ratio. Pharmaceutically acceptable loxymethyl, 1-(alkanoyloxy)ethyl having from 4 to 9 carbon salts are well known in the art. For example, Berge et al. atoms, 1-methyl-1-(alkanoyloxy)-ethyl having from 5 to 10 describes pharmaceutically acceptable salts in detail in J. carbon atoms, alkoxycarbonyloxymethyl having from 3 to 6 Pharmaceutical Sciences (1977) 66:1-19. Pharmaceutically carbon atoms, 1-(alkoxycarbonyloxy)ethyl having from 4 to acceptable salts of the compounds provided herein include 65 7 carbon atoms, 1-methyl-1-(alkoxycarbonyloxy)ethyl hav those derived from Suitable inorganic and organic acids and ing from 5 to 8 carbon atoms, N-(alkoxycarbonyl)aminom bases. Examples of pharmaceutically acceptable, nontoxic ethyl having from 3 to 9 carbon atoms, 1-(N-(alkoxycarbo US 8,546,432 B2 15 16 nyl)amino)ethyl having from 4 to 10 carbon atoms, formation and crystallization of chiral salts, or prepared by 3-phthalidyl, 4-crotonolactonyl, gamma-butyrolacton-4-yl, asymmetric syntheses. See, for example, Enantiomers, Race di-N,N-(C-C)alkylamino(C-C)alkyl (such as (3-dim mates and Resolutions (Jacques, Ed., Wiley InterScience, ethylaminoethyl), carbamoyl-(C-C)alkyl, N,N-di(C-C) New York, 1981); Wilen et al., Tetrahedron 33:2725 (1977): alkylcarbamoyl-(C-C)alkyl and piperidino-, pyrrolidino Stereochemistry of Carbon Compounds (E. L. Eliel, Ed., or morpholino(C-C)alkyl. McGraw-Hill, N.Y., 1962); and Tables of Resolving Agents Similarly, if a disclosed compound or a pharmaceutically and Optical Resolutions p. 268 (E. L. Eliel, Ed., Univ. of Notre Dame Press, Notre Dame, Ind. 1972). acceptable form of the compound contains an alcohol func In certain embodiments, the pharmaceutically acceptable tional group, a prodrug can be formed by the replacement of form thereof is a polymorph. As used herein, "polymorph” the hydrogen atom of the alcohol group with a group Such as 10 refers to a compound having more than one crystal structure, (C-C)alkanoyloxymethyl, 1-((C-C)alkanoyloxy)ethyl, e.g., resulting from differences in molecular packing and/or 1-methyl-1-((C-C)alkanoyloxy)ethyl (C-C)alkoxycarbo molecular conformation of the compound in the Solid state. nyloxymethyl, N-(C-C)alkoxycarbonylaminomethyl, The disclosure also embraces isotopically labeled com Succinoyl, (C-C)alkanoyl, C.-amino(C-C)alkanoyl, aryla pounds which are identical to those recited herein, except that cyl and C-aminoacyl, or C.-aminoacyl-C-aminoacyl, where 15 one or more atoms are replaced by an atom having an atomic each C-aminoacyl group is independently selected from the mass or mass number different from the atomic mass or mass naturally occurring L-amino acids, P(O)(OH), —P(O)(O number usually found in nature. Examples of isotopes that (C-C)alkyl) or glycosyl (the radical resulting from the can be incorporated into disclosed compounds include iso removal of a hydroxyl group of the hemiacetal form of a topes of hydrogen, carbon, nitrogen, oxygen, phosphorus, carbohydrate). fluorine and chlorine, such as H, H, C, C, N, O, O, If a disclosed compound or a pharmaceutically acceptable P. P. SS, F, and C1, respectively. form of the compound incorporates an amine functional Certain isotopically-labeled disclosed compounds (e.g., group, a prodrug can be formed by the replacement of a those labeled with Hand ''C) are useful in compound and/or hydrogen atom in the amine group with a group Such as substrate tissue distribution assays. Tritiated (i.e., H) and R-carbonyl, RO-carbonyl, NRR'-carbonyl where RandR'are 25 carbon-14 (i.e., ''C) isotopes can allow for ease of prepara each independently (C-Co.)alkyl, (C-C)cycloalkyl, ben tion and detectability. Further, substitution with heavier iso topes such as deuterium (i.e., H) can afford certain therapeu Zyl, or R-carbonyl is a natural O-aminoacyl or natural C-ami tic advantages resulting from greater metabolic stability (e.g., noacyl-natural C-aminoacyl, C(OH)C(O)OY' wherein Y is increased in Vivo half-life or reduced dosage requirements). H, (C-C)alkyl or benzyl, -C(OY)Y wherein Y is (C- Isotopically labeled disclosed compounds can generally be C.) alkylandY is (C-C)alkyl, carboxy(C-C)alkyl, amino 30 prepared by following procedures analogous to those dis (C-C)alkyl or mono-N or di-N,N-(C-C)alkylami closed in the Exemplification section herein by substituting noalkyl, C(Y)Y wherein Y is H or methyl and Y is an isotopically labeled reagent for a non-isotopically labeled mono-N- or di-N,N-(C-C)alkylamino, morpholino, pip reagent. eridin-1-yl or pyrrolidin-1-yl. In certain embodiments, the pharmaceutically acceptable 35 DETAILED DESCRIPTION form thereof is a tautomer. As used herein, the term “tau tomer' includes two or more interconvertable compounds 1. Brief Description of Figures resulting from at least one formal migration of a hydrogen atom and at least one change in Valency (e.g., a single bond to FIG. 1 illustrates a schematic diagram of Subcutaneous a double bond, a triple bond to a single bond, or vice versa). 40 mouse Xenograft model for assaying papillomaviruses. The exact ratio of the tautomers depends on several factors, FIG. 2 illustrates a schematic diagram of cutaneous mouse including temperature, solvent, and pH. Tautomerizations Xenograft model for assaying papillomaviruses. (i.e., the reaction providing a tautomeric pair) can be cata lyzed by acid or base, or can occur without the action or 2. Compounds presence of an external agent. Exemplary tautomerizations 45 include, but are not limited to, keto-to-enol; amide-to-imide; Without being limited by a particular theory, the present lactam-to-lactim; enamine-to-imine; and enamine-to-(a dif disclosure is based on the discovery that tetrazolones are ferent) enamine tautomerizations. inhibitors of human fatty acid synthase (FASN) and thus are In certain embodiments, the pharmaceutically acceptable useful in the treatment of FASN-mediated diseases, disorders form thereof is an isomer. As used herein, the term "isomer' 50 or conditions. Further, without being limited by a particular includes any and all geometric isomers and stereoisomers. theory, in certain embodiments, the compounds provided For example, "isomers' include cis- and trans-isomers, E herein can inhibit long chain fatty acid elongase (ELOVL) and Z-isomers, R- and S-enantiomers, diastereomers, (D)- such as ELOVL 6. Thus, in some embodiments, compounds isomers, (L)-isomers, racemic mixtures thereof, and other provided herein are useful in the treatment of ELOVL-medi mixtures thereof, as falling within the scope of this disclosure. 55 ated diseases, disorders or conditions. For instance, an isomer?enantiomer can, in Some embodi For example, in one aspect, provided herein is a compound ments, be provided substantially free of the corresponding of formula (I): enantiomer, and may also be referred to as "optically enriched.” “Optically-enriched, as used herein, means that (I) the compound is made up of a significantly greater proportion 60 O of one enantiomer. In certain embodiments, the compound O provided herein is made up of at least about 90% by weight of one enantiomer. In other embodiments the compound is made s--\ F N RB up of at least about 95%, 98%, or 99% by weight of one NFN I enantiomer. Enantiomers can be isolated from racemic mix 65 tures by any method known to those skilled in the art, includ ing chiral high pressure liquid chromatography (HPLC), the US 8,546,432 B2 17 18 or a pharmaceutically acceptable form thereof; membered heteroaliphatic, Co carbocyclyl, 3-14 mem wherein: bered heterocyclyl, C. aryl, and 5-14 membered het R is selected from Cocarbocyclyl, 3-14 membered het eroaryl; each instance of R is, independently, selected from erocyclyl, C-aryland 5-14 membered heteroaryl; hydrogen, OH, -OR.", N(R), —CN, -C(=O) R’ is selected from Co alkyl, Coalkenyl, Co alky RC', C(=O)N(RC), CORC, SORC, nyl, 3-14 membered heteroaliphatic, Co carbocyclyl, 3-14 C(-NR)OR', C(NRC)N(R), SON(R), membered heterocyclyl, C-aryl and 5-14 membered het SORC, SOORC, SORC, C(S)N(RC), eroaryl; C(O)SR, C(—S)SR, P(=O).R.", P(=O) R is selected from hydrogen, OH, -OR.", (R') - P(=O)N(R), -P(=O)(NR), Co alkyl, - ON(R), N(R), C(=O)R', CHO, 10 C2-o perhaloalkyl, Coalkenyl, C2-io alkynyl, 3-14 mem CORC, C(=O)N(R), C( NRC2)ORC, bered heteroaliphatic, Cocarbocyclyl, 3-14 membered het C(-NR)N(R), SOR', S(–O)R', Si erocyclyl, C-aryl, and 5-14 membered heteroaryl, or two (R)s. C1-io alkyl, C-10 perhaloalkyl, C2-io alkenyl, Co R groups are joined to form a 3-14 membered heterocyclyl alkynyl, 3-14 membered heteroaliphatic, Co carbocyclyl, or 5-14 membered heteroaryl ring: 15 or R and R together with the nitrogen (N) atom to which 3-14 membered heterocyclyl, C-aryl, and 5-14 membered each is attached are joined to form a 5-14 membered ring; heteroaryl; wherein: wherein: R’ is substituted with the group: each instance of R is, independently, selected from Co alkyl, Co perhaloalkyl, Coalkenyl, C2-io alkynyl, 3-14 wherein: membered heteroaliphatic, Co carbocyclyl, 3-14 mem L is a covalent bond or a divalent Cohydrocarbon chain, bered heterocyclyl, C. aryl, and 5-14 membered het wherein one, two or three methylene units of L are optionally eroaryl; and and independently replaced with one or more —O— —S , each instance of R is, independently, selected from NR , -(C—NR) C(=O) , C(=S) , hydrogen, OH, -OR.", N(R), —CN, -C(=O) * - S(=O) , S(=O) , divalent carbocyclyl, divalent RC, C(=O)N(RC), CORC, SORC, C(NRC) heterocyclyl, divalent aryl or divalent heteroaryl group; ORC1, C(-NR)N(RC), SON(RC). SORC, RP is selected from CN, NO, N, SOH, SOOR, SOR', C(—S)N(R), C(=O)SR, - SOH, C(=O)R’7, COH, CHO, C(OR), C(—S)SRC, P(=O).R.", - P(=O)(R'), —P(=O)N(R) -P(=O)(NR), Co alkyl, Co 30 perhaloalkyl, Coalkenyl, Co alkynyl, 3-14 membered heteroaliphatic, Cocarbocyclyl, 3-14 membered heterocy clyl, Caryl, and 5-14 membered heteroaryl, or two R. C( NR)N(R), OC( NR)N(R), NRC groups are joined to form a 3-14 membered heterocyclyl or ( NR)N(R), C(O)NR'SOR7, 35 NRSOR7, SON(R). SOR7, SOOR7, 5-14 membered heteroaryl ring: OSOR7, S(=O)R’7, OS(–O)R’7, C(—S)N or RandR together with the nitrogen (N) atom to which (R), C(=O)SR 7, C(—S)SR', SC( S)SR-7, each is attached are joined to form a 5-14 membered hetero P(=O)R’, OP(=O)R’, P(=O)(R7), OP cyclyl or heteroaryl ring. (—O)(R7), OP(=O)(OR), P(=O)N(R), In one embodiment, provided herein is a compound of 40 OP(=O)N(R), P(=O)(NR), OP(=O) formula (I): (NR), NRP(O)(OR), NRP( -O)(NR), –B(OR), BR7(OR), and tetrazolyl: each instance of R is, independently, selected from Co (I) O alkyl, Co perhaloalkyl, Coalkenyl, Co alkynyl, 3-14 45 membered heteroaliphatic, Co carbocyclyl, 3-14 mem R bered heterocyclyl, C. aryl, and 5-14 membered het NN lf- B eroaryl; YN-R NFN I each instance of R is, independently, selected from RC hydrogen, OH, OR7, N(R), CN, C(=O)R’7, 50 C(=O)N(R), COR7, SOR7, C(-NR) OR7, C(—NR)N(R), SON(R), SOR, or a pharmaceutically acceptable form thereof; SOOR, SOR7, C(-S)N(R), C(=O)SR, wherein: C(—S)SR, P(=O)R’7, P(=O)(R7), P(=O), R" is selected from Caryl and 5-14 membered het N(R) - P(=O)CNR), Co alkyl, Co perhaloalkyl, eroaryl; 55 Coalkenyl, Co alkynyl, 3-14 membered heteroaliphatic, R’ is selected from Caryl and 5-14 membered het C. carbocyclyl, 3-14 membered heterocyclyl, Caryl, eroaryl; and 5-14 membered heteroaryl, or two R' groups are joined R is selected from -OH, -OR', ON(R), to form a 3-14 membered heterocyclyl or 5-14 membered N(R), C(=O)R', CHO,-COR', C(=O)N heteroaryl ring; and (R), C( NRC)ORC, C(-NR)N(R), 60 each instance of R is, independently, selected from —SOR", S(=O)R', Si(R'), Coalkyl, Coper hydrogen, Coalkyl, Coperhaloalkyl, Coalkenyl, Co haloalkyl, Coalkenyl, Co alkynyl, 3-14 membered het alkynyl, 3-14 membered heteroaliphatic, Co carbocyclyl, eroaliphatic, Cocarbocyclyl, 3-14 membered heterocyclyl, 3-14 membered heterocyclyl, C-aryl, and 5-14 membered Caryl, and 5-14 membered heteroaryl, with the proviso heteroaryl, or two R' groups are joined to form a 3-14 mem that R is not —CH: 65 bered heterocyclyl or 5-14 membered heteroaryl ring. each instance of R is, independently, selected from Co In one embodiment, provided herein is a compound of alkyl, Co perhaloalkyl, Coalkenyl, C2-io alkynyl, 3-14 formula (I): US 8,546,432 B2 19 20 In certain embodiments, R is 5-14 membered heteroaryl. (I) In certain embodiments, R is 5-10 membered heteroaryl. In certain embodiments, R is 5-6 membered heteroaryl. In cer tain embodiments, R is 5,6-bicyclic heteroaryl. In certain embodiments, R is 6,6-bicyclic heteroaryl. In certain embodiments, R is a 5-membered heteroaryl group. Exemplary 5-membered heteroaryl groups include, but are not limited to, pyrrolyl, furanyl, thiophenyl, imida Zolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, or a pharmaceutically acceptable form thereof; 10 triazolyl, oxadiazolyl, thiadiazolyl and tetrazolyl. wherein: R is selected from Cocarbocyclyl, 3-14 membered het In certain embodiments, R is a 6-membered heteroaryl erocyclyl, C-aryland 5-14 membered heteroaryl; group. Exemplary 6-membered heteroaryl groups include, R’ is selected from Co alkyl, Coalkenyl, Co alky but are not limited to, pyridinyl, pyridazinyl, pyrimidinyl, nyl, 3-14 membered heteroaliphatic, Co carbocyclyl, 3-14 15 pyrazinyl, triazinyl and tetrazinyl. membered heterocyclyl, C-aryl and 5-14 membered het In certain embodiments, R is a 5,6-bicyclic heteroaryl eroaryl; group. Exemplary 5,6-bicyclic heteroaryl groups include, but R is selected from hydrogen, OH, -OR.", are not limited to, indolyl, isoindolyl, indazolyl, benztriaz - ON(R), N(R), C(=O)R', CHO, olyl, benzothiophenyl, isobenzothiophenyl, benzofuranyl, CO.R.", C(=O)N(R), C( NRC)ORC, benzoisofuranyl, benzimidazolyl, benzoxazolyl, benzisoX C(-NRC)N(RCP). SORC, S(=O)R', Si azolyl, benzoxadiazolyl, benzthiazolyl, benzisothiazolyl, (R)s. C1-io alkyl, C-10 perhaloalkyl, C2-io alkenyl, Co benzthiadiazolyl, indolizinyl, and purinyl. alkynyl, 3-14 membered heteroaliphatic, Co carbocyclyl, In certain embodiments, R is a 6,6-bicyclic heteroaryl 3-14 membered heterocyclyl, C-aryl, and 5-14 membered group. Exemplary 6,6-bicyclic heteroaryl groups include, but heteroaryl; 25 are not limited to, naphthyridinyl, pteridinyl, quinolinyl, iso each instance of R is, independently, selected from Co quinolinyl, cinnolinyl, quinoxalinyl, phthalazinyl and alkyl, Co perhaloalkyl, Coalkenyl, C2-io alkynyl, 3-14 quinazolinyl. membered heteroaliphatic, Co carbocyclyl, 3-14 mem In certain embodiments, R is a group of the formula (i): bered heterocyclyl, C. aryl, and 5-14 membered het eroaryl; and 30 each instance of R' is, independently, selected from hydrogen, OH, -OR', N(R), —CN, -C(=O) (i) RC', C(=O)N(RC), COR', SORC, C(-NR)OR', C(-NR)N(R), SON(R), SORC, SOORC, SORC, C(—S)N(RC), 35 C(=O)SR, C(—S)SR, P(=O).R.", P(=O) (R'). -P(=O)N(R), -P(=O)(NR), Co alkyl, Co perhaloalkyl, Coalkenyl, Co alkynyl, 3-14 mem bered heteroaliphatic, Cocarbocyclyl, 3-14 membered het erocyclyl, Caryl, and 5-14 membered heteroaryl, or two 40 R° groups are joined to form a 3-14 membered heterocyclyl wherein each group W. R. W. R. W. R. W. R., and or 5-14 membered heteroaryl ring: W. Rindependently represents either a nitrogen atom (N) or R and R together with the nitrogen (N) atom to which or C R', C R. C. R., C R', or C R. respectively: each is attached are joined to form a 5-14 membered ring. and Group R' 45 wherein R. R. R. R. and Rare independently selected As described generally above, R is selected from Cso from the group consisting of hydrogen, halogen, —CN. carbocyclyl, 3-14 membered heterocyclyl, Caryl, and NO, N, -SOH, -SOH, -OH, -OR', ON 5-14 membered heteroaryl. (R'), N(R'), N(OR)R', SH, SR'', In certain embodiments, R is Co carbocyclyl. Exem -SSR, C(=O)R'', -COH, CHO, C(OR), plary carbocyclyl groups include, but are not limited to, 50 COR'', OC(=O)R', OCOR', C(O)N(R'), cyclopropyl (C), cyclobutyl (C), cyclopentyl (Cs), cyclo OC(=O)N(R'), NRC(=O)R'', NR'COR'', pentenyl (Cs), cyclohexyl (C), cyclohexenyl (C), cyclo NR'C(=O)N(R'), C( NR-2)OR1, OC hexadienyl (C), cycloheptyl (C7), cycloheptadienyl (C7). (—NR)R', OC(—NR)OR'', C(-NR)N(R'), cycloheptatrienyl (C7) and cyclooctyl (Cs). OC(—NR'2)N(R'), NR1-C( NR1)N(R-2), In certain embodiments, R is 3-14 membered heterocy 55 C(=O)NR'SOR'', NRSOR', SON(R'), clyl. Exemplary heterocyclyl groups include, but are not lim SOR', SOOR1, OSOR', S(=O)R'', OS ited to, azirdinyl, oxiranyl, thiorenyl, aZetidinyl, oxetanyl. (=O)R', Si(R'), OSi(R'), C(=S)N(R'), thietanyl, tetrahydrofuranyl, dihydrofuranyl, tetrahy C(=O)SR'', C(—S)SR', SC(—S)SR'', drothiophenyl, dihydrothiophenyl, pyrrolidinyl, dihydropyr - P(=O), R', OP(=O).R'', P(=O)(R'), OP rolyl, dioxolanyl, oxathiolanyl, dithiolanyl, piperidinyl, tet 60 (=O)(R'), OP(=O)(OR") - P(=O)N(R'), rahydropyranyl, dihydropyridinyl, thianyl, piperazinyl, – OP(=O)N(R'), -P(=O)(NR), OP(=O) morpholinyl, dithianyl, dioxanyl, azepanyl, oxepanylthiepa (NR), NR'P(=O)(OR), NR'P(=O)(NR'), nyl, azocanyl, oxecanyl and thiocanyl. P(R'), P(R'), OP(R'), OP(R'), In certain embodiments, R is Cola aryl. Exemplary aryl —B(OR") —BR' (OR"),Coalkyl, Coperhaloalkyl, groups include, but are not limited to, phenyl, naphthyl and 65 Coalkenyl, Co alkynyl, 3-14 membered heteroaliphatic, anthracy1. In certain embodiments, R is phenyl (Caryl). In Clo carbocyclyl, 3-14 membered heterocyclyl, Caryl, certain embodiments, R is naphthyl (Co aryl). and 5-14 membered heteroaryl; or one or more of R' and R. US 8,546,432 B2 21 22 R° and R. RandR or R and Rare joined to form a Co NR'COR'', NR'C(=O)N(R'), C(-NR') carbocyclyl, 3-14 membered heterocyclyl, Caryl or 5-14 OR-11, OC( NR12)R1, OC(—NR'2)OR'', membered heteroaryl ring; C(-NR')N(R'), OC(—NR)N(R'), NRC each instance of R' is, independently, selected from Co (—NR)N(R'), C(=O)NR'SOR'', alkyl, Co perhaloalkyl, Coalkenyl, C2-io alkynyl, 3-14 NR'SOR', SON(R'). SOR', SOOR'', membered heteroaliphatic, Co carbocyclyl, 3-14 mem OSOR', S(=O)R'', OS(=O)R'', C. alkyl, bered heterocyclyl, C. aryl, and 5-14 membered het Co perhaloalkyl, Coalkenyl, C2-io alkynyl, 3-14 mem eroaryl; bered heteroaliphatic, Cocarbocyclyl, 3-14 membered het each instance of R' is, independently, selected from erocyclyl, C-aryl, and 5-14 membered heteroaryl; or one hydrogen, -OH,--OR', N(R'), —CN, C(=O)R', 10 or more of R' and R, R and R. RandR' or Rand Rare C(=O)N(R'), COR', SOR'', C(—NR) joined to form a Co carbocyclyl 3-14 membered heterocy OR'', C(-NR')N(R'), SON(R'), SOR", clyl, Caryl or 5-14 membered heteroaryl ring. SOOR', SOR'', C(—S)N(R'), C(=O)SR', In certain embodiments, R. R. R. R. and Rare inde -C(=S)SR', -P(=O).R'', -P(=O)(R'), -P(=O), pendently selected from the group consisting of hydrogen, N(R'), -P(=O)(NR'), Co alkyl, Co perhaloalkyl, 15 halogen, CN, OR'', N(R'), -COH, -COR', Coalkenyl, Co alkynyl, 3-14 membered heteroaliphatic, —C(=O)N(R'), -SOR', Co alkyl, Co alkynyl, Clo carbocyclyl, 3-14 membered heterocyclyl, Caryl, 3-14 membered heterocyclyl, and Caryl; or one or more and 5-14 membered heteroaryl, or two R' groups are joined of R' and R, R and R. RandR or Rand Rare joined to to form a 3-14 membered heterocyclyl or 5-14 membered form a 5-14 membered heteroaryl ring. heteroaryl ring; and In certain embodiments, R. R. R. R. and Rare inde each instance of R is, independently, selected from pendently selected from the group consisting of hydrogen, hydrogen, Coalkyl, C-operhaloalkyl, Coalkenyl, Co halogen, —OR'', N(R'), -COH, -C(=O)N(R'), alkynyl, 3-14 membered heteroaliphatic, Co carbocyclyl, —SOR', and 3-14 membered heterocyclyl: or R and R 3-14 membered heterocyclyl, C-aryl, and 5-14 membered are joined to form a 5-14 membered heteroaryl ring. heteroaryl, or two R' groups are joined to form a 3-14 mem 25 In certain embodiments, R. R. R. R. and Rare inde bered heterocyclyl or 5-14 membered heteroaryl ring. pendently selected from the group consisting of hydrogen, In certain embodiments, the group of formula (i) represents halogen, —OR', and –C(=O)N(R'); or R and Rare a C-aryl group or a 6-14 membered heteroaryl group. In joined to form a 5-14 membered heteroaryl ring. certain embodiments, the group of formula (i) represents a In certain embodiments, R. R. R. R. and Rare inde 6-14 membered heteroaryl group. In certain embodiments, 30 pendently selected from the group consisting of hydrogen, the group of formula (i) represents a C- aryl group. In halogen, —OR', and –C(=O)N(R'); or R and Rare certain embodiments, the Caryl group of formula (i) rep joined to form a 5-14 membered heteroaryl ring. resents a phenyl group. In certain embodiments, R', R. R. R. and Rare inde As used herein, when one or more of R', R. R. RandR pendently selected from the group consisting of hydrogen, is referred to as “not hydrogen', it is meant that one or more 35 halogen, and—OR". In certain embodiments, R', R. R. R' of R', R. R. R. and R is independently selected from a and Rare independently selected from the group consisting group consisting of halogen, —CN, —NO. —N, -SOH, of hydrogen, fluoro, chloro, and—OR". In certain embodi - SOH, OH, OR'', ON(R'), N(R'), ments, R', R. R. RandR are independently selected from N(OR)R', SH, SR', SSR, C(=O)R', the group consisting of hydrogen, fluoro, chloro, and —OMe. -COH, -CHO, C(OR), COR'', OC(=O) 40 In certain embodiments, R', R. R. R. and Rare indepen R'', OCOR'', C(=O)N(R'), OC(=O)N(R'), dently selected from the group consisting of hydrogen, fluoro NR1-C(O)R1, NR COR1, NR1-C(O)N and—OR''. In certain embodiments, R. R. R. RandR (R'), C(-NR')OR', OC(-NR')R'', OC are independently selected from the group consisting of (—NR)OR', C(-NR')N(R'), OC(—NR')N hydrogen, fluoro and —OMe. In certain embodiments, R', (R'), NR'C(—NR'2)N(R'), C(=O) 45 R. R. Rand Rare independently selected from the group NR'SOR'', NRSOR', SON(R'), SOR', consisting of hydrogen and fluoro. In certain embodiments, SOOR'', OSOR', S(=O)R'', OS(=O)R'', R", R. R. R. and R are independently selected from the Si(R'), OSi(R'), C(=S)N(R'), C(=O) group consisting of hydrogen and chloro. SR'', C(—S)SR', SC(S)SR'', P(=O).R.", OP In certain embodiments, R and Rare joined to form a (=O).R'', P(=O)(R'), OP(=O)(R'), OP(=O) 50 5-14 membered heteroaryl ring. (OR), P(=O)N(R'), OP(=O)N(R'), In certain embodiments, R is a group of the formula (ii): P(=O)(NR'2), OP(=O)(NR'2), NR P(=O) (OR), NRP(=O)(NR), P(R) P(R'), OP(R'), OP(R'), B(OR"), or BR (OR 1), (ii) Co alkyl, Co perhaloalkyl, Coalkenyl, Co alkynyl, 55 3-14 membered heteroaliphatic, Co carbocyclyl. 3-14 membered heterocyclyl, Caryl, and 5-14 membered het eroaryl; or one or more of R' and R, RandR, R and Ror R" and Rare joined to form a Cso carbocyclyl, 3-14 mem bered heterocyclyl, C-aryl or 5-14 membered heteroaryl 60 r1ng. In certain embodiments, R', R. R. R. and Rare inde pendently selected from the group consisting of hydrogen, wherein R', R. R. R. and Rare as defined above and halogen, —CN, NO, SOH, -SOH, -OH,--OR', herein. N(R'), C(=O)R', COH, CHO, C(OR), 65 In certain embodiments, the group of formula (ii) repre COR'', OC(=O)R'', OCOR'', C(=O)N sents a C-14 aryl group. In certain embodiments, the C-14 (R'), OC(=O)N(R'), NR'C(=O)R', aryl group of formula (ii) represents a phenyl group. US 8,546,432 B2 23 24 In certain embodiments, R is a monosubstituted, disubsti -continued tuted or trisubstituted group of the formula (ii). In certain (ii-e) embodiments, R is a monosubstituted or disubstituted group of the formula (ii). In certain embodiments, R is a monosubstituted group of 5 the formula (ii). For example, in certain embodiments, R is an ortho-sub stituted group of the formula (ii), e.g., wherein R'-R are (ii-f) hydrogen, and R is not hydrogen, e.g., of the formula (ii-a). In certain embodiments, R' is a meta-substituted group of the formula (ii), e.g., wherein R'-R and Rare hydrogen and R" is not hydrogen, e.g., of the formula (ii-b). In certain embodiments, R is a para-substituted group of the formula (ii), e.g., wherein R. R. Rand Rare hydrogen (ii-g) and R is not hydrogen, e.g., of the formula (ii-c). 15

(ii-a) 2O (ii-h)

(ii-b) as

(ii-i)

(ii-c) 30

35 In certain embodiments, R is a disubstituted group of the For example, in certain embodiments, R is a 2,6-disubsti formula (ii). tuted group as described herein, e.g., of the formula (ii-d): For example, in certain embodiments, R is a 2,6-disubsti tuted group of the formula (ii), e.g., wherein R, RandR are 40 hydrogen, and R' and Rare not hydrogen, e.g., of the for (ii-d) mula (ii-d). In certain embodiments, R is a 2,5-disubstituted group of the formula (ii), e.g., wherein R. RandRare hydrogen, and R" and Rare not hydrogen, e.g., of the formula (ii-e). 45 In certain embodiments, R is a 2.4-disubstituted group of the formula (ii), e.g., wherein R. RandRare hydrogen, and R" and Rare not hydrogen, e.g., of the formula (ii-f). In certain embodiments, R is a 2,3-disubstituted group of the formula (ii), e.g., wherein R', RandRarehydrogen, and 50 wherein R' and Rare as defined above and herein. RandR are not hydrogen, e.g., of the formula (ii-g). In certain embodiments, one of R' and R is halogen, In certain embodiments, R is a 3,4-disubstituted group of CN, OR'', N(R'), COH, COR', C(=O) the formula (ii), e.g., wherein R', RandRarehydrogen, and N(R'), SOR', Co alkyl, Co alkynyl, 3-14 mem R° and Rare not hydrogen, e.g., of the formula (ii-h). bered heterocyclyl, and Caryl, and the other of RandR In certain embodiments, R is a 3.5-disubstituted group of 55 is halogen, CN, OR', N(R'). -COH,-COR', the formula (ii), e.g., wherein R', RandRare hydrogen, and —C(=O)N(R') - SOR', Co alkyl, Co alkynyl, R° and Rare not hydrogen, e.g., of the formula (ii-i). 3-14 membered heterocyclyl, and Caryl. In certain embodiments, one of R' and R is halogen, —OR', Co alkyl, or C(=O)N(R'), and the other of (ii-d) 60 R" and R is halogen, —OR', Co alkyl, or —C(=O)N (R'). In certain embodiments, each of R' and R is indepen dently halogen. For example, each of R' and R is indepen 65 dently selected from fluoro and chloro. In certain embodiments, R is a trisubstituted group of the formula (ii). US 8,546,432 B2 25 26 For example, in certain embodiments, R is a 2.4.6-trisub In certain embodiments, R is a 3-pyridinyl wherein stituted group of the formula (ii), e.g., wherein RandR are W R is N, and W. R. W. R. W. R., and W R are hydrogen, and R', R and Rare not hydrogen, e.g., of the C R', C R., C R and C R. respectively, e.g., of the formula (iii). formula (iv). In certain embodiments, R is a 2.3,6-trisubstituted group In certain embodiments, R is a 4-pyridinyl wherein of the formula (ii), e.g., wherein R and Rare hydrogen, and W R is N, and W. R. W. R. W. R, and W R are R", Rand Rare not hydrogen, e.g., of the formula (ii-k). C R', C R., C R and C R. respectively, e.g., of the In certain embodiments, R is a 2,4,5-trisubstituted group formula (v). of the formula (ii), e.g., wherein R and Rare hydrogen, and R", RandR are not hydrogen, e.g., of the formula (i-1). 10 In certain embodiments, R is a 2,3,4-trisubstituted group (iii) of the formula (ii), e.g., wherein R and Rare hydrogen, and R", Rand Rare not hydrogen, e.g., of the formula (ii-m). In certain embodiments, R is a 3,4,5-trisubstituted group 15 of the formula (ii), e.g., wherein R' and Rare hydrogen, and R. RandR are not hydrogen, e.g., of the formula (ii-n).

(iv) (ii-)

25 (v) (ii-k)

30

R5 (ii-I) RI 35 wherein R', R. R. R. and Rare as defined above and R3 herein. In certain embodiments, R is a monosubstituted or disub stituted pyridinyl. R4 40 In certain embodiments, R is a monosubstituted pyridinyl. (ii-m) In certain embodiments, R is a monosubstituted pyridinyl R2 of the formula (iii) wherein R. R. Rare hydrogen and R is not hydrogen, e.g., of the formula (iii-a). R3 In certain embodiments, R is a monosubstituted pyridinyl 45 of the formula (iii) wherein R. R. Rare hydrogen and R is not hydrogen, e.g., of the formula (iii-b). C, In certain embodiments, R is a monosubstituted pyridinyl (ii-n) R2 of the formula (iii) wherein R. R. Rare hydrogen and R is not hydrogen, e.g., of the formula (iii-c). 50 In certain embodiments, R is a monosubstituted pyridinyl of the formula (iii) wherein R. R. Rare hydrogen and R is not hydrogen, e.g., of the formula (iii-d). R4 55 (iii-a) R2 In certain embodiments, R is heteroaryl selected from a 5-6-membered heteroaryl, a 5,6-bicyclic heteroaryl or a 6.6- bicyclic heteroaryl. In certain embodiments, R is a 6-membered heteroaryl. In certain embodiments, R is a 6-membered heteroaryl selected 60 from pyridinyl. In certain embodiments, R is 2-pyridinyl, (iii-b) 3-pyridinyl or 4-pyridinyl. In certain embodiments, R is a 2-pyridinyl wherein W. R' is N, and W. R. W. R. W. R, and W R are 65 C. R. C. R., C R and C R. respectively, e.g., of the formula (iii). US 8,546,432 B2 27 28 -continued (iii-c) (V-a) Z N\ S R4 (iii-d) (v-b) Z N \ 10 S.

R5

15 In certain embodiments, R is a monosubstituted pyridinyl of the formula (iv) wherein R. R. Rare hydrogen and R' is In certain embodiments, R is a disubstituted pyridinyl. not hydrogen, e.g., of the formula (iv-a). In certain embodiments, R is a disubstituted pyridinyl of In certain embodiments, R is a monosubstituted pyridinyl the formula (iii) wherein R and Rare hydrogen and R and of the formula (iv) wherein R. R. Rare hydrogen and R is Rare not hydrogen, e.g., of the formula (iii-e). not hydrogen, e.g., of the formula (iv-b). In certain embodiments, R is a disubstituted pyridinyl of In certain embodiments, R is a monosubstituted pyridinyl the formula (iii) wherein R and Rare hydrogen and Rand of the formula (iv) wherein R', R. Rare hydrogen and R is Rare not hydrogen, e.g., of the formula (iii-f). not hydrogen, e.g., of the formula (iv-c). 25 In certain embodiments, R is a disubstituted pyridinyl of In certain embodiments, R is a monosubstituted pyridinyl the formula (iii) wherein R and Rare hydrogen and Rand of the formula (iv) wherein R. R. Rare hydrogen and R is Rare not hydrogen, e.g., of the formula (iii-g). not hydrogen, e.g., of the formula (iv-d). In certain embodiments, R is a disubstituted pyridinyl of the formula (iii) wherein R and Rare hydrogen and Rand 30 R" are not hydrogen, e.g., of the formula (iii-h). (iv-a) In certain embodiments, R is a disubstituted pyridinyl of the formula (iii) wherein R* and Rare hydrogen and Rand Rare not hydrogen, e.g., of the formula (iii-i). 35 In certain embodiments, R is a disubstituted pyridinyl of the formula (iii) wherein R and Rare hydrogen and Rand C. Rare not hydrogen, e.g., of the formula (iii-j). (iv-b) N 3 40 (iii-e) OS.

(iv-c) 45 cy (iii-f)

R4 C 50 Y

(iv-d) (iii-g) Y \ 55 Y

CR5 60 In certain embodiments, R is a monosubstituted pyridinyl of the formula (v) wherein R. R. Rare hydrogen and R' is not hydrogen, e.g., of the formula (V-a). Y In certain embodiments, R is a monosubstituted pyridinyl 65 of the formula (v) wherein R. R. Rare hydrogen and R is C not hydrogen, e.g., of the formula (V-b). US 8,546,432 B2 29 30 -continued -continued 2 (iii-i) (iv-) R N R3 Z N Y \ \ S. S.

(iii-) R3 Z N\ 10 In certain embodiments, R is a disubstituted pyridinyl of S. the formula (v) wherein Rand Rare hydrogen and R' and R4 Rare not hydrogen, e.g., of the formula (V-c). In certain embodiments, R is a disubstituted pyridinyl of 15 the formula (v) wherein R* and Rare hydrogen and R' and In certain embodiments, R is a disubstituted pyridinyl of Rare not hydrogen, e.g., of the formula (v-d). the formula (iv) wherein Rand Rare hydrogen and R' and Rare not hydrogen, e.g., of the formula (iv-e). In certain embodiments, R is a disubstituted pyridinyl of In certain embodiments, R is a disubstituted pyridinyl of the formula (v) wherein Rand Rare hydrogen and R' and the formula (iv) wherein Rand Rare hydrogen and R' and R" are not hydrogen, e.g., of the formula (v-e). Rare not hydrogen, e.g., of the formula (iv-f). In certain embodiments, R is a disubstituted pyridinyl of In certain embodiments, R is a disubstituted pyridinyl of the formula (v) wherein R' and Rare hydrogen and R and the formula (iv) wherein R* and Rare hydrogen and R' and Rare not hydrogen, e.g., of the formula (V-f). Rare not hydrogen, e.g., of the formula (iv-g). In certain embodiments, R is a disubstituted pyridinyl of 25 the formula (iv) wherein R' and Rare hydrogen and Rand (v-c) Rare not hydrogen, e.g., of the formula (iv-h). R1 In certain embodiments, R is a disubstituted pyridinyl of Z the formula (iv) wherein R' and Rare hydrogen and Rand N Rare not hydrogen, e.g., of the formula (iv-i). 30 In certain embodiments, R is a disubstituted pyridinyl of S. the formula (iv) wherein R' and Rare hydrogen and Rand C!R5 Rare not hydrogen, e.g., of the formula (iv-j).

35 (iv.-e) (v-d) R1 R2 N R1

Y \ N Z S 40 S. R5 (iv-f) R1 N 45 (v-e) Y \ RI S. R4 % \ (iv-g) RI 50 S. R R3 / \ S. (v-f) (iv-h) 55 R2 R3 2 \ N % \ S. S. 60 R5 R4 (iv-i) R3 / \ In certain embodiments, R is a 5,6-bicyclic heteroaryl. S. 65 For example, in certain embodiments, R is a 5,6-bicyclic R4 heteroaryl group of the formula (vi) (which is a subset of a group of the formula (ii-g)): US 8,546,432 B2 31 32 In certain embodiments, R is a heteroaryl group of the

(vi) formulae (vi-a) or (vi-b):

(vi-a)

10 wherein R. R. Rare as defined above and herein and R' and Rare joined to form a 5-membered heteroaryl ring: X, Y and Z are independently selected from CR'', O, S, N, or NR-15; 15 each instance of R' is, independently, selected from (vi-b) hydrogen, halogen, —CN, NO. —N. —SOH, -SOH, OH, OR1, ON(R-7), N(R-7), N(OR1)R', SH, SR, SSR, C(=O)R, COH, CHO, C(OR"). COR1, OC(=O)R1, OCOR', C(=O)N(R-7), OC(=O)N(R-7), NR-7C(=O) R16, NR-17COR1, NR7C(=O)N(R7), C(-NR 7)OR1, OC(-NR 7)R1, OC(—NR-7) OR, C(-NR7)N(R7), OC(-NR7)N(R-7), 25 RA4 NR-7C( NR-7)N(R-7), C(=O)NR''SOR, NR 7SOR1, SON(R-7), SOR', SOORA, OSOR', S(—O)R, OS(—OR, Si(R), wherein R', R, R are as defined above and X and Z are OSi(R), C(=S)N(R7), C(=O)SR, C(=S) independently selected from O, S and NR. SR', SC(=S)SR-, - P(=O).R1, OP(=O).R.", 30 In certain embodiments, wherein R is a heteroaryl group - P(=O)(R'), OP(=O)(R), OP(=O)(OR), of the formulae (vi-a) or (vi-b), X and Z are O (i.e., benzox azolyl). In certain embodiments, X and Z are S (i.e., benz thiazolyl). In certain embodiments, X and Z are NR' (i.e., imidazolyl). OP(R), OP(R), B(OR), or BR(OR), 35 Coalkyl, Co perhaloalkyl, Coalkenyl, Co alkynyl, In certain embodiments, R is a heteroaryl group of the 3-14 membered heteroaliphatic, Co carbocyclyl, 3-14 formulae (vi-c) or (vi-d): membered heterocyclyl, Caryl, and 5-14 membered het eroaryl; 40 (vi-c) each instance of R' is, independently, selected from Co R2 alkyl, Co perhaloalkyl, Coalkenyl, C2-io alkynyl, 3-14 R1 membered heteroaliphatic, Co carbocyclyl, 3-14 mem R3 bered heterocyclyl, C. aryl, and 5-14 membered het eroaryl; 45 each instance of R and R7 is, independently, selected X from hydrogen, OH, -OR, NOR7), —CN, N 2 C(=O)R1, C(=O)N(R-7), CO.R., SOR-7, N RA4 (vi-d) C(-NR')OR, C(-NR7)N(R7), SON(R'), R2 SOR', SOOR', SOR, C(-S)N(R-7), 50 C(=O)SR1, C(—S)SR', P(=O).R', P(=O) RI (R), - P(=O)N(R'), P(=O)(NR), C-io alkyl, R3 Co perhaloalkyl, Coalkenyl, Co alkynyl, 3-14 mem bered heteroaliphatic, Cocarbocyclyl, 3-14 membered het erocyclyl, Caryl, and 5-14 membered heteroaryl, or two 55 RA4 N R7 groups are joined to form a 3-14 membered heterocyclyl N-X or 5-14 membered heteroaryl ring: each instance of R is, independently, selected from hydrogen, Coalkyl, C-operhaloalkyl, Coalkenyl, Co wherein R. R. Rare as defined above and X is indepen alkynyl, 3-14 membered heteroaliphatic, Co carbocyclyl, 60 dently selected from O, S and NR. 3-14 membered heterocyclyl, C-aryl, and 5-14 membered heteroaryl, or two R' groups are joined to form a 3-14 mem In certain embodiments, wherein R is a heteroaryl group bered heterocyclyl or 5-14 membered heteroaryl ring; and of the formulae (vi-c) or (vi-d), X is O (i.e., benzisoxazolyl). the dashed line represents a double or single bond. In certain embodiments, X is S (i.e., benzisothiazolyl). In In certain embodiments, R' is hydrogen. In certain embodi 65 certain embodiments, X is NR' (i.e., indazolyl). ments, R is hydrogen. In certain embodiments, R is hydro In certain embodiments, R is a heteroaryl group of the gen. In certain embodiments, R', R and Rare hydrogen. formulae (vi-e), (vi-f) or (vi-g): US 8,546,432 B2 33 34 Clo carbocyclyl, 3-14 membered heterocyclyl, C. aryl (vi-e) and 5-14 membered heteroaryl. R2 In certain embodiments, R is an acyclic group, i.e., RI Selected from Co alkyl, Coalkenyl, C2-io alkynyl and R3 3-14 membered heteroaliphatic. In certain embodiments, R. is Co alkyl. In certain embodiments, R is a substituted Co alkyl, e.g., a Co aralkyl group. In certain embodi Ra-6 ments, R is a Caralkyl, e.g., for example, a substituted or unsubstituted benzyl group (C. aralkyl) or substituted or Y RA4 10 (vi-f) unsubstituted phenylethyl group (C. aralkyl). In certain R2 embodiments, R is a Coheteroaralkyl. In certain embodi RI ments, R is alkenyl. In certain embodiments, R is alkynyl. R3 In certain embodiments, R is 3-14 membered het eroaliphatic. 15 Alternatively, in certain embodiments, R is a cyclic group, NS i.e., selected from Co carbocyclyl, 3-14 membered hetero RA4 N-2 cyclyl, C-aryl and 5-14 membered heteroaryl. In certain embodiments, R is Co carbocyclyl or 3-14 RA4 membered heterocyclyl. (vi-g) In certain embodiments, R is Co carbocyclyl. Exem R2 plary carbocyclyl groups include, but are not limited to, RI cyclopropyl (C), cyclobutyl (C), cyclopentyl (Cs), cyclo R3 pentenyl (Cs), cyclohexyl (C), cyclohexenyl (C), cyclo 25 hexadienyl (C), cycloheptyl (C7), cycloheptadienyl (C7). NS cycloheptatrienyl (C7) and cyclooctyl (Cs). X In certain embodiments, R is 3-14 membered heterocy la clyl. Exemplary heterocyclyl groups include, but are not lim RA4 ited to, azirdinyl, oxiranyl, thiorenyl, aZetidinyl, oxetanyl. RA4 30 thietanyl, tetrahydrofuranyl, dihydrofuranyl, tetrahy drothiophenyl, dihydrothiophenyl, pyrrolidinyl, dihydropyr wherein R', R, R and Rare as defined above and X,Y rolyl, dioxolanyl, oxathiolanyl, dithiolanyl, piperidinyl, tet and Z are independently selected from O, S and NR". rahydropyranyl, dihydropyridinyl, thianyl, piperazinyl, In certain embodiments, wherein R is a heteroaryl group morpholinyl, dithianyl, dioxanyl, azepanyl, oxepanylthiepa of the formulae (vi-e), (vi-f) or (vi-g), Y is O (i.e., benzofura 35 nyl, azocanyl, oxecanyl and thiocanyl. nyl or isobenzofuranyl). In certain embodiments, Y is S (i.e., In certain embodiments, R is Carylor 5-14 membered benzothiophenyl or isobenzothiophenyl). In certain embodi heteroaryl. ments, Y is NR' (i.e., indolyl or isoindolyl). In certain embodiments, R is Cola aryl. Exemplary aryl In certain embodiments, R is a heteroaryl group of the groups include, but are not limited to, phenyl, naphthyl and formula (vi-h): 40 anthracy1. In certain embodiments, R is phenyl (Caryl). In certain embodiments, R is naphthyl (Co aryl). In certain embodiments, R is 5-14 membered heteroaryl. (vi-f) In certain embodiments, R is 5-10 membered heteroaryl. In R2 RI certain embodiments, R is 5-6 membered heteroaryl. In cer 45 tain embodiments, R is a 5,6-bicyclic heteroaryl. In certain R3 embodiments, R is a 6.6-bicyclic heteroaryl. In certain embodiments, R is a 5-membered heteroaryl group. Exemplary 5-membered heteroaryl groups include, but are not limited to, pyrrolyl, furanyl, thiophenyl, imida N N 50 Zolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, y 1 triazolyl, oxadiazolyl, thiadiazolyl and tetrazolyl. In certain embodiments, R is a 6-membered heteroaryl wherein R', R. Rare as defined above and Y is indepen group. Exemplary 6-membered heteroaryl groups include, dently selected from O, S and NR. but are not limited to, pyridinyl, pyridazinyl, pyrimidinyl, In certain embodiments, wherein R is a heteroaryl group 55 pyrazinyl, triazinyl and tetrazinyl. of the formula (vi-e),Y is O (i.e., benzoxadiazolyl). In certain In certain embodiments, R is a 5,6-bicyclic heteroaryl embodiments, Y is S (i.e., benzthiadiazolyl). In certain group. Exemplary 5,6-bicyclic heteroaryl groups include, but embodiments, Y is NR' (i.e., benztriazolyl). are not limited to, indolyl, isoindolyl, indazolyl, benztriaz Group R' olyl, benzothiophenyl, isobenzothiophenyl, benzofuranyl, As described generally above, R is selected from Co 60 benzoisofuranyl, benzimidazolyl, benzoxazolyl, benzisoX alkyl, Co alkenyl, Co alkynyl, 3-14 membered het azolyl, benzoxadiazolyl, benzthiazolyl, benzisothiazolyl, eroaliphatic, Co carbocyclyl, 3-14 membered heterocyclyl, benzthiadiazolyl, indolizinyl, and purinyl. C. aryl and 5-14 membered heteroaryl; or R and R. In certain embodiments, R is a 6,6-bicyclic heteroaryl together with the nitrogen (N) atom to which each is attached group. Exemplary 6,6-bicyclic heteroaryl groups include, but are joined to form a 5-14 membered ring. 65 are not limited to, naphthyridinyl, pteridinyl, quinolinyl, iso In certain embodiments, R is selected from Co alkyl, quinolinyl, cinnolinyl, quinoxalinyl, phthalazinyl and Coalkenyl, Co alkynyl, 3-14 membered heteroaliphatic, quinazolinyl.

US 8,546,432 B2 37 38 In certain embodiments, RP is—CO.H. In certain embodi In certain embodiments, -L-RP and Rare not vicinal R” ments, L is a covalent bond. Substituents (i.e., not attached to two adjacent atoms on the In certain embodiments, wherein R is substituted with group R, e.g., meta or para to each other). In certain embodi -L-R, R is further substituted with the group: ments, -L-RP and —R are meta to each other. In certain embodiments, -L-RP and—R' are para to each other. RE In certain embodiments, the R is a group of the formula wherein: (vii): R is selected from halogen, -OH, -OR''. - ON (Rf ')2. N(R), N(OR712)R?.12, –SH, SR? 10, SSR, OC(=O)R’, OCOR'', OC(=O)N 10 (vii) (Rf ')2. NRC(-OR, NRI COR? 10, R6 NRC(O)N(R), OC( NR)R’9, OC N ( NR OR? o, OC(-NR' 1)N(R? )2. NRI IC W a's -R ( NR)N(R), NR?. SOR? 10, OSOR, N 4 OS(—O)R', Si(R'), OSi(R'), SC(S) 15 W -W SR 9, OP(=O).R., OP(=O)(R0), OP(=O) I R9 (OR'), OP(=O)N(R'''), OP(=O)(NR'''), NRI 'P(=O) (OR), NRI 'P(=O) (NR'2), P(R'2) P(R2), OP(R2), OP(R'2), 3-14 wherein each group W. R. W. R. W. R. W. R. and membered heterocyclyl and 5-14 membered heteroaryl, W. R' independently represents either a nitrogenatom (N) wherein the point of attachment of the 3-14 membered het or C R. C. R. C. R. C. R. or C R', respectively: erocyclyl or 5-14 membered heteroaryl group is on a nitrogen and atom; wherein R. R. R. RandR'' are independently selected each instance of R' is, independently, selected from from the group consisting of hydrogen, halogen, —CN. Co alkyl, Co perhaloalkyl, Coalkenyl, Co alkynyl, 25 NO, N, SOH, -SOH, OH, -OR', ON 3-14 membered heteroaliphatic, Co carbocyclyl, 3-14 (R'), N(R'), N(OR )R’s, –SH, SRI s membered heterocyclyl, Caryl, and 5-14 membered het SSR, C(=O)R’, COH, CHO, C(OR), eroaryl; COR'', OC(=O)R’ OCOR', C(=O)N each instance of R'' is, independently, selected from (R'), OC(=O)N(R'), NRC(O)R’, hydrogen, OH, -OR', N(R') –CN, -C(=O) 30 OR,NRCO.R. OC( NR)R’,NRC(O)N(R'), OC( NR)OR'',CNR) R?.10, C(=O)N(R'), CORI, SOR, C( NR)N(R), OC( NR)N(R), NRC C(-NR'2)OR, C(-NR'2)N(Ri), SON ( NR)N(R'), C(O)NR'SOR', (R2). SOR2, SOOR2, SOR1, C(S)N NR’SOR', SON(R). SOR', SOOR'', (R2), C(=O)SR'', C(—S)SR', P(=O)R’, OSOR', S(-OR'', OS(—O)R', Si(R'), P(=O)(R0), P(=O)N(R), P(=O)(NR'2), 35 Co alkyl, Co perhaloalkyl, Coalkenyl, Co alkynyl, SR',9Si(R'), SC(S)SR'', C(S)N(R). P(O).R'', CSOSR. OP(=O).R'', C(S) 3-14 membered heteroaliphatic, Co carbocyclyl, 3-14 P(=O)(R'), OP(=O)(R'), OP(=O)(OR), membered heterocyclyl, Caryl, and 5-14 membered het eroaryl, or two R' groups are joined to form a 3-14 mem bered heterocyclyl or 5-14 membered heteroaryl ring; and 40 each instance of R' is, independently, selected from OP(R), OP(R), B(OR), or BR' (OR), hydrogen, Coalkyl, Coperhaloalkyl, Coalkenyl, Co Coalkyl, Co perhaloalkyl, Coalkenyl, Co alkynyl, alkynyl, 3-14 membered heteroaliphatic, Co carbocyclyl, 3-14 membered heteroaliphatic, Co carbocyclyl, 3-14 3-14 membered heterocyclyl, C-aryl, and 5-14 membered membered heterocyclyl, C. aryl, 5-14 membered het heteroaryl, or two R' groups are joined to form a 3-14 45 eroaryl, -L-RP and—R: or one or more of RandR, R7 and membered heterocyclyl or 5-14 membered heteroaryl ring. R. R. and R or R and R'' are joined to form a Co In certain embodiments, R is selected from halogen, carbocyclyl, 3-14 membered heterocyclyl, Caryl or 5-14 OH, OR, ON(R), N(R'''), N(OR'') membered heteroaryl ring; or R'' and Rarejoined to form a R? 12, –SH, SR? 10, SSR? 12, Si(R), 3-14 membered heterocyclyl or 5-14 membered heteroaryl OSi(R) P(R) P(R), OP(R), OP 50 r1ng, (R'), 3-14 membered heterocyclyl and 5-14 membered each instance of R' is, independently, selected from Co heteroaryl, wherein the point of attachment of the 3-14 mem alkyl, Co perhaloalkyl, Coalkenyl, Co alkynyl, 3-14 bered heterocyclyl or 5-14 membered heteroaryl group is on membered heteroaliphatic, Co carbocyclyl, 3-14 mem a nitrogen atom. bered heterocyclyl, C. aryl, and 5-14 membered het In certain embodiments, R’ is selected from halogen, 55 eroaryl; –OH, -OR', N(R''), 3-14 membered heterocyclyl each instance of R’ is, independently, selected from and 5-14 membered heteroaryl, wherein the point of attach hydrogen, OH, OR', N(R'), CN, C(=O)R', ment of the 3-14 membered heterocyclyl or 5-14 membered C(=O)N(R), COR', SOR'', C(-NR) heteroaryl group is on a nitrogen atom. OR'', C(—NR)N(R), SON(R), SOR, In certain embodiments, R’ is selected from halogen, 60 SOOR, SOR'', C(—S)N(R), C(=O)SR, —OR'' and N(R'). In certain embodiments, R is C(—S)SR, P(=O).R'', P(=O)(R), P(=O), halogen. In certain embodiments, R' is —OR''. In certain N(R'), -P(=O)(NR), Co alkyl, Co perhaloalkyl, embodiments, R is N(R'). Coalkenyl, Co alkynyl, 3-14 membered heteroaliphatic, In certain embodiments, -L-RP and -R are vicinal R” Clo carbocyclyl, 3-14 membered heterocyclyl, Caryl, Substituents (i.e., attached to two adjacent atoms on the group 65 and 5-14 membered heteroaryl, or two R' groups are joined R”; e.g., ortho to each other). In certain embodiments, -L-RP to form a 3-14 membered heterocyclyl or 5-14 membered and -R are ortho to each other. heteroaryl ring; US 8,546,432 B2 39 40 each instance of R is, independently, selected from In certain embodiments, at least one of R. R. R. R. and hydrogen, Coalkyl, Coperhaloalkyl, Coalkenyl, Co R" is the group -L-RP as defined above and herein. In certain alkynyl, 3-14 membered heteroaliphatic, Co carbocyclyl, embodiments, at least one of R. R. R. R. and R' is the 3-14 membered heterocyclyl, C-aryl, and 5-14 membered group R as defined herein. heteroaryl, or two R' groups are joined to form a 3-14 mem 5 In certain embodiments, the group of the formula (viii) bered heterocyclyl or 5-14 membered heteroaryl ring: represents a C- aryl group. In certain embodiments, the and L, RP and R', are as defined above and herein. Caryl group of the formula (viii) represents a phenyl As used herein, when one or more of R. R. R. RandR' group. is referred to as “not hydrogen', it is meant that one or more In certain embodiments, R is a monosubstituted, disubsti of R. R. R. RandR' is independently selected from the 10 tuted or trisubstituted group of the formula (viii). In certain group consisting of halogen, —CN. —NO. —N, -SOH, embodiments, R is a monosubstituted or disubstituted group SOH, OH, OR', ON(R'), N(R'), of the formula (viii). N(OR’)R’, SH, SR', SSR, C(O)R’, In certain embodiments, R is a monosubstituted group of -COH, -CHO, C(OR), COR'', OC(=O) the formula (viii). R'', OCOR'', C(=O)N(R), OC(=O)N(R), 15 For example, in certain embodiments, R is an ortho-sub NRC(O)R'', NRCOR'', NRC(O)N stituted group of formula (viii), e.g., wherein R-R are (R), C( NR'2)OR, OC( NR'2)R’, OC hydrogen, and R' is not hydrogen, e.g., of the formula (viii ( NR)OR'', C( NR)N(R), OC( NR)N a). (R), NRC(—NR)N(R), C(=O) In certain embodiments, R is a meta-substituted group of NRSOR, NRSO.R., SONCRP). SOR, the formula (viii), e.g., wherein R-R and R' are hydrogen SOOR'', OSOR', S(–O)R’, OS(—O)R’", and R is not hydrogen, e.g., of the formula (viii-b). Si(R'), OSi(R'), C(=S)N(R'), C(=O) In certain embodiments, R is a para-substituted group of SR'', C(—S)SR', SC(=S)SR'', P(=O).R'', the formula (viii), e.g., wherein R. R. RandR'' are hydro OP(=O).R'', P(=O)(R'), OP(=O)(R'), OP 25 gen and R is not hydrogen, e.g., of the formula (viii-c). (=O)(OR’), - P(=O)N(R'), – OP(=O)N(R'), P(=O)(NR), OP(=O)(NR), NRP(=O) (OR), NRPG O)(NR), P(R') P(R), (viii-a) OP(R'), OP(R), B(OR), BR(OR’),-L- R. R. Coalkyl, Coperhaloalkyl, Coalkenyl, Co 30 alkynyl, 3-14 membered heteroaliphatic, Co carbocyclyl, 3-14 membered heterocyclyl, C-aryl, and 5-14 membered heteroaryl; or wherein one or more of R and R. RandR, RandR or RandR'arejoined to forma Ciocarbocyclyl, (viii-b) 3-14 membered heterocyclyl, Caryl or 5-14 membered 35 heteroaryl ring, or wherein R'' and R are joined to form a 3-14 membered heterocyclyl or 5-14 membered heteroaryl r1ng. In certain embodiments, at least one of R. R. R. R. and (viii-c) R' is the group -L-RP as defined above and herein. In certain 40 embodiments, at least one of R. R. R. R. and R' is the group —R as defined herein. In certain embodiments, the group of formula (vii) repre sents a Caryl or a 6-14 membered heteroaryl group. In 45 certain embodiments, the group of formula (vii) represents a In certain embodiments, R is a disubstituted group of the 6-14 membered heteroaryl group. In certain embodiments, formula (viii). the group of formula (vii) represents a C- aryl group. In For example, in certain embodiments, R is a 2,6-disubsti certain embodiments, the group of formula (vii) represents a tuted group of the formula (viii), e.g., wherein R. RandR phenyl group. 50 are hydrogen, and R and R'' are not hydrogen, e.g., of the In certain embodiments, W. R. W. R. W. R. W. R. formula (viii-d). and W R' represent C R. C. R. C. R. C. R. or In certain embodiments, R is a 2.5-disubstituted group of C R', respectively. For example, in certain embodiments, the formula (viii), e.g., wherein R, R and Rare hydrogen, R’ is a group of the formula (viii): and RandR'' are not hydrogen, e.g., of the formula (viii-e). 55 In certain embodiments, R is a 2,4-disubstituted group of the formula (viii), e.g., wherein R, R and Rare hydrogen, (viii) and RandR'' are not hydrogen, e.g., of the formula (viii-f). In certain embodiments, R is a 2,3-disubstituted group of formula (viii), e.g., wherein R. R7 and Rare hydrogen, and 60 RandR'' are not hydrogen, e.g., of the formula (viii-g). In certain embodiments, R is a 3,4-disubstituted group of the formula (viii), e.g., wherein R. R7 and R'' are hydrogen, and Rand Rare not hydrogen, e.g., of the formula (viii-h). 65 In certain embodiments, R is a 3,5-disubstituted group of wherein R. R. R. R. and R'' are as defined above and the formula (viii), e.g., wherein R. R7 and R'' are hydrogen, herein. and R7 and Rare not hydrogen, e.g., of the formula (viii-i). US 8,546,432 B2 41 42

(viii-d) (viii-) R

R10 (viii-e) R7 10 (viii-k)

15 R10 (viii-f) s R8 R10 (viii-l) R10 R7 (viii-g) 25 s R8 9 R10 R R10 (viii-h) R8 30 (viii-m) R9 R8 (viii-i) R7 35 R9 R10 (viii-n)

40 R9

In certain embodiments, R is a trisubstituted group of the formula (viii). 45 For example, in certain embodiments, R is a 2.4.6-trisub stituted group of formula (viii), e.g., wherein R7 and Rare In certain embodiments, R is heteroaryl selected from a hydrogen, and R. RandR'' are not hydrogen, e.g., of the 5-6-membered heteroaryl, a 5,6-bicyclic heteroaryl, or a 6.6- formula (viii-j). bicyclic heteroaryl. In certain embodiments, R is a 2.3,6-trisubstituted group 50 In certain embodiments, R is a 6-membered heteroaryl. In of the formula (viii), e.g., wherein R and R are hydrogen, certain embodiments, R is a 6-membered heteroaryl selected and R', R and R are not hydrogen, e.g., of the formula from pyridinyl. In certain embodiments, R is 2-pyridinyl, (viii-k). 3-pyridinyl or 4-pyridinyl. In certain embodiments, R is a 2,4,5-trisubstituted group 55 In certain embodiments, R is a 2-pyridinyl wherein of the formula (viii), e.g., wherein R and Rare hydrogen, W R is N, and W. R. W. R. W. R. and W R'' are and R, R and R'' are not hydrogen, e.g., of the formula C R', C R., C R and C R', respectively, e.g., of the (viii-l). formula (ix). In certain embodiments, R is a 2,3,4-trisubstituted group In certain embodiments, R is a 3-pyridinyl wherein of the formula (viii), e.g., wherein R and Rare hydrogen, 60 W. R7 is N, and W. R. W. R. W. R. and W R'' are and R, R and R'' are not hydrogen, e.g., of the formula C. R. C. R., C R and C R', respectively, e.g., of the (viii-m). formula (X). In certain embodiments, R is a 3,4,5-trisubstituted group In certain embodiments, R is a 4-pyridinyl wherein of the formula (viii), e.g., wherein RandR'' are hydrogen, 65 W R is N, and W. R. W. R. W. R. and W R'' are and R. R. and R are not hydrogen, e.g., of the formula C. R. C. R., C R and C R', respectively, e.g., of the (viii-n). formula (xi). US 8,546,432 B2 43 44 -continued (ix) R7 (ix-d) Na Na R8 N-l 9 R10 R10 R (x) 10 R In certain embodiments, R is a monosubstituted pyridinyl 2N R8 of the formula (x) wherein R. R. R'' are hydrogen and R is not hydrogen, e.g., of the formula (X-a). N-l In certain embodiments, R is a monosubstituted pyridinyl 9 15 of the formula (x) wherein R. R. R'' are hydrogen and R is R10 R not hydrogen, e.g., of the formula (X-b). (xi) R7 In certain embodiments, R is a monosubstituted pyridinyl R of the formula (x) wherein R. R. R'' are hydrogen and R is e not hydrogen, e.g., of the formula (X-c). N In certain embodiments, R is a monosubstituted pyridinyl Nl of the formula (x) wherein R. R. Rare hydrogen and R' is 9 not hydrogen, e.g., of the formula (X-d). R10 R 25 wherein R. R. R. R. and R' are as defined above and (X-a) herein. In certain embodiments, at least one of R. R. R. R. and R" is the group -L-RP as defined above and herein. In certain embodiments, at least one of R. R. R. R. and R' is the 30 group —R', as defined herein. In certain embodiments, R is a monosubstituted or disub (x-b) stituted pyridinyl. In certain embodiments, R is a monosubstituted pyridinyl. In certain embodiments, R is a monosubstituted pyridinyl 35 of the formula (ix) wherein R. R. R'' are hydrogen and R' is not hydrogen, e.g., of the formula (ix-a). In certain embodiments, R is a monosubstituted pyridinyl (x-c) of the formula (ix) wherein R. R. R'' are hydrogen and R' 40 is not hydrogen, e.g., of the formula (ix-b). In certain embodiments, R is a monosubstituted pyridinyl of the formula (ix) wherein R. R. R'' are hydrogen and R' is not hydrogen, e.g., of the formula (ix-c). In certain embodiments, R is a monosubstituted pyridinyl (x-d) of the formula (ix) wherein R. R. Rare hydrogen and R' 45 is not hydrogen, e.g., of the formula (ix-d).

(ix-a) 50 In certain embodiments, R is a monosubstituted pyridinyl of the formula (xi) wherein R. R. Rare hydrogen and R' is not hydrogen, e.g., of the formula (Xi-a). 55 In certain embodiments, R is a monosubstituted pyridinyl (ix-b) of the formula (v) wherein R. R. R'' are hydrogen and R is not hydrogen, e.g., of the formula (xi-b).

60 (ix-c)

65 US 8,546,432 B2 45 46 -continued -continued (xi-b) (ix-) Ne Nl N-l

In certain embodiments, R is a disubstituted pyridinyl. In certain embodiments, R is a disubstituted pyridinyl of 10 the formula (x) wherein R and Rare hydrogen and Rand In certain embodiments, R is a disubstituted pyridinyl of R" are not hydrogen, e.g., of the formula (x-e). the formula (ix) wherein Rand Rare hydrogen and R7 and In certain embodiments, R is a disubstituted pyridinyl of R" are not hydrogen, e.g., of the formula (ix-e). the formula (x) wherein RandR'' are hydrogen and Rand In certain embodiments, R is a disubstituted pyridinyl of Rare not hydrogen, e.g., of the formula (x-f). the formula (ix) wherein R and Rare hydrogen and Rand 15 In certain embodiments, R is a disubstituted pyridinyl of the formula (x) wherein R and R'' are hydrogen and Rand R" are not hydrogen, e.g., of the formula (ix-f). Rare not hydrogen, e.g., of the formula (x-g). In certain embodiments, R is a disubstituted pyridinyl of In certain embodiments, R is a disubstituted pyridinyl of the formula (ix) wherein R7 and Rare hydrogen and Rand the formula (x) wherein R and Rare hydrogen and Rand R" are not hydrogen, e.g., of the formula (ix-g). R" are not hydrogen, e.g., of the formula (x-h). In certain embodiments, R is a disubstituted pyridinyl of In certain embodiments, R is a disubstituted pyridinyl of the formula (ix) wherein RandR'' are hydrogen and Rand the formula (x) wherein RandR'' are hydrogen and Rand Rare not hydrogen, e.g., of the formula (ix-h). Rare not hydrogen, e.g., of the formula (x-i). In certain embodiments, R is a disubstituted pyridinyl of In certain embodiments, R is a disubstituted pyridinyl of the formula (ix) wherein RandR'' are hydrogen and R7 and 25 the formula (x) wherein Rand Rare hydrogen and R and Rare not hydrogen, e.g., of the formula (ix-i). R" are not hydrogen, e.g., of the formula (x-j). In certain embodiments, R is a disubstituted pyridinyl of the formula (ix) wherein RandR'' are hydrogen and Rand (X-e) Rare not hydrogen, e.g., of the formula (ix-j).

(ix-e)

35

40 (ix-f) (x-g)

45 (ix-g)

50

(ix-h)

55 (x-i)

(ix-i) 60

65

US 8,546,432 B2 49 50 In certain embodiments, X is N. In certain embodiments, X C(=O)N(R), COR', SOR'', C(-NR) is NR''. In certain embodiments, X is O. In certain embodi OR'', C(—NR’)N(R'), SON(R'). SOR'', ments, X is S. In certain embodiments, X is CR'R''. SOOR, S(—O)R'', C(-S)N(R), C(=O) In certain embodiments, R is Cso carbocyclyl or 5-10 SR, C( S)SR, P(=O).R'', P(=O)(R'), membered heterocyclyl of the formula (xiii): - P(=O)N(R'), -P(=O)(NR), Co alkyl, C perhaloalkyl, Coalkenyl, Co alkynyl, 3-14 membered heteroaliphatic, Co carbocyclyl. 3-14 membered heterocy (xiii) clyl, Caryl, and 5-14 membered heteroaryl, or two R' groups are joined to form a 3-14 membered heterocyclyl or 10 5-14 membered heteroaryl ring: each instance of R is, independently, selected from hydrogen, Coalkyl, C-operhaloalkyl, Coalkenyl, Co alkynyl, 3-14 membered heteroaliphatic, Co carbocyclyl, 3-14 membered heterocyclyl, C-aryl, and 5-14 membered 15 heteroaryl, or two R' groups are joined to form a 3-14 mem wherein: bered heterocyclyl or 5-14 membered heteroaryl ring: X is N, NRO, O, S or CRR’; and L. RandR are as defined above and herein. p is 0, 1 or 2: In certain embodiments, at least one of R. R. R. R. R’, R, R-7, R,R,R,R, and R is the group-L-RP as defined above and herein. In certain embodiments, at least one of at least one of R, R, R,R,R,R, R-7, R, R. R. R', and R° is selected from -R as defined herein. 25 In certain embodiments, p is 0. In certain embodiments, p is 1. In certain embodiments, p is 2. In certain embodiments, X is N. In certain embodiments, X is NR''. In certain embodiments, X is O. In certain embodi ments, X is S. In certain embodiments, X is CR'R''. 30 For example, in certain embodiments, X is O. In certain embodiments, R is a 5-10 membered heterocyclyl of the formulae (xii-a) or (xiii-a):

35 (xii-a)

OP(R), OP(R), B(OR), or BR' (OR), Coalkyl, Co perhaloalkyl, Coalkenyl, Co alkynyl, 40 3-14 membered heteroaliphatic, Co carbocyclyl, 3-14 membered heterocyclyl, C. aryl, 5-14 membered het eroaryl, -L-RP and -R: or one or more of RandR, R’ (xiii-a) and R', RandR, RandR, Rand R7, RandR, and Rand R', are joined to form a double bond or a Co 45 carbocyclyl, 3-14 membered heterocyclyl, Caryl or 5-14 membered heteroaryl ring; optionally wherein X is N, then N and R' or N and Rare joined to form a double bond; R is selected from hydrogen, OH, -OR'', R27 N(R). CN, C(=O)R’, C(=O)N(R), 50 COR'', SOR'', C( NR’)OR'', C( NR’)N (R), SON(R), SOR, SOOR, S(–O) wherein p, R2, R22, R2, R2, R2, R2, R27, R2 and R2 R'', C(—S)N(R), C(=O)SR, C(-S)SR, are as defined above and herein. P(=O).R.' s - P(=O) (R), P(=O)N(R )2. In certain embodiments, X is NR. For example, in certain —P(=O)(NR), Coalkyl, Coperhaloalkyl, Coalk 55 embodiments, R is heterocyclyl of the formulae (xii-b) or enyl, Co alkynyl, 3-14 membered heteroaliphatic, Co (xiii-b): carbocyclyl, 3-14 membered heterocyclyl, C. aryl, and 5-14 membered heteroaryl, or RandR' or R' and Rare joined to form a 3-14 membered heterocyclyl or 5-14 mem (xii-b) bered heteroaryl ring: 60 wherein: each instance of R' is, independently, selected from Co alkyl, Co perhaloalkyl, Coalkenyl, Co alkynyl, Co carbocyclyl, 3-14 membered heterocyclyl, C. aryl, and 5-14 membered heteroaryl; 65 each instance of R’ is, independently, selected from hydrogen, OH, OR', N(R'), CN, C(=O)R', US 8,546,432 B2 51 52 -continued – OP(=O)N(R) - P(=O)(NR), OP(=O) (xiii-b) (NR'2), NR2P(=O)(OR), NR2P(=O)(NR'2), P(R), P(R), OP(R), – OP(R), B(OR), or - BR" (OR), Co alkyl, Co perhaloalkyl, Coalkenyl, Co alkynyl, 3-14 mem bered heteroaliphatic, Cocarbocyclyl, 3-14 membered het erocyclyl, Caryl, 5-14 membered heteroaryl, -L-RP and R: or one or more of R7 and R, R and R, R and R. RandR, R and R, RandR, RandR, and 10 R" and R', are joined to form a double bond or a Co wherein p, R2, R22, R2, R2, R2, R2, R27, R2, R2 and carbocyclyl, 3-14 membered heterocyclyl, Caryl or 5-14 R" are as defined above and herein. membered heteroaryl ring; optionally wherein Q is N, then N In certain embodiments, X is CR'R''. For example, in and R' or N and Rare joined to form a double bond; certain embodiments, R is Cso carbocyclyl of the formula 15 R' is selected from hydrogen, OH, -OR.", (xii-c): N(R) - CN, C(=O)R'', C(=O)N(R), COR', SOR", C(-NR)OR', C(-NR)N (R), SON(R). SOR, SOOR, SOR, (xii-c) C(=S)N(R), C(=O)SR, C(—S)SR, - P(=O).R.", P(=O)(R') - P(=O)N(R), —P(=O)(NR), Co alkyl, Coperhaloalkyl, Coalk enyl, Co alkynyl, 3-14 membered heteroaliphatic, Co carbocyclyl, 3-14 membered heterocyclyl, C. aryl, and 5-14 membered heteroaryl, optionally wherein RandR' or 25 R" and Rarejoined to forma3-14 membered heterocyclyl, R27 or 5-14 membered heteroaryl ring: wherein: wherein p. R2, R22, R23, R24, R25, R26, R27, R2, R29, R31 each instance of R is, independently, selected from Co and R° are as defined above and herein. alkyl, Co perhaloalkyl, Coalkenyl, Co alkynyl, Co Joined Groups RandR 30 carbocyclyl, 3-14 membered heterocyclyl, C. aryl, and As described generally above, in certain embodiments, R. 5-14 membered heteroaryl; and R together with the nitrogen (N) atom to which each is each instance of R is, independently, selected from attached are joined to form a 5-14 membered ring. hydrogen, OH, OR, N(R), —CN, C(=O)R'', For example, in certain embodiments, RandR together C(=O)N(R), COR', SOR", C(-NR) with the nitrogen (N) atom to which each is attached are 35 OR'', C(—NR)N(R), SON(R), SOR, joined to form a 5-14 membered ring of the formula (xiv): - SOOR, S(=O)R'', C(=S)N(R), C(=O) SR, C(-S)SR, P(O).R'', P(=O)(R'), - P(=O)N(R), -P(=O)(NR), Co alkyl, C (xiv) 40 perhaloalkyl, Coalkenyl, Co alkynyl, 3-14 membered heteroaliphatic, Co carbocyclyl. 3-14 membered heterocy clyl, Caryl, and 5-14 membered heteroaryl, or two R. groups are joined to form a 3-14 membered heterocyclyl or 5-14 membered heteroaryl ring: 45 each instance of R is, independently, selected from hydrogen, Coalkyl, C-operhaloalkyl, Coalkenyl, Co alkynyl, 3-14 membered heteroaliphatic, Co carbocyclyl, 3-14 membered heterocyclyl, C-aryl, and 5-14 membered wherein: heteroaryl, or two R groups are joined to form a 3-14 mem Q is N, NRO, O, S, or CR'R'' 50 bered heterocyclyl or 5-14 membered heteroaryl ring: m is 0, 1 or 2; and and L. RandR' are as defined above and herein. each instance of R, R2, R,R,R,R, R-7, R, R In certain embodiments, at least one of R', R', R. R. and R' is independently selected from hydrogen, halogen, R. R. R. R7, R, R and R is the group -L-RP as defined above and herein. In certain embodiments, at least 55 O of R, R4, R2, R-43, R14, R15, R16, R17, R18, RandR is selected from —R as defined herein. In certain embodiments, m is 0. In certain embodiments, m is 1. In certain embodiments, m is 2. In certain embodiments, Q is N. In certain embodiments, Q 60 is NR". In certain embodiments, Q is O. In certain embodi ments, Q is S. In certain embodiments, Q is CR'R''. In certain embodiments, R'' and Rare joined to form a double bond and RandRarejoined to form a Caryl or 5-14 membered heteroaryl. For example, in certain embodi 65 ments, R” and R together with the nitrogen (N) atom to which each is attached are joined to form a 5-14 membered ring of the formula (XV): US 8,546,432 B2 53 54 each instance of R is, independently, selected from (xv) hydrogen, OH, OR, N(R), CN, C(=O)R, C(=O)N(R), CO.R., SOR, C(-NR) OR, C(-NR)N(R), SON(R), SOR, SOOR, SOR, C(-S)N(R), C(=O)SR, C(—S)SR, P(=O).R., P(=O)(R), P(=O), N(R) - P(=O)CNR), Co alkyl, Co perhaloalkyl, Coalkenyl, Co alkynyl, 3-14 membered heteroaliphatic, C. carbocyclyl, 3-14 membered heterocyclyl, Caryl, 10 and 5-14 membered heteroaryl, or two R' groups are joined to form a 3-14 membered heterocyclyl or 5-14 membered heteroaryl ring; and wherein Q, m, W, R, R2, R., R., R., R., R, R7, R8 each instance of R is, independently, selected from and Rare as defined above and herein. hydrogen, Coalkyl, Coperhaloalkyl, Coalkenyl, Co 15 alkynyl, 3-14 membered heteroaliphatic, Co carbocyclyl, In certain embodiments, Q is CR'R''. R and R' are 3-14 membered heterocyclyl, C-aryl, and 5-14 membered joined to form a double bond and R and R' are joined to heteroaryl, or two R' groups are joined to form a 3-14 mem form a Caryl or 5-14 membered heteroaryl. For example, bered heterocyclyl or 5-14 membered heteroaryl ring. in certain embodiments, RandR together with the nitrogen In certain embodiments, at least one of R. R. R. R. (N) atom to which each is attached are joined to form a group R7, R. R. R7, RandR is the group-L-RP as defined of the formula (xvi): above and herein. In certain embodiments, at least one of R". R,R,R, R-7, R, R, R7, RandR is selected from —R as defined herein. (xvii) In certain embodiments, m is 0. In certain embodiments, m 25 is 1. In certain embodiments, m is 2. Group R. As described generally above, R is selected from hydro gen, OH, -OR'', ON(R), N(R), —C(=O) R", CHO, COR', C(=O)N(R), C(-NR) 30 ORC1, C(—NRC)N(RCP). SOR', S(=O)RC, —Si(R)s. Clio alkyl, Co perhaloalkyl, Co alkenyl, Coalkynyl, 3-14 membered heteroaliphatic, Cocarbocy clyl. 3-14 membered heterocyclyl, C-aryl, and 5-14 mem wherein m, W. R. R. R. R. R7 and Rare as defined bered heteroaryl; above and herein; and 35 wherein: wherein R. R. Rand Rare independently selected each instance of R is, independently, selected from Co from the group consisting of hydrogen, halogen, —CN. alkyl, Co perhaloalkyl, Coalkenyl, C2-io alkynyl, Cso NO, N, -SOH, -SOH, -OH, -OR. - ON carbocyclyl, 3-14 membered heterocyclyl, C. aryl, and (RS), N(RS), N(OR)R, SH, SR, 5-14 membered heteroaryl; and SSR, C(=O)R, COH, CHO, C(OR), 40 each instance of R is, independently, selected from COR, OC(=O)R, OCOR, C(=O)N hydrogen, OH, -OR.", N(R), —CN, -C(=O) (RS), OC(=O)N(R), NRC(O)R*, RC', C(=O)N(RC), CORC, SORC, NRSCOR, NRC(O)N(R), C( NRS) C(-NR)OR', C(-NR)N(R), SON(R), OR*, OC( NRS)R’, OC( NRS)OR, SORC, SOOR, SOR", C(—S)N(R), C(-NRS)N(R), OC(—NRS)N(R), NRC 45 C(=O)SRC, C(—S)SRC, P(=O).R.", P(=O) (—NR)N(R), C(=O)NRSOR, (R'), - P(=O)N(R), P(=O)NR), C-io alkyl, NRSO.R., SON(R), SOR, SOOR, Co perhaloalkyl, Coalkenyl, Co alkynyl, 3-14 mem OSOR, S(—O)R’, OS(—OR, Si(R), bered heteroaliphatic, Cocarbocyclyl, 3-14 membered het –OSi(R), C(=S)N(R), C(=O)SR, C(=S) erocyclyl, C-aryl, and 5-14 membered heteroaryl, or two SR, SC(S)SR, P(=O).R, OP(=O).R., 50 R° groups are joined to form a 3-14 membered heterocyclyl P(=O)(R), OP(=O)(R), OP(=O)(OR), or 5-14 membered heteroaryl ring: or R and R together with the nitrogen (N) atom to which each is attached are joined to form a 5-14 membered ring. In certain embodiments, R is selected from Co alkyl, OP(R), OP(R), B(OR), or BR(OR), 55 Co perhaloalkyl, Coalkenyl, C2-io alkynyl, 3-14 mem Coalkyl, Co perhaloalkyl, Coalkenyl, Co alkynyl, bered heteroaliphatic, Cocarbocyclyl, 3-14 membered het 3-14 membered heteroaliphatic, Co carbocyclyl, 3-14 erocyclyl, Caryl, and 5-14 membered heteroaryl. membered heterocyclyl, C. aryl, 5-14 membered het In certain embodiments, R is an unsubstituted group, e.g., eroaryl, -L-RP and R. or one or more of Rand R7, R7 selected from unsubstituted Co alkyl, unsubstituted Co and R', and RandR' are joined to form a Cso carbocy 60 alkenyl, unsubstituted Co alkynyl, unsubstituted 3-14 clyl. 3-14 membered heterocyclyl, C-aryl or 5-14 mem membered heteroaliphatic, unsubstituted Co carbocyclyl, bered heteroaryl ring: unsubstituted 3-14 membered heterocyclyl, unsubstituted each instance of R is, independently, selected from Co C. aryl and unsubstituted 5-14 membered heteroaryl. alkyl, Co perhaloalkyl, Coalkenyl, C2-io alkynyl, 3-14 However, in certain embodiments, R is an unsubstituted membered heteroaliphatic, Co carbocyclyl, 3-14 mem 65 group wherein—CH and —CH2CH are excluded. bered heterocyclyl, C. aryl, and 5-14 membered het In certain embodiments, R is a group having 2 or more eroaryl; carbon atoms, e.g., selected from C2-io alkyl, C2-o perha US 8,546,432 B2 55 56 loalkyl, Coalkenyl, Co alkynyl, 3-14 membered het stituted Cocarbocyclyl, unsubstituted 3-14 membered het eroaliphatic, Cocarbocyclyl, 3-14 membered heterocyclyl, erocyclyl, unsubstituted C. aryl and unsubstituted 5-14 Caryl, and 5-14 membered heteroaryl. In certain embodi membered heteroaryl. ments, R is an unsubstituted group having 2 or more carbon In certain embodiments, R is Cso carbocyclyl. In certain atoms. However, in certain embodiments, R is a group hav embodiments, R is Cao carbocyclyl. In certain embodi ing 2 or more carbon atoms wherein —CH2CH is excluded. ments, R is Cso carbocyclyl. In certain embodiments, R is In certain embodiments, R is a group having 3 or more Css carbocyclyl. In certain embodiments, R is Co car carbon atoms, e.g., selected from Cao alkyl, Cao perha bocyclyl selected from cyclopropyl (C), cyclobutyl (C), loalkyl, Coalkenyl, Co alkynyl, 3-14 membered het cyclopentyl (Cs), cyclopentenyl (Cs), cyclohexyl (C), cyclo eroaliphatic, Co carbocyclyl, 3-14 membered heterocyclyl, 10 hexenyl (C), cyclohexadienyl (C), cycloheptyl (C7), cyclo Caryl, and 5-14 membered heteroaryl. In certain embodi heptadienyl (C7), cycloheptatrienyl (C7) and cyclooctyl (Cs). ments, R is an unsubstituted group having 3 or more carbon In certain embodiments, R is Cso carbocyclyl selected atoms. However, in certain embodiments, R is a group hav from cyclopenty1 and cyclohexyl. In certain embodiments, ing 3 or more carbon atoms wherein —CH(CH) is R is an unsubstituted Cocarbocyclyl. excluded. 15 In certain embodiments, R is 3-14 membered heterocy In certain embodiments, R is a group having 4 or more clyl. In certain embodiments, R is 5-10 membered hetero carbon atoms, e.g., selected from Cao alkyl, Cao perha cyclyl. In certain embodiments, R is 5-6 membered hetero loalkyl, Cao alkenyl, Cao alkynyl, 5-14 membered het cyclyl. In certain embodiments, R is 3-14 membered eroaliphatic, Cso carbocyclyl, 5-14 membered heterocyclyl, heterocyclyl selected from azirdinyl, oxiranyl, thiorenyl, aze Caryl, and 5-14 membered heteroaryl. In certain embodi tidinyl, oxetanyl, thietanyl, tetrahydrofuranyl, dihydrofura ments, R is an unsubstituted group having 4 or more carbon nyl, tetrahydrothiophenyl, dihydrothiophenyl, pyrrolidinyl, atOmS. dihydropyrrolyl, dioxolanyl, oxathiolanyl, dithiolanyl, pip In certain embodiments, R is an acyclic group, e.g., eridinyl, tetrahydropyranyl, dihydropyridinyl, thianyl, piper Selected from Co alkyl, Coalkenyl, C2-io alkynyl and azinyl, morpholinyl, dithianyl, dioxanyl, azepanyl, oxepanyl 3-14 membered heteroaliphatic. In certain embodiments, R. 25 thiepanyl, azocanyl, oxecanyl and thiocanyl. In certain is an unsubstituted acyclic group, e.g., selected from unsub embodiments, R is 3-14 membered heterocyclyl selected stituted Co alkyl, unsubstituted Coalkenyl, unsubsti from tetrahydropyranyl. In certain embodiments, R is an tuted Co alkynyl and unsubstituted 3-14 membered het unsubstituted 3-14 membered heterocyclyl. eroaliphatic. However, in certain embodiments, R is an In certain embodiments, R is Cola aryl. In certain acyclic group, wherein—CH and —CHCH are excluded. 30 embodiments, R is a Caryl selected from phenyl, naph In certain embodiments, R is Clio alkyl. In certain thyl and anthracyl. In certain embodiments, R a Cola aryl embodiments, R is an unsubstituted Co alkyl. In certain selected from phenyl. In certain embodiments, R is an embodiments, R is Coalkyl, wherein —CH is excluded. unsubstituted Caryl. In certain embodiments, R is Clio alkyl, wherein In certain embodiments, R is 5-14 membered heteroaryl. —CHCH is excluded. In certain embodiments, R is Co. 35 In certain embodiments, R is 5-10 membered heteroaryl. In alkyl, wherein—CH(CH) is excluded. certain embodiments, R is 5-6 membered heteroaryl. In cer In certain embodiments, R is Clo alkyl, e.g., selected tain embodiments, R is a 5-membered heteroaryl, e.g., from ethyl, n-propyl, isopropyl. n-butyl, tert-butyl, sec-butyl, selected from pyrrolyl, furanyl, thiophenyl, imidazolyl pyra iso-butyl, n-pentyl, pentan-3-yl, amyl, neopentyl, 3-methyl Zolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, triazolyl, 2-butanyl, tertiary amyland n-hexyl. In certain embodiments, 40 oxadiazolyl, thiadiazolyl and tetrazolyl. In certain embodi R is an unsubstituted Clo alkyl. In certain embodiments, ments, R is a 6-membered heteroaryl, e.g., selected from R is Co alkyl, wherein—CHCH is excluded. In certain pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl and embodiments, R is Co alkyl, wherein -CH(CH4), is tetrazinyl. In certain embodiments, R is an unsubstituted excluded. 5-14 membered heteroaryl. In certain embodiments, R is Co alkyl, e.g., selected 45 Exemplary Combinations of Groups R, R and R. from n-propyl, isopropyl. n-butyl, tert-butyl, sec-butyl, iso Various combinations of R", R and/or R are contem butyl, n-pentyl, pentan-3-yl, amyl, neopentyl, 3-methyl-2- plated herein, and are described in more detail below and butanyl, tertiary amyl and n-hexyl. In certain embodiments, herein. R is an unsubstituted Co alkyl. In certain embodiments, For example, in certain embodiments, both R and R are R is Coalkyl, wherein—CH(CH), is excluded. 50 cyclic, i.e., R is selected from Cso carbocyclyl 3-14 mem In certain embodiments, R is Caio alkyl, e.g., selected bered heterocyclyl, Caryland 5-14 membered heteroaryl, from n-butyl, tert-butyl, Sec-butyl, iso-butyl, n-pentyl, pen and R is selected from Cso carbocyclyl, 3-14 membered tan-3-yl, amyl, neopentyl, 3-methyl-2-butanyl, tertiary amyl heterocyclyl, Caryl, and 5-14 membered heteroaryl. In and n-hexyl. In certain embodiments, R is an unsubstituted certain embodiments, R is a group having 2 or more carbon Caio alkyl. 55 atoms. In certain embodiments, R is a group having 3 or In certain embodiments, R is Coalkenyl. In certain more carbon atoms. In certain embodiments, R is a group embodiments, R is an unsubstituted Coalkenyl. In certain having 4 or more carbon atoms. In certain embodiments, R. embodiments, R is Coalkenyl selected from allyl. is an unsubstituted cyclic group. In certain embodiments, R is Co alkynyl. In certain In certain embodiments, R is cyclic and R is acyclic, i.e., embodiments, R is an unsubstituted Co alkynyl. 60 R’ is selected from Cso carbocyclyl, 3-14 membered het In certain embodiments, R is 3-14 membered het erocyclyl, Caryland 5-14 membered heteroaryl, andR is eroaliphatic. In certain embodiments, R is an unsubstituted selected from -OH, -OR", ON(R), N(R), 3-14 membered heteroaliphatic. C(=O)R', -CHO, CO.R.", C(=O)N(R), In certain embodiments, R is a cyclic group, e.g., selected C(-NR)OR', C(-NR)N(R). SOR", from Co carbocyclyl, 3-14 membered heterocyclyl, C 65 —S(=O)R', Si(R), Co alkyl, Co perhaloalkyl, aryland 5-14 membered heteroaryl. In certain embodiments, Coalkenyl, Co alkynyl, 3-14 membered heteroaliphatic, R is an unsubstituted cyclic group, e.g., selected from unsub where RandR areas defined above and herein. In certain US 8,546,432 B2 57 58 embodiments, R is an acyclic group having 2 or more carbon atoms. In certain embodiments, R is an acyclic group having (II-a) 3 or more carbon atoms. In certain embodiments, R is an acyclic group having 4 or more carbon atoms. In certain R2 embodiments, R is an unsubstituted acyclic group. 3-y' a In certain embodiments, R and R are independently RNw? \ l e R8 selected from Caryl and 5-14 membered heteroaryl. In y-s-s y certain embodiments, R is Caryl, and R is Caryl or R S W \= IN N W 5-14 membered heteroaryl. In certain embodiments, R is 10 R5 RC R9 5-14 membered heteroaryl, and R is Caryl or 5-14 mem bered heteroaryl. In certain embodiments, R is Caryl or 2 (II-b) 5-14 membered heteroaryl, and R is Caryl. In certain R R1 R7 embodiments, R is Caryl or 5-14 membered heteroaryl, 3 2-w^ O O I and R is 5-14 membered heteroaryl. 15 In certain embodiments, both R" and Rare Caryl. In Wsw y N N - N-NC certain embodiments, both R and Rare phenyl (Caryl). In R4 W NRN f W certain embodiments, R is Cola aryl and R is Cso car R5 RC I bocyclyl. R10 In certain embodiments, R is C. aryl and R is 5-14 (II-c) membered heteroaryl. R2 \ R In certain embodiments, R is Caryl and R is 3-14 WN w O O membered heterocyclyl. RNw? \ l e R8 In certain embodiments, R is Caryl and R and R' 25 together with the nitrogen (N) atom to which each is attached wSt y- N N } are joined to form a 5-14 membered ring. R V \={ N w-l R5 RC I In certain embodiments, both R and R are 5-14 mem R10 bered heteroaryl. 30 In certain embodiments, R is 5-14 membered heteroaryl and R is Cocarbocyclyl. or a pharmaceutically acceptable form thereof; In certain embodiments, R is 5-14 membered heteroaryl wherein R. W. R. W. R. W. R. W. R. W. R. and R is Caryl. W. R. W. R. W. R. and W. R'' are as defined above In certain embodiments, R is 5-14 membered heteroaryl 35 and herein. and R is 3-14 membered heterocyclyl. In certain embodiments, at least one of R. R. Rand R' In certain embodiments, R is 5-14 membered heteroaryl, of the formulae (II-a), (II-b) or (II-c) is the group -L-RP as and RandR together with the nitrogen (N) atom to which defined above and herein. In certain embodiments, at least one of R. R. RandR' of the formulae (II-a), (II-b) or (II-c) each is attached are joined to form a 5-14 membered ring. 40 is further selected from the group R', as defined above and In certain embodiments, the compound is of the formula herein. (II): In certain embodiments, the compound is of the formula (III): (II) 45 R2 \ RI R7 (III) W-N/ O R6 I R3% W SP NW sy y l - W2 Yv-R 50 O O R I Wsw NV /N -R 4. R3 leW. R4 V NRN I w-" R5 RC I R9 V--- / N-N- Yv-R4. R10 R4 NFN I w-" R5 RC I R9 55 R10 or a pharmaceutically acceptable form thereof; wherein R. W. R. W. R. W. R. W. R. W. R. W. R. W. R7, W. R. W. R., and W. Rare as defined or a pharmaceutically acceptable form thereof; above and herein. wherein R. R. R. R. R. R. W. R. W. R. W. R. In certain embodiments, at least one of R. R. R. Rand 60 W. R. and W. R'' are as defined above and herein. R' of the formula (II) is the group -L-RP as defined above In certain embodiments, at least one of R. R. R. Rand and herein. In certain embodiments, at least one of R. R. R. R' of the compound of formula (III) is the group -L-RP as R and R' of the formula (II) is further selected from the defined above and herein. In certain embodiments, at least group —R as defined above and herein. 65 one of R. R. R. RandR' of the compound of formula (III) In certain embodiments, the compound is of the formulae is further selected from the group R as defined above and (II-a), (II-b) or (II-c): herein. US 8,546,432 B2 59 60 In certain embodiments, the compound is of the formulae Cso carbocyclyl; and R-R" are independently selected (III-a), (III-b) or (III-c): from H. Coalkyl, Co alkyloxy, Caryloxy, COOH, and COR". In certain embodiments, R'-Rare indepen dently H. methyl, methoxy, CN, and SOMe: R is unsubsti 5 tuted C- alkyl or unsubstituted Cse cycloalkyl; and R-R' (III-a) are independently selected from H. methyl, methoxy, phe R2 noxy, COOH, and COMe. RI In certain embodiments, the compound is of the formulae O O (IV-a), (IV-b), (IV-c) or (IV-d): R3 l - e w-R N N I V / N R4 R5 NRN t N "\ (IV-a) 2 (III-b) R R1 R2 O R7 15 R O O I S \l R8 N - N R3 N\ 1 |N - Y-NC R4 R5 NRN t R9 R4 NRN I W (IV-b) R5 RC I R2 R10 R1 R7 (III-c) O 2 R RI is? W. J f 25 O O N R4 NNRN - I R5 RC R3 N N l ?y R10 R4 NRN YN-\ff W (IV-c) R5 RC I 2 R10 R R1 O R3 O or a pharmaceutically acceptable form thereof; l R8 wherein RC, R, R2, R. R. R. W. R7, W. R. W. R. and W R'' are as defined above and herein. 35 N R4 NNFN N f In certain embodiments, at least one of R. R. Rand R' R5 RC of the formulae (III-a), (III-b) or (III-c) is the group -L-RP as R10 defined above and herein. In certain embodiments, at least (IV-d) one of R. R. RandR' of formulae (III-a), (III-b) or (III-c) R2 is further selected from the group R', as defined above and 40 herein. ex N O O 6 R7 In certain embodiments, the compound is of the formula R11 l 2\X (IV): 2 N N cool \ f N N 45 NRN I (IV) R2 or a pharmaceutically acceptable form thereof; RI O 6 R7 R wherein RC, R, R,R,R,R,R, R7, R, R, and Rare 3 O 50 as defined above and herein. R l R8 In certain embodiments, at least one of R. R. Rand R' N N of the formulae (IV-a), (IV-b) or (IV-c) is the group -L-RP as R4 VNFN R / IN defined above and herein. In certain embodiments, at least R5 RC R9 R10 one of R. R. RandR' of the formulae(IV-a), (IV-b),(IV-c) 55 or (IV-d) is further selected from the group R as defined above and herein. or a pharmaceutically acceptable form thereof; In one embodiment, provided herein is a compound of wherein R,R,R,R,R,R,R,R,R,R, and Rare formula (IV-d), or a pharmaceutically acceptable form as defined above and herein. thereof. In one embodiment where the compound is of for In certain embodiments, at least one of R. R. R. Rand 60 mula (IV-d), R is Coalkyl or Cocarbocyclyl. In one R' of the formula (IV) is the group -L-RP as defined above embodiment, R is ethyl, isopropyl, cyclopentyl or cyclo and herein. In certain embodiments, at least one of R. R. R. hexyl. R and R' of the formula (IV) is further selected from the In another embodiment where the compound is of formula group R', as defined above and herein. In certain embodi (IV-d), R' and Rare each independently hydrogen, halogen, ments, R-R are independently H, Co alkyl, Clio alky 65 —CN, OR' or -SOR', wherein R' is Coalkyl. In loxy, C. aryloxy, CN, SON(R7), SOR', and another embodiment, R' and R are each independently —SO,OR'; R is unsubstituted Coalkyl or unsubstituted hydrogen, fluoro, methoxy, —CN or —SOCH. US 8,546,432 B2 61 62 In another embodiment where the compound is of formula wherein R, X,Y,Z, R', R,R,R,R,R, and R'' are as (IV-d), R and R7 are each independently hydrogen, halogen defined above and herein. or —O R', wherein R' is Co alkyl or Caryl. In In certain embodiments, at least one of R. R. Rand R' another embodiment, R and R are each independently of the formulae (V-a), (V-b) or (V-c) is the group -L-RP as hydrogen, fluoro, methoxy or phenyloxy. defined above and herein. In certain embodiments, at least In certain embodiments, the compound is of the formula one of R. R. RandR' of the formulae (V-a), (V-b) or (V-c) (V): is further selected from the group R as defined above and herein. (V) 10 In certain embodiments, the compound is of the formula (VI):

(VI) 15 R2 R1 R7 O 6 R3 O R

N N - R8 W V / N or a pharmaceutically acceptable form thereof; N NFN I 9 wherein R, X,Y,Z, R,R,R,R,R,R,R, and Rare N-N RC R as defined above and herein. Y R10 In certain embodiments, at least one of R. R. R. Rand R' of the formula (V) is the group -L-RP as defined above 25 or a pharmaceutically acceptable form thereof; and herein. In certain embodiments, at least one of R. R. R. R and R' of the formula (V) is further selected from the wherein R. Y. R. R. R. R. R. R. R. and R'' are as group —R as defined above and herein. defined above and herein. In certain embodiments, the compound is of the formulae In certain embodiments, at least one of R. R. R. Rand (V-a), (V-b) or (V-c): 30 R' of the formula (VI) is the group -L-RP as defined above and herein. In certain embodiments, at least one of R. R. R. R and R' of the formula (VI) is further selected from the (V-a) group R', as defined above and herein. In certain embodi ments, R-R are independently H, Co alkyl, Clio alky 35 loxy, C aryloxy, CN, SON(R7), SOR', and —SO,OR'; R is unsubstituted Coalkyl or unsubstituted R8 Cso carbocyclyl; and R-R" are independently selected from H. Co alkyl, Co alkyloxy, Caryloxy, COOH, 40 and CO.R". In certain embodiments, R'-R are indepen R9 dently H, methyl, methoxy, and CN; R is unsubstituted Cs. cycloalkyl; and R-R'' are independently selected from H. methyl, methoxy, phenoxy, COOH, and COMe. In certain embodiments, the compound is of the formulae (V-b) 45 (VI-a), (VI-b) or (VI-c): R7

(VI-a) R2 R1 O R10 S \, lif R8 55 N - N (V-c) ^ NRN l 9 N-N R R R2 (VI-b) 60 R1 R7 R8 * ( \lO N - N R10 65 ^ R = i \-N R R10 or a pharmaceutically acceptable form thereof; US 8,546,432 B2 63 64 -continued embodiments, a compound of formula (I) is any of the com (VI-c) pounds provided in Tables 1, 2 or 3 having an activity of 'A' R2 or A*. In certain embodiments, a compound of formula (I) RI is any of the compounds provided in Tables 1, 2 or 3 having an O 5 activity of “B” or “B”. In certain embodiments, a compound R8 of formula (I) is any of the compounds provided in Tables 1, ( S J f 2 or 3 having an activity of “C” or “C”. / NNRN - fN In certain embodiments, compounds provided herein \,-N R 10 include any of the compounds provided in Tables 1, 2 or 3 substituted with a group -L-RP, as defined above and herein, and having an activity of “A”, “A*”, “B” or “B*”. or a pharmaceutically acceptable form thereof; For example, in certain embodiments, the compound of wherein R. Y. R. R. R. R. R. R., and R'' are as formula (I) is a compound selected from the group consisting defined above and herein. 15 of: In certain embodiments, at least one of R. R. Rand R' of the formulae (VI-a), (VI-b) or (VI-c) is the group -L-RP as defined above and herein. In certain embodiments, at least one of R7, R, R and R' of the formulae (VI-a), (VI-b) or (VI-c) is further selected from the group R', as defined above and herein. Exemplary Compounds NFN I Exemplary compounds are set forth in the Exemplification Et COH, and listed in Tables 1, 2, 3 and 4 provided therein. O In certain embodiments, a compound of formula (I) is 25 selected from any of the compounds provided in Tables 1, 2, -l CO2H, 3 or 4. In certain embodiments, a compound of formula (I) is N N selected from any of the compounds provided in Table 1. In NFN | certain embodiments, a compound of formula (I) is selected Et OMe O from any of the compounds provided in Table 2. In certain 30 O embodiments, a compound of formula (I) is selected from any ul OMe of the compounds provided in Table 3. In certain embodi N N ments, a compound of formula (I) is selected from any of the V / N NRN compounds provided in Table 4. SOMe COH, In certain embodiments, a compound of formula (I) is 35 selected from any of the compounds provided in Tables 1, 2 or 3. In certain embodiments, a compound of formula (I) is selected from any of the compounds provided in Tables 1 or 2. O In certain embodiments, a compound of formula (I) is O selected from any of the compounds provided in Tables 1 or 3. 40 -l CO2H, N N In certain embodiments, a compound of formula (I) is V / N selected from any of the compounds provided in Tables 1 or 4. NRN In certain embodiments, a compound of formula (I) is OPh Et O selected from any of the compounds provided in Tables 2 or 3. O In certain embodiments, a compound of formula (I) is 45 -l CO2H, selected from any of the compounds provided in Tables 2 or 4. N N Activities provided from the FASN NADPH Consumption V / N Assay are designated in Tables 1, 2, 3 and 4 with a star (*). NFN wherein “A*” refers to compounds having an ICs of less than OMe Et 60 nM; “B” refers to compounds having an ICs of 60 nM to 50 250 nM, inclusive: “C” refers to compounds having an ICso l o, MeO of greater than 250 nM to 1000 nM, inclusive: “D*” refers to N - compounds having an ICs of greater than 1000 nM to 10,000 V / N NRN | nM, inclusive; and “E*” refers to compounds having an ICso OMe Et COH, of greater than 10,000 nM, as measured by the assay. 55 O Activities provided from the FASN Scintillation Proximity O Flashplate Assay are provided in Tables 1,2,3 and 4, wherein -l COBn, “A” refers to compounds having an ICs of less than 15 N N V / N nM/mL: “B” refers to compounds having an ICs of 15 nM to NRN 100 nM, inclusive: “C” refers to compounds having an ICs of 60 OMe Et greater than 100 nM to 200 nM, inclusive: “D” refers to O compounds having an ICs of greater than 200 nM to 5000 O nM, inclusive; and “E” refers to compounds having an ICs of -l COBn, N N greater than 5000 nM, as measured by the assay. V / N In certain embodiments, a compound of formula (I) is any 65 NRN I of the compounds provided in Tables 1, 2 or 3 having an OMe Et OMe activity of “A”, “A*”, “B”, “B”, “C” or “C**. In certain

US 8,546,432 B2 83 84 -continued -continued F O O OMe, l O OMe, N N N - N 5 \ / N HN NRN O iPr COBn NH2 CO2H N

10

F O O or a pharmaceutically acceptable form thereof. l - YNefYa OMe, In certain embodiments, the compound of formula (I) is a N N N 15 compound selected from the group consisting of: N E I iPr COBn O PhMeN NE F F O O o, MeO O 2O

N - OMe, N /N N NRN I F COH, O iPr CO2H PhMeN 25 O O F OMe, O O N N \ / N 30 OMe, NRN I N N O iPr CO2H V / N BMeN NRN F F CO2H O O 35 OMe, N N N F NFN I O O iPr COBn O OMe, BMeN F 40 N N O \ / N O NRN | -K OMe, F iPr CO2H N N F N O \= 45 O N CO2H NH2, N N V / N NFN f O F iPr CO2H 50 OMe, O l N Ya e l OMe, N El f N / N ( e N CO2H NRN S 55 F CO2H MeO

OMe 60 F O - N Y a E-l f N ( -N COPMB, / l N S NRN f 65 F iPr COH, US 8,546,432 B2 85 -continued -continued F F O O MeO O -l OMe, OMe, N N 5 N N V / N V / N NRN f NRN F iPr CO2H OMe CO2H F O o, MeO l OMe, 10 N N F O V / N NRN -l O MeO F CO2H N N - 15 V / N NFN f OMe iPr COH, F O O -l NH2, 2O F N N O V / N O NRN OMe, F CO2H N N \= i 25 /-/ CO2H F O NO O

l NMe2, O N N 30 O V / N NRN -l OMe, F COH N N W V / N N NRN N N CO2H 35 S-1 F O MeO -l OMe, N N V / N 40 NFN O F CO2H O OMe, N N V / N F O 45 HN NRN l o, MeO NH2 CO2H N - V / N NRN I F iPr COH, 50 or a pharmaceutically acceptable form thereof. O O In other embodiments, the compound of formula (I) is a OMe, compound selected from the group consisting of N N V / N 55 NRN f F OMe iPr CO2H O O O l MeO N N N - 60 \ / N V / N C--O-F NFN NRN -Q O OMe COH,

65

US 8,546,432 B2 89 90 -continued exchangers, alumina, aluminum Stearate, lecithin, serum pro F teins, such as human serum albumin, buffer Substances Such O as phosphates, glycine, Sorbic acid, or potassium Sorbate, l O partial glyceride mixtures of Saturated vegetable fatty acids, N N -K water, salts or electrolytes, such as protamine Sulfate, diso V / N NRN f dium hydrogen phosphate, potassium hydrogen phosphate, OMe iPr COH, Sodium chloride, Zinc salts, colloidal silica, magnesium tri F silicate, polyvinyl pyrrolidone, polyacrylates, waxes, poly O -l O MeO ethylene-polyoxypropylene-block polymers, wool fat, Sugars 10 Such as lactose, glucose and Sucrose; starches such as corn N N -K starch and potato starch; cellulose and its derivatives Such as V / N sodium carboxymethyl cellulose, ethyl cellulose and cellu NRN f OMe iPr COH, lose acetate; powdered tragacanth; malt, gelatin; talc, excipi O ents such as cocoa butter and Suppository waxes; oils such as O 15 peanut oil, cottonseed oil; safflower oil; sesame oil, olive oil; -l OMe, corn oil and soybean oil; glycols; Such a propylene glycol or N N polyethylene glycol; esters such as ethyl oleate and ethyl W \ / N N NFN laurate; agar, buffering agents such as magnesium hydroxide Ys -N CO2H and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's Solution; ethyl alcohol, and phos phate buffer solutions, as well as other non-toxic compatible lubricants such as Sodium lauryl Sulfate and magnesium Stear ate, as well as coloring agents, releasing agents, coating or a pharmaceutically acceptable form thereof. agents, Sweetening, flavoring and perfuming agents, preser 25 Vatives and antioxidants can also be present in the pharma 3. Pharmaceutical Compositions and Formulations ceutically acceptable composition, according to the judgment of the formulator. In certain embodiments, provided herein is a pharmaceu In some embodiments, a compound of formula (I) is tical composition comprising at least one compound of for administered at about 0.01 mg/kg to about 200 mg/kg, such as 30 at about 0.1 mg/kg to about 100 mg/kg, further Such as at mula (I) or a pharmaceutically acceptable form thereof, and about 0.5 mg/kg to about 50 mg/kg. one or more pharmaceutically acceptable excipients. A “subject’ to which administration is contemplated In some embodiments, provided herein is a pharmaceutical includes, but is not limited to, humans (i.e., a male or female composition comprising at least one compound of formula (I) of any age group, e.g., a pediatric Subject (e.g., infant, child, or a pharmaceutically acceptable form thereof, as provided in 35 adolescent) or adult Subject (e.g., young adult, middle-aged Tables 1, 2, 3, or 4 and one or more pharmaceutically accept adult or senior adult)) and/or other primates (e.g., cynomol able excipients. In other embodiments, provided herein is a gus monkeys, rhesus monkeys); mammals, including com pharmaceutical composition comprising at least one com mercially relevant mammals such as cattle, pigs, horses, pound of formula (I) or a pharmaceutically acceptable form sheep, goats, cats, and/or dogs; and/or birds, including com thereof, as provided in Tables 1, 2, or 3 having an activity of 40 mercially relevantbirds such as chickens, ducks, geese, and/ “A”, “A”, “B” or “B” and one or more pharmaceutically or turkeys. acceptable excipients. In other embodiments, provided herein The formulations of the pharmaceutically acceptable com is a pharmaceutical composition comprising at least one com positions described herein can be prepared by any method pound of formula (I) or a pharmaceutically acceptable form known or hereafter developed in the art of pharmacology. In thereof, as provided in Tables 1, 2 or 3 having an activity of 45 general. Such preparatory methods include the step of bring “A” or “A” and one or more pharmaceutically acceptable ing the compound of formula (I) into association with one or excipients. more pharmaceutically acceptable excipients and then, if nec As described above, the pharmaceutical compositions pro essary and/or desirable, shaping and/or packaging the prod vided herein can comprise a “pharmaceutically acceptable uct into a desired single- or multi-dose unit. excipient’, which, as used herein, includes any and all sol 50 A pharmaceutical composition provided herein can be pre vents, diluents, or other liquid vehicle, dispersion or Suspen pared, packaged, and/or sold in bulk, as a single unit dose, sion aids, Surface active agents, isotonic agents, thickening or and/or as a plurality of single unit doses. As used herein, a emulsifying agents, preservatives, solid binders, lubricants “unit dose' is a discrete amount of the pharmaceutical com and the like, as Suited to the particular dosage form desired. position comprising a predetermined amount of at least one Remington's Pharmaceutical Sciences, 16th Ed., E. W. Mar 55 compound of formula (I). The amount of the compound of tin (Mack Publishing Co., Easton, Pa., 1980) discloses vari formula (I) is generally equal to the dosage of the compound ous pharmaceutically acceptable excipients used informulat of formula (I) which would be administered to a subject ing pharmaceutically acceptable compositions and known and/or a convenient fraction of Such a dosage such as, for techniques for the preparation thereof. Except insofar as any example, one-half or one-third of Such a dosage. conventional excipient medium is incompatible with the 60 The relative amounts of the compound of formula (I), the compounds provided herein, such as by producing any unde pharmaceutically acceptable excipient, and/or any additional sirable biological effect or otherwise interacting in a delete ingredients in a pharmaceutical composition provided herein rious manner with any other component(s) of the pharmaceu will vary, depending upon the identity, size, and/or condition tically acceptable composition, the excipients use is of the subject treated and further depending upon the route by contemplated to be within the scope of this disclosure. Some 65 which the composition is to be administered. By way of examples of materials which can serve as pharmaceutically example, the composition can comprise between 0.1% and acceptable excipients include, but are not limited to, ion 100% (w/w) of the compound of formula (I). US 8,546,432 B2 91 92 In some embodiments, the pharmaceutically acceptable ethoxylated castor oil, polyoxymethylene Stearate, and Solu excipient is at least 95%, 96%, 97%, 98%, 99%, or 100% tol), Sucrose fatty acid esters, polyethylene glycol fatty acid pure. In some embodiments, the excipient is approved for use esters (e.g. Cremophor), polyoxyethylene ethers, (e.g. poly in humans and for veterinary use. In some embodiments, the oxyethylene lauryl ether Brij 30), poly(vinyl-pyrrolidone), excipient has been approved by United States Food and Drug diethylene glycol monolaurate, triethanolamine oleate, Administration. In some embodiments, the excipient is phar Sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyl maceutical grade. In some embodiments, the excipient meets laurate, sodium lauryl sulfate, Pluronic F 68, Poloxamer 188, the standards of the United States Pharmacopoeia (USP), the cetrimonium bromide, cetylpyridinium chloride, benzalko European Pharmacopoeia (EP), the British Pharmacopoeia, nium chloride, docusate Sodium, etc. and/or combinations and/or the International Pharmacopoeia. 10 thereof. Pharmaceutically acceptable excipients used in the manu Exemplary binding agents include, but are not limited to, facture of pharmaceutically acceptable compositions include, Starch (e.g. cornstarch and starch paste); gelatin: Sugars (e.g. but are not limited to, inert diluents, dispersing and/or granu Sucrose, glucose, dextrose, dextrin, molasses, lactose, lacti lating agents, Surface active agents and/or emulsifiers, disin tol, mannitol, etc.); natural and synthetic gums (e.g. acacia, tegrating agents, binding agents, preservatives, buffering 15 Sodium alginate, extract of Irish moss, panwar gum, ghatti agents, lubricating agents, and/or oils. One or more Such gum, mucilage of isapol husks, carboxymethylcellulose, excipients can optionally be included in the formulations. methylcellulose, ethylcellulose, hydroxyethylcellulose, Excipients such as cocoa butter and Suppository waxes, col hydroxypropyl cellulose, hydroxypropyl methylcellulose, oring agents, coating agents, Sweetening, flavoring, and per microcrystalline cellulose, cellulose acetate, poly(vinyl-pyr fuming agents can be present in the pharmaceutically accept rolidone), magnesium aluminum silicate (Veegum), and larch able composition, according to the judgment of the arabogalactan); alginates; polyethylene oxide; polyethylene formulator. glycol; inorganic calcium salts; silicic acid; polymethacry Exemplary pharmaceutically acceptable excipients lates; waxes; water, alcohol; etc.; and combinations thereof. include, but are not limited to, diluents such as calcium car Exemplary preservatives can include antioxidants, chelat bonate, Sodium carbonate, calcium phosphate, dicalcium 25 ing agents, antimicrobial preservatives, antifungal preserva phosphate, calcium sulfate, calcium hydrogen phosphate, tives, alcohol preservatives, acidic preservatives, and other Sodium phosphate lactose, Sucrose, cellulose, microcrystal preservatives. Exemplary antioxidants include, but are not line cellulose, kaolin, mannitol, Sorbitol, inositol, Sodium limited to, alpha tocopherol, ascorbic acid, acorbyl palmitate, chloride, dry starch, cornstarch, powdered Sugar, etc., and butylated hydroxyanisole, butylated hydroxytoluene, mono combinations thereof. 30 thioglycerol, potassium metabisulfite, propionic acid, propyl Exemplary granulating and/or dispersing agents include, gallate, sodium ascorbate, sodium bisulfite, sodium met but are not limited to, potato starch, corn starch, tapioca abisulfite, and sodium sulfite. Exemplary chelating agents Starch, sodium starch glycolate, clays, alginic acid, guar gum, include ethylenediaminetetraacetic acid (EDTA), citric acid citrus pulp, agar, bentonite, cellulose and wood products, monohydrate, disodium edetate, dipotassium edetate, edetic natural Sponge, cation-exchange resins, calcium carbonate, 35 acid, fumaric acid, malic acid, phosphoric acid, Sodium ede silicates, Sodium carbonate, cross-linked poly(Vinyl-pyrroli tate, tartaric acid, and trisodium edetate. Exemplary antimi done) (crospovidone), sodium carboxymethyl starch (sodium crobial preservatives include, but are not limited to, benza starch glycolate), carboxymethyl cellulose, cross-linked lkonium chloride, benzethonium chloride, benzyl alcohol, Sodium carboxymethyl cellulose (croScarmellose), methyl bronopol, cetrimide, cetylpyridinium chloride, chlorhexi cellulose, pregelatinized starch (starch 1500), microcrystal 40 dine, chlorobutanol, chlorocresol, chloroxylenol, cresol, line starch, water insoluble starch, calcium carboxymethyl ethyl alcohol, glycerin, hexetidine, imidurea, phenol, phe cellulose, magnesium aluminum silicate (Veegum), sodium noxyethanol, phenylethyl alcohol, phenylmercuric nitrate, lauryl Sulfate, quaternary ammonium compounds, etc., and propylene glycol, and thimerosal. Exemplary antifungal pre combinations thereof. servatives include, but are not limited to, butyl paraben, Exemplary Surface active agents and/or emulsifiers 45 methyl paraben, ethyl paraben, propyl paraben, benzoic acid, include, but are not limited to, natural emulsifiers (e.g. acacia, hydroxybenzoic acid, potassium benzoate, potassium Sor agar, alginic acid, sodium alginate, tragacanth, chondruX. bate, Sodium benzoate, Sodium propionate, and Sorbic acid. cholesterol, Xanthan, pectin, gelatin, egg yolk, casein, wool Exemplary alcohol preservatives include, but are not limited fat, cholesterol, wax, and lecithin), colloidal clays (e.g. ben to, ethanol, polyethylene glycol, phenol, phenolic com tonite aluminum silicate and Veegum magnesium alumi 50 pounds, bisphenol, chlorobutanol, hydroxybenzoate, and num silicate), long chain amino acid derivatives, high phenylethyl alcohol. Exemplary acidic preservatives include, molecular weight alcohols (e.g. Stearyl alcohol, cetyl alcohol, but are not limited to, vitamin A, vitamin C, vitamin E, beta oleyl alcohol, triacetin monostearate, ethylene glycol distear carotene, citric acid, acetic acid, dehydroacetic acid, ascorbic ate, glyceryl monoStearate, and propylene glycol monostear acid, Sorbic acid, and phytic acid. Other preservatives ate, polyvinyl alcohol), carbomers (e.g. carboxy polymethyl 55 include, but are not limited to, tocopherol, tocopherol acetate, ene, polyacrylic acid, acrylic acid polymer, and carboxyvinyl deteroxime mesylate, cetrimide, butylated hydroxyanisol polymer), carrageenan, cellulosic derivatives (e.g. carboxym (BHA), butylated hydroxytoluened (BHT), ethylenediamine, ethylcellulose sodium, powdered cellulose, hydroxymethyl sodium lauryl sulfate (SLS), sodium lauryl ether sulfate cellulose, hydroxypropyl cellulose, hydroxypropyl methyl (SLES), sodium bisulfite, sodium metabisulfite, potassium cellulose, methylcellulose), Sorbitan fatty acid esters (e.g. 60 sulfite, potassium metabisulfite, Glydant Plus, Phenonip, polyoxyethylene Sorbitan monolaurate Tween 20, polyoxy methylparaben, Germall 115, Germaben II, Neolone, ethylene sorbitan Tween 60, polyoxyethylene sorbitan Kathon, and Euxyl. In certain embodiments, the preservative monooleate Tween 80, sorbitan monopalmitate Span 40, is an anti-oxidant. In other embodiments, the preservative is a sorbitan monostearate Span 60, sorbitan tristearate Span chelating agent. 65, glyceryl monooleate, sorbitan monooleate Span 80), 65 Exemplary buffering agents include, but are not limited to, polyoxyethylene esters (e.g. polyoxyethylene monostearate citrate buffer solutions, acetate buffer solutions, phosphate Myri 45, polyoxyethylene hydrogenated castor oil, poly buffer solutions, ammonium chloride, calcium carbonate, US 8,546,432 B2 93 94 calcium chloride, calcium citrate, calcium glubionate, cal embodiment, the oral Suspension can comprise a compound cium gluceptate, calcium gluconate, D-gluconic acid, cal of formula (I) and between about 0.1 and 2% carboxymeth cium glycerophosphate, calcium lactate, propanoic acid, cal ylcellulose. cium levulinate, pentanoic acid, dibasic calcium phosphate, Injectable preparations, for example, sterile injectable phosphoric acid, tribasic calcium phosphate, calcium hydrox aqueous or oleaginous Suspensions can beformulated accord ide phosphate, potassium acetate, potassium chloride, potas ing to the known art using Suitable dispersing or wetting sium gluconate, potassium mixtures, dibasic potassium phos agents and Suspending agents. The sterile injectable prepara phate, monobasic potassium phosphate, potassium phosphate tion can be a sterile injectable solution, Suspension or emul mixtures, sodium acetate, sodium bicarbonate, sodium chlo sion in a nontoxic parenterally acceptable diluent or solvent, 10 for example, as a solution in 1,3-butanediol. Among the ride, Sodium citrate, sodium lactate, dibasic sodium phos acceptable vehicles and solvents that can be employed are phate, monobasic sodium phosphate, Sodium phosphate mix water, Ringer's solution, U.S.P. and isotonic sodium chloride tures, tromethamine, magnesium hydroxide, aluminum Solution. In addition, Sterile, fixed oils are conventionally hydroxide, alginic acid, pyrogen-free water, isotonic saline, employed as a solvent or Suspending medium. For this pur Ringer's solution, ethyl alcohol, etc., and combinations 15 pose any bland fixed oil can be employed including synthetic thereof. mono- or diglycerides. In addition, fatty acids such as oleic Exemplary lubricating agents include, but are not limited acid are used in the preparation of injectables. to, magnesium Stearate, calcium Stearate, Stearic acid, silica, The injectable formulations can be sterilized, for example, talc, malt, glyceryl behanate, hydrogenated vegetable oils, by filtration through a bacterial-retaining filter, or by incor polyethylene glycol, Sodium benzoate, Sodium acetate, porating sterilizing agents in the form of sterile solid compo Sodium chloride, leucine, magnesium lauryl Sulfate, sodium sitions which can be dissolved or dispersed in sterile water or lauryl Sulfate, etc., and combinations thereof. other sterile injectable medium prior to use. Injectable com Exemplary oils include, but are not limited to, almond, positions can contain from about 0.1 to about 5% w/w of the apricot kernel, avocado, babassu, bergamot, black current compound of formula (I). Seed, borage, cade, camomile, canola, caraway, carnauba, 25 In order to prolong the effect of a drug, it is often desirable castor, cinnamon, cocoa butter, coconut, cod liver, coffee, to slow the absorption of the drug from subcutaneous or corn, cotton seed, emu, eucalyptus, evening primrose, fish, intramuscular injection. This can be accomplished by the use flaxseed, geraniol, gourd, grape seed, hazel nut, hyssop, iso of a liquid Suspension of crystalline or amorphous material propyl myristate, jojoba, kukui nut, lavandin, lavender, with poor water solubility. The rate of absorption of the drug lemon, litsea cubeba, macademia nut, mallow, mango seed, 30 then depends upon its rate of dissolution which, in turn, can meadowfoam seed, mink, nutmeg, olive, orange, orange depend upon crystal size and crystalline form. Alternatively, roughy, palm, palm kernel, peach kernel, peanut, poppy seed, delayed absorption of a parenterally administered drug form pumpkin seed, rapeseed, rice bran, rosemary, Safflower, san can be accomplished by dissolving or Suspending the drug in dalwood, Sasquana, savoury, sea buckthorn, Sesame, shea an oil vehicle. butter, silicone, soybean, Sunflower, tea tree, thistle, tsubaki, 35 Compositions for rectal or vaginal administration are typi Vetiver, walnut, and wheat germ oils. Exemplary oils include, cally Suppositories which can be prepared by mixing the but are not limited to, butyl Stearate, caprylic triglyceride, conjugates provided herein with Suitable non-irritating capric triglyceride, cyclomethicone, diethyl sebacate, dime excipients such as cocoa butter, polyethylene glycol or a thicone 360, isopropyl myristate, mineral oil, octyldode Suppository wax which are solid at ambient temperature but canol, oleyl alcohol, silicone oil, and combinations thereof. 40 liquid at body temperature and therefore melt in the rectum or Liquid dosage forms for oral and parenteral administration vaginal cavity and release the compound of formula (I). include, but are not limited to, pharmaceutically acceptable Solid dosage forms for oral administration include cap emulsions, microemulsions, Solutions, Suspensions, syrups Sules, tablets, pills, powders, and granules. In Such solid dos and elixirs. In addition to the compound of formula (I), the age forms, the compound of formula (I) is mixed with at least liquid dosage forms can comprise inert diluents commonly 45 one inert, pharmaceutically acceptable excipient such as used in the art such as, for example, water or other solvents, Sodium citrate or dicalcium phosphate and/or a) fillers or solubilizing agents and emulsifiers such as ethyl alcohol, extenders such as starches, lactose, Sucrose, glucose, manni isopropyl alcohol, ethyl carbonate, ethylacetate, benzyl alco tol, and silicic acid, b) binders such as, for example, car hol, benzyl benzoate, propylene glycol. 1,3-butylene glycol, boxymethylcellulose, alginates, gelatin, polyvinylpyrrolidi dimethylformamide, oils (in particular, cottonseed, ground 50 none. Sucrose, and acacia, c) humectants such as glycerol. d) nut, corn, germ, olive, castor, and sesame oils), glycerol, disintegrating agents such as agar, calcium carbonate, potato tetrahydrofurfuryl alcohol, polyethylene glycols and fatty or tapioca starch, alginic acid, certain silicates, and sodium acid esters of sorbitan, and mixtures thereof. Besides inert carbonate, e) solution retarding agents such as paraffin, f) diluents, the oral compositions can include adjuvants such as absorption accelerators such as quaternary ammonium com wetting agents, emulsifying and Suspending agents, Sweeten 55 pounds, g) wetting agents such as, for example, cetyl alcohol ing, flavoring, and perfuming agents. In certain embodiments and glycerol monostearate, h) absorbents such as kaolin and for parenteral administration, the conjugates provided herein bentonite clay, and i) lubricants such as talc, calcium Stearate, are mixed with Solubilizing agents such as Cremophor, alco magnesium Stearate, Solid polyethylene glycols, sodium lau hols, oils, modified oils, glycols, polysorbates, cyclodextrins, ryl Sulfate, and mixtures thereof. In the case of capsules, polymers, and combinations thereof. For example, in certain 60 tablets and pills, the dosage form can comprise buffering embodiments, the oral Suspension can comprise at least one agents. The unit dose formulation, for example, a tablet, can compound of formula (I) and carboxymethylcellulose. In contain from about 0.05% to about 95% by weight of the Some embodiments, the oral Suspension can comprise at least compound of formula (I). one compound of formula (I), carboxymethylcellulose, and Solid compositions of a similar type can be employed as DMSO. In one embodiment, the oral suspension can com 65 fillers in Soft and hard-filled gelatin capsules using Such phar prise a compound of formula (I) and 0.5% carboxymethyl maceutically acceptable excipients as lactose or milk Sugar as cellulose/5% DMSO/0.5% Tween (PKPD#5). In another well as high molecular weight polyethylene glycols and the US 8,546,432 B2 95 96 like. The Solid dosage forms of tablets, dragees, capsules, Solutions and/or Suspensions. Topically-administrable for pills, and granules can be prepared with coatings and shells mulations can, for example, comprise from about 1% to about Such as enteric coatings and other coatings well known in the 10% (w/w) compound of formula (I), although the concen pharmaceutical formulating art. They can optionally com tration of the compound of formula (I) can be as high as the prise opacifying agents and can be of a composition that they solubility limit of the compound of formula (I) in the solvent. release the compound of formula (I) only. In some embodi In some embodiments, topically-administrable formulations ments, the compound of formula (I) can be released in a can, for example, comprise from about 1% to about 9% (w/w) certain part of the intestinal tract, optionally, in a delayed compound of formula (I), such as from about 1% to about 8% manner. Examples of embedding compositions which can be (w/w), further such as from about 1% to about 7% (w/w), used include polymeric Substances and waxes. Solid compo 10 further such as from about 1% to about 6% (w/w), further sitions of a similar type can be employed as fillers in Soft and such as from about 1% to about 5% (w/w), further such as hard-filled gelatin capsules using Such excipients as lactose or from about 1% to about 4% (w/w), further such as from about milk Sugar as well as high molecular weight polyethylene 1% to about 3% (w/w), and further such as from about 1% to glycols and the like. about 2% (w/w) compound of formula (I). Formulations for The compound of formula (I) can be in micro-encapsulated 15 topical administration can further comprise one or more of form with one or more pharmaceutically acceptable excipi the additional pharmaceutically acceptable excipients ents as noted above. In Such solid dosage forms, the com described herein. pound of formula (I) can be admixed with at least one inert A pharmaceutical composition provided herein can be pre diluent such as Sucrose, lactose or starch. Such dosage forms pared, packaged, and/or sold in a formulation Suitable for can comprise, as is normal practice, additional Substances pulmonary administration via the buccal cavity. Such a for other than inert diluents, e.g., tableting lubricants and other mulation can comprise dry particles which comprise the com tableting aids such a magnesium Stearate and microcrystal pound of formula (I) and which have a diameter in the range line cellulose. In the case of capsules, tablets and pills, the from about 0.5 to about 7 nanometers, such as from about 1 to dosage forms can comprise buffering agents. about 6 nanometers, further such as from about 2 to about 5 Dosage forms for topical and/or transdermal administra 25 nanometers, and further such as from about 3 to about 4 tion of a compound of formula (I) provided hereincan include nanometers. Such pharmaceutical compositions are conve ointments, pastes, creams, lotions, gels, powders, Solutions, niently in the form of dry powders for administration using a sprays, inhalants and/or patches. Generally, the compound of device comprising a dry powder reservoir to which a stream formula (I) is admixed under sterile conditions with one or of propellant can be directed to disperse the powder and/or more pharmaceutically acceptable excipients and/or any 30 using a self propelling solvent/powder dispensing container needed preservatives and/or buffers as may be required. Addi Such as a device comprising the compound of formula (I) tionally, the use of transdermal patches, which often have the dissolved and/or suspended in a low-boiling propellant in a added advantage of providing controlled delivery of a com sealed container. Such powders comprise particles wherein at pound of formula (I) to the body, is contemplated herein. Such least 98% of the particles by weight have a diameter greater dosage forms can be prepared, for example, by dissolving 35 than 0.5 nanometers and at least 95% of the particles by and/or dispensing the compound of formula (I) in the proper number have a diameter less than 7 nanometers. Alternatively, medium. Alternatively or additionally, the rate can be con at least 95% of the particles by weight have a diameter greater trolled by either providing a rate controlling membrane and/ than 1 nanometer and at least 90% of the particles by number or by dispersing the compound of formula (I) in a polymer have a diameter less than 6 nanometers. Dry powder compo matrix and/or gel. 40 sitions can include a solid fine powder diluent such as Sugar Suitable devices for use in delivering intradermal pharma and can be provided in a unit dose form. ceutically acceptable compositions described herein include Low boiling propellants generally include liquid propel short needle devices such as those described in U.S. Pat. Nos. lants having a boiling point of below 65° F. at atmospheric 4,886,499; 5,190,521; 5,328,483; 5,527,288; 4,270,537; pressure. Generally, the propellant can constitute 50% to 5,015,235; 5,141.496; and 5,417,662. Intradermal composi 45 99.9% (w/w) of the pharmaceutical composition, and the tions can be administered by devices which limit the effective active ingredient can constitute 0.1% to 20% (w/w) of the penetration length of a needle into the skin, such as those pharmaceutical composition. The propellant can further com described in PCT publication WO99/34850 and functional prise additional excipients such as a liquid non-ionic and/or equivalents thereof. Jet injection devices which deliver liquid Solid anionic Surfactant and/or a Solid diluent (which may vaccines to the dermis via a liquid jet injector and/or via a 50 have a particle size of the same order as particles comprising needle which pierces the stratum corneum and produces a jet the compound of formula (I)). which reaches the dermis are suitable. Jet injection devices Pharmaceutical compositions provided hereinformulated are described, for example, in U.S. Pat. Nos. 5,480,381: for pulmonary delivery can provide the compound of formula 5,599,302; 5,334,144; 5,993,412: 5,649,912; 5,569, 189: (I) in the form of droplets of a solution and/or Suspension. 5,704,911; 5,383,851; 5,893,397; 5,466.220; 5,339,163; 55 Such formulations can be prepared, packaged, and/or sold as 5,312,335; 5,503,627; 5,064,413: 5,520,639; 4,596,556; aqueous and/or dilute alcoholic Solutions and/or Suspensions, 4,790,824; 4.941,880; 4.940,460; and PCT publications WO optionally sterile, comprising the compound of formula (I), 97/37705 and WO 97/13537. Ballistic powder/particle deliv and can be administered using any nebulization and/or atomi ery devices which use compressed gas to accelerate vaccine Zation device. Such formulations can further comprise one or in powder form through the outer layers of the skin to the 60 more additional excipients including, but not limited to, a dermis are suitable. Alternatively or additionally, conven flavoring agent such as Saccharin Sodium, a volatile oil, a tional Syringes can be used in the classical mantoux method of buffering agent, a Surface active agent, and/or a preservative intradermal administration. such as methylhydroxybenzoate. The droplets provided by Formulations suitable for topical administration include, this route of administration can have an average diameter in but are not limited to, liquid and/or semi liquid preparations 65 the range from about 0.1 to about 200 nanometers. Such as liniments, lotions, oil in water and/or water in oil The formulations described herein as being useful for pull emulsions such as creams, ointments and/or pastes, and/or monary delivery are useful for intranasal delivery of a phar US 8,546,432 B2 97 98 maceutical composition provided herein. Another formula means for proper administration, such as, for example, gradu tion Suitable for intranasal administration is a coarse powder ated cups, Syringes, needles, cleaning aids, and the like. In comprising the compound of formula (I) and having an aver certain embodiments, a kit can include instructions for proper age particle from about 0.2 to 500 micrometers. Such a for administration and/or preparation for proper administration. mulation is administered, for example, by rapid inhalation The instructions would direct the consumer or medical through the nasal passage from a container of the powder held personnel to administer the dosage form according to admin close to the nostrils. istration modes known to those skilled in the art. Such kits Formulations Suitable for nasal administration can, for could be packaged and sold in single or multiple kit units. An example, comprise from about as little as 0.1% (w/w) and as example of such a kit is a so-called blister pack. Blister packs much as 100% (w/w) of the compound of formula (I), and can 10 are well known in the packaging industry and are being comprise one or more of the additional excipients described widely used for the packaging of pharmaceutical unit dosage herein. A pharmaceutical composition provided hereincan be forms (tablets, capsules, and the like). Blister packs generally prepared, packaged, and/or sold in a formulation Suitable for consist of a sheet of relatively stiff material covered with a foil buccal administration. Such formulations can, for example, of a preferably transparent plastic material. During the pack be in the form of tablets and/or lozenges made using conven 15 aging process, recesses are formed in the plastic foil. The tional methods, and can, for example, comprise 0.1 to 20% recesses have the size and shape of the tablets or capsules to (w/w) of the compound of formula (I), the balance compris be packed. Next, the tablets or capsules are placed in the ing an orally dissolvable and/or degradable composition and, recesses and the sheet of relatively stiff material is sealed optionally, one or more of the additional pharmaceutically against the plastic foil at the face of the foil which is opposite acceptable excipients described herein. In some embodi from the direction in which the recesses were formed. As a ments, formulations Suitable for buccal administration can result, the tablets or capsules are sealed in the recesses comprise a powder and/or an aerosolized and/or atomized between the plastic foil and the sheet. The strength of the Solution and/or Suspension comprising the compound of for sheet is such that the tablets or capsules can be removed from mula (I). Such powdered, aerosolized, and/or aerosolized the blister pack by manually applying pressure on the formulations, when dispersed, can have an average particle 25 recesses whereby an opening is formed in the sheet at the and/or droplet size in the range from about 0.1 to about 200 place of the recess. The tablet or capsule can then be removed nanometers, and can further comprise one or more of the via said opening. additional pharmaceutically acceptable excipients described It can be desirable to provide a memory aid on the kit, e.g., herein. in the form of numbers next to the tablets or capsules whereby A pharmaceutical composition provided herein can be pre 30 the numbers correspond with the days of the regimen which pared, packaged, and/or sold in a formulation Suitable for the tablets or capsules so specified should be ingested. ophthalmic administration. Such formulations can, for Another example of such a memory aid is a calendar printed example, be in the form of eye drops including, for example, on the card, e.g., as follows “First Week, Monday, a 0.1/1.0% (w/w) solution and/or suspension of the com Tuesday,...etc....Second Week, Monday,Tuesday,...etc. pound of formula (I) in an aqueous or oily liquid carrier. Such 35 Other variations of memory aids will be readily apparent. A drops can further comprise buffering agents, salts, and/or one "daily dose” can be a single tablet or capsule or several pills or more other of the additional pharmaceutically acceptable or capsules to be taken on a given day. excipients described herein. Other opthalmically-adminis trable formulations which are useful include those which 4. Uses and Methods of Treatment comprise the compound of formula (I) in microcrystalline 40 form and/or in a liposomal preparation. Ear drops and/or eye 4.1 Definitions drops are contemplated as being within the scope of this As used herein, and unless otherwise specified, the terms disclosure. “treat,” “treating and “treatment contemplate an action that General considerations in the formulation and/or manufac occurs while a Subject is suffering from the specified disease, ture of pharmaceutical compositions can be found, for 45 disorder or condition, which reduces the severity of the dis example, in Remington. The Science and Practice of Phar ease, disorder or condition, or retards or slows the progression macy 21 Ed., (Lippincott Williams & Wilkins, 2005). of the disease, disorder or condition. Although the descriptions of pharmaceutical compositions As used herein, unless otherwise specified, the terms “pre provided herein are principally directed to pharmaceutical vent.” “preventing and “prevention contemplate an action compositions which are Suitable for administration to 50 that occurs before a subject begins to suffer from the specified humans, it will be understood by the skilled artisan that such disease, disorder or condition, which inhibits or reduces the compositions are generally suitable for administration to ani severity of the disease, disorder or condition. mals of all sorts. Modification of pharmaceutical composi As used herein, and unless otherwise specified, the terms tions Suitable for administration to humans in order to render “manage.” “managing” and “management' encompass pre the compositions suitable for administration to various ani 55 venting the recurrence of the specified disease, disorder or mals is well understood, and the ordinarily skilled veterinary condition in a subject who has already suffered from the pharmacologist can design and/or perform such modification disease, disorder or condition, and/or lengthening the time with merely ordinary, if any, experimentation. that a subject who has suffered from the disease, disorder or Further provided herein are kits comprising one or more condition remains in remission. The terms encompass modu compounds of formula (I) (or pharmaceutically acceptable 60 lating the threshold, development and/or duration of the dis forms thereof), and/or an pharmaceutical composition as ease, disorder or condition, or changing the way that a subject described above. Kits are typically provided in a suitable responds to the disease, disorder or condition. container (e.g., for example, a foil, plastic, or cardboard pack As used herein “inhibition”, “inhibiting”, “inhibit” and age). In certain embodiments, a kit can include one or more “inhibitor', and the like, refer to the ability of a compound to pharmaceutically acceptable excipients, pharmaceutical 65 reduce, slow, halt or prevent activity of a particular biological additives, therapeutically active agents, and the like, as process (e.g., FASN activity) in a cell relative to vehicle. In described herein. In certain embodiments, a kit can include certain embodiments, the inhibition results in reduction of the US 8,546,432 B2 99 100 activity by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, Compounds of formula (I) provided herein can be inhibi 50%. 60%, 70%, 80%, 90% or more of the activity without tors of FASN. A “FASN-mediated disorder” as used herein, such inhibition. refers to a disease, disorder or condition which is treatable by As used herein, and unless otherwise specified, a “thera inhibition of FASN activity. FASN-mediated disorders peutically effective amount of a compound is an amount 5 include, but are not limited to, hyperproliferative disorders: sufficient to provide a therapeutic benefit in the treatment or inflammatory disorders; obesity related disorders, such as, management of a disease, disorder or condition, or to delay or but not limited to, Type II diabetes mellitus and fatty liver minimize one or more symptoms associated with the disease, disease; microbial infections, such as, but not limited to, viral, disorder or condition. A therapeutically effective amount of a bacterial, fungal, parasitic, and protozoal infections; and compound means an amount of therapeutic agent, alone or in 10 complications thereof. combination with other therapies, which provides atherapeu In certain embodiments, the FASN-mediated disorder is a tic benefit in the treatment or management of the disease, hyperproliferative disorder. In certain embodiments, the disorder or condition. The term “therapeutically effective hyperproliferative disorder is cancer. To date, aberrant FASN amount can encompass an amount that improves overall 15 activity has been observed in a variety of hyperproliferative therapy, reduces or avoids symptoms or causes of disease or disorders which include, but are not limited to: condition, or enhances the therapeutic efficacy of another (i) bladder cancer (see Visca et al., Anticancer Res. (2003) therapeutic agent. 23:335-339); As used herein, and unless otherwise specified, a “prophy (ii) brain cancer (e.g., meningioma, see: Haase et al., lactically effective amount of a compound is an amount 20 Neuro-Oncology (2010) Advance Access published Feb. 5, Sufficient to prevent a disease, disorder or condition, or one or 2010, 1-11; e.g., glioma: see Zhao et al., Br. J. Cancer (2006) more symptoms associated with the disease, disorder or con 95:869-878; e.g., meduloblastoma: see Slade et al., Antican dition, or prevent its recurrence. A prophylactically effective cer Res. (2003) 23:1235-1243); amount of a compound means an amount of therapeutic (iii) breast cancer (see Alo et al., Cancer (1996) 77:474 agent, alone or in combination with other agents, which pro- 25 482; Pizer et al., Cancer Res. (1996) 56:2745-2747: Pizer et vides a prophylactic benefit in the prevention of the disease, al., Cancer Res. (2000) 60:213-218; Milgraum et al., Clin. disorder or condition. The term “prophylactically effective Cancer Res. (1997) 3:2115-2120; Lupu and Menendez, amount can encompass an amount that improves overall Endocrinology (2006) 147:4056-4066; Alo et al. Oncol. Rep. prophylaxis or enhances the prophylactic efficacy of another (2000) 7:1383-1388: Wang et al., Cancer Lett. (2001) 167: prophylactic agent. 30 99-104; Liu et al., Mol. Cancer. Ther. (2008) 7:263-270; and 4.2 Embodiments Kuhajda et al., PNAS (2000) 97:3450-3454; e.g., mammary In one embodiment, provided herein are methods for treat cancer: see Hennigar et al., Biochim. Biophys. Acta (1998) ing, preventing and/or managing a FASN-mediated disorder, 1392:85-100 and Alliet al., Oncogene (2005) 24:39-46): disease or condition comprising administering to a subject in (iv) colorectal cancer (see Rashid et al., Am. J. Pathol. need thereof a therapeutically or prophylactically effective 35 (1997) 150:201-208; Huang et al., World J. Gastroenterol. amount of at least one compound of formula (I) or a pharma (2000) 6:295-297; Zhan et al., Clin. Cancer Res. (2008) ceutically acceptable form thereof, or a pharmaceutical com 14:5735-5742); position comprising at least one compound of formula (I) or (V) esophageal cancer (see Nemoto et al., Pathobiology a pharmaceutically acceptable form thereof. (2001) 69:297-303); In another embodiment, provided herein are methods for 40 (vi) endometrial cancer (see Pizer et al., Cancer (1998) inhibiting FASN in a subject comprising administering to a 83:528-537; Pizeret al., Int. J. Gynecol. Pathol. (1997) 16:45 subject in need thereof a therapeutically effective amount of 51; Lupu and Menendez, Endocrinology (2006) 147:4056 at least one compound of formula (I) or a pharmaceutically 406; and Sebastiani et al., Gynecologic Oncology (2004) acceptable form thereof, or a pharmaceutical composition 92: 101-105); comprising at least one compound of formula (I) or a phar- 45 (viii) gastric cancer (see Kusakabe et al. Histopathology maceutically acceptable form thereof. (2002) 40:71-79); In another embodiment, provided herein is a method of (ix) gastrointestinal stromal tumor (see Rossi et al., J. inhibiting activation of the FASN pathway in vitro or ex vivo, Pathol. (2006). 209:369-375); comprising contacting a FASN protein with at least one com (X) kidney cancer (e.g., nephroblastoma/Wilms tumor: see pound of formula (I) or a pharmaceutically acceptable form 50 Camassei et al., Med. Pediatr. Oncol. (2003)40:302-308); thereof, or a pharmaceutical composition comprising at least (xi) liver cancer (see Evertet al., Lab. Invest. (2005)85:99 one compound of formula (I) or a pharmaceutically accept 108); able form thereof, in an amount sufficient to reduce the acti (xii) lung cancer (see Piyathilake et al., Human Pathol. vation of the FASN pathway. (2000)31:1068-1073 and Visca et al., Anticancer Res. (2004) In another embodiment, provided herein is the use of at 55 24:4169-4173); least one compound of formula (I) or a pharmaceutically (xiii) mesothelioma (see Gabrielson et al., Clin. Cancer acceptable form thereof, or a pharmaceutical composition Research (2001) 7:153-157); comprising at least one compound of formula (I) or a phar (xiv) multiple myeloma (see Wang et al., J. Zhejiang Univ. maceutically acceptable form thereof, for the treatment of a Sci B (2008) 9:441-447); FASN-mediated disorder, disease or condition in a subject. 60 (XV) neuroblastoma (see Slade et al., Anticancer Res. In another embodiment, provided herein is the use of at (2003) 23:1235-1243); least one compound of formula (I) or a pharmaceutically (xvi) oral cancer (see Krontiras et al., Head Neck (1999) acceptable form thereof, or a pharmaceutical composition 21:325-329; and Agostini et al., Oral Oncol. (2004) 40:728 comprising at least one compound of formula (I) or a phar 735; see also e.g., oral squamous cell carcinoma (OSCC): maceutically acceptable form thereof, in the manufacture of a 65 Silva et al., Oral Diseases (2007) 14:376-382); medicament. In certain embodiments, the medicament is use (xvii) ovarian cancer (see Pizer et al., Cancer Res. (1996) ful for treating a FASN-mediated disorder in a subject. 56:1189-1193; Alo et al., Oncol. Rep. (2000) 7:1383-1388; US 8,546,432 B2 101 102 Wang et al., Oncogene (2005) 24:3574-3582: Gansler et al., (NHL), follicular lymphoma, diffuse large B-cell lymphoma Hum. Pathol. (1997) 28:686-692; and Zhou et al., Cancer (DLBCL), mantle cell lymphoma (MCL)), leiomyosarcoma Res. (2007) 2964-2971); (LMS), mastocytosis (e.g., systemic mastocytosis), multiple (Xviii) pancreatic cancer (e.g., pancreatic andenocarci myeloma (MM), myelodysplastic syndrome (MDS), noma, intraductal papillary mucinous neoplasm (IPMN): see mesothelioma, myeloproliferative disorder (MPD) (e.g., Walter et al., Cancer Epidemiol. Biomarkers Prev. (2009) polycythemia Vera (PV), essential thrombocytosis (ET), 18:2380-2385): agnogenic myeloid metaplasia (AMM) a.k.a. myelofibrosis (xix) Pagets disease of the Vulva (see Alo et al., Int. J. (MF), chronic idiopathic myelofibrosis, chronic myelocytic Gynecol. Pathol. (2005) 24:404–408): leukemia (CML), chronic neutrophilic leukemia (CNL), (XX) prostate cancer (see Pizer et al., Proc Am. Assoc. 10 hypereosinophilic syndrome (HES)), neuroblastoma, neu Cancer Res. (2000) 41:655; Swinnen et al., Int. J. Cancer rofibroma (e.g., neurofibromatosis (NF) type 1 or type 2, (2002)98:19-22; Epstein et al., Urology (1995)45:81–86: De Schwannomatosis), neuroendocrine cancer (e.g., gastroen Schriver et al., Cancer Res. (2003) 63:3799-3804; Pizer et teropancreatic neuroendoctrine tumor (GEP-NET), carcinoid al., Prostate (2001) 47:102-110; Furuya et al., Anticancer tumor), osteosarcoma, oral cancer (e.g., oral squamous cell Res. (1997) 17:4589-4593: Shurbaji et al., Hum. Pathol. 15 carcinoma (OSCC)), ovarian cancer (e.g., cystadenocarci (1996) 27:917-921; Migita et al., J. Nat. Cancer Inst. (2009) noma, ovarian embryonal carcinoma, ovarian adenocarci 101:519-532: Rossi et al., Mol. Cancer. Res. (2003) 1:707 noma), Paget’s disease of the Vulva, Paget’s disease of the 715; and Shah et al., Hum. Pathol. (2006) 37:401-409): penis, papillary adenocarcinoma, pancreatic cancer (e.g., (XXi) retinoblastoma (see Camassei et al., Investig. Opthal pancreatic andenocarcinoma, intraductal papillary mucinous mol. Vis. Sci. (2003) 44:2399-2403; and Slade et al., Antican neoplasm (IPMN)), pinealoma, primitive neuroectodermal cer Res. (2003) 23:1235-1243); tumor (PNT), prostate cancer (e.g., prostate adenocarci (XXii) soft tissue sarcoma (e.g., malignant fibrous histiocy noma), rhabdomyosarcoma, retinoblastoma, salivary gland toma (MFH), liposarcoma, malignant peripheral nerve sheath cancer, skin cancer (e.g., squamous cell carcinoma (SCC), tumor (MPNST), chondrosarcoma: see Takahiro et al., Clin. keratoacanthoma (KA), melanoma, basal cell carcinoma), Cancer Res. (2003) 9:2204-2212): 25 Small bowel cancer (e.g., appendix cancer), Soft tissue sar (XXiii) skin cancer (e.g., melanoma: see Innocenzi et al., J. coma (e.g., malignant fibrous histiocytoma (MFH), liposar Cutan. Pathol. (2003)30:23-28; Kapur et al., Modern Pathol coma, malignant peripheral nerve sheath tumor (MPNST), ogy (2005) 18:1107-1112 and Carvalho et al., Int. J. Cancer chondrosarcoma, fibrosarcoma, myxosarcoma), sebaceous (2008) 123:2557-2565); and gland carcinoma, Sweat gland carcinoma, synovioma, tes (xxiv) thyroid cancer (see Vald et al., Mod. Path. (1999) 30 ticular cancer (e.g., seminoma, testicular embryonal carci 12:70A: Sekiguchi et al., Biomed. Pharmacother. (2001) noma), thyroid cancer (e.g., papillary carcinoma of the thy 55:466-474; e.g., papillary thyroid carcinoma (PTC): see roid, papillary thyroid carcinoma (PTC), medullary thyroid Uddin et al., J. Clin. Endocrinol. Metab. (2008) 93:4088 cancer), and Waldenström's macroglobulinemia. 4097). In certain embodiments, the hyperproliferative disorder is It is envisioned that aberrant FASN activity plays a role in 35 selected from bladder cancer, brain cancer, breast cancer, other hyperproliferative disorders. Exemplary hyperprolif colorectal cancer, esophageal cancer, endometrial cancer, erative diseases, disorders, conditions or cancers include, but gastric cancer, gastrointestinal stromal tumor, kidney cancer, are not limited to, acoustic neuroma, adenocarcinoma, adre liver cancer, lung cancer, mesothelioma, multiple myeloma, nal gland cancer, angiosarcoma (e.g., lymphangiosarcoma, neuroblastoma, oral cancer, ovarian cancer, pancreatic can lymphangioendotheliosarcoma, hemangiosarcoma), benign 40 cer, prostate cancer, Paget’s disease of the Vulva, retinoblas monoclonal gammopathy, biliary cancer (e.g., cholangiocar toma, soft tissue sarcoma, skin cancer or thyroid cancer. cinoma), bladder cancer, breast cancer (e.g., adenocarcinoma In certain embodiments, the cancer is selected from of the breast, papillary carcinoma of the breast, mammary mesothelioma, multiple myeloma, neuroblastoma, Paget’s cancer, medullary carcinoma of the breast), brain cancer (e.g., disease, retinoblastoma, leukemia, myelodisplastic Syn meningioma; glioma, e.g., astrocytoma, oligodendroglioma: 45 drome, or soft tissue sarcoma. medulloblastoma), bronchus cancer (e.g., bronchogenic car In certain embodiments, the brain cancer is meningioma, cinoma), cervical cancer (e.g., cervical adenocarcinoma), glioma or meduloblastoma. choriocarcinoma, chordoma, craniopharyngioma, colorectal In certain embodiments, the oral cancer is oral squamous cancer (e.g., colorectal adenocarcinoma), epithelial carci cell carcinoma. noma, ependymoma, endotheliosarcoma (e.g., Kaposi's sar 50 In certain embodiments, the pancreatic cancer is pancreatic coma, multiple idiopathic hemorrhagic sarcoma), endome andenocarcinoma or intraductal papillary mucinous neo trial cancer, esophageal cancer (e.g., adenocarcinoma of the plasm. esophagus, Barrett's adenocarinoma), Ewing sarcoma, famil In certain embodiments, the Soft tissue carcinoma is malig iar hypereosinophilia, gastric cancer (e.g., stomachadenocar nant fibrous histiocytoma, liposarcoma, malignant peripheral cinoma), gastrointestinal stromal tumor (GIST), head and 55 nerve sheath tumor, or chondrosarcoma. neck cancer, heavy chain disease (e.g., alpha chain disease, In certain embodiments, the skin cancer is melanoma. gamma chain disease, muchain disease), hemangioblastoma, In certain embodiments, the thyroid cancer is papillary inflammatory myofibroblastic tumors, immunocytic amyloi thyroid carcinoma. dosis, kidney cancer (e.g., nephroblastoma a.k.a. Wilms In certain embodiments, the FASN-mediated disorder is an tumor, renal cell carcinoma), liver cancer (e.g., hepatocellular 60 inflammatory disorder. The term “inflammatory disorder cancer (HCC) such as hepatocellular carcinoma, malignant refers to a disease or condition characterized by one or more hepatoma), lung cancer (e.g., Small cell lung cancer (SCLC), symptoms of pain, heat, redness, Swelling, and loss of func non-Small cell lung cancer (NSCLC), adenocarcinoma of the tion. Inflammatory disorders are meant to encompass inflam lung), leukemia (e.g., acute lymphocytic leukemia (ALL), mation associated with immune system disorders as well as acute myelocytic leukemia (AML), chronic myelocytic leu 65 inflammation associated with non-immune system disorders. kemia (CML), chronic lymphocytic leukemia (CLL)), lym Inflammatory disorders are meant to encompass acute phoma (e.g., Hodgkin lymphoma, non-Hodgkin lymphoma inflammation and chronic inflammation. To date, aberrant US 8,546,432 B2 103 104 FASN activity has been observed in inflammatory bowel alcoholic cirrhosis, endotoxin mediated liver injury) or diseases such as ulcerative colitis (see Consolazio et al., Ana chronic renal failure. Thus, In some embodiments, disclosed tomic Pathology (2006) 126:113-118; Rashid et al., Am. J. methods are applicable to obese subjects, diabetic Subjects, Pathol. (1997) 150:201-208). It is envisioned that aberrant and alcoholic Subjects, and are generally useful as part of a FASN activity plays a role in other inflammatory disorders. program to treat an obesity-related disorder or a complication Exemplary inflammatory disorders include, but are not thereof. limited to, inflammation associated with acne, anemia (e.g., An "obesity-related disorder as used herein, includes, but aplastic anemia, haemolytic autoimmune anaemia), asthma, is not limited to, obesity, undesired weight gain (e.g., from arteritis (e.g., polyarteritis, temporal arteritis, periarteritis medication-induced weight gain, from cessation of Smoking) nodosa, Takayasu's arteritis), arthritis (e.g., crystallinearthri 10 tis, osteoarthritis, psoriatic arthritis, gouty arthritis, reactive and an over-eating disorder (e.g., binge eating, bulimia, com arthritis, rheumatoid arthritis and Reiters arthritis), ankylos pulsive eating, or a lack of appetite control each of which can ing spondylitis, amylosis, amyotrophic lateral Sclerosis, optionally lead to undesired weight gain or obesity). “Obe autoimmune diseases, allergies or allergic reactions, Alzhe sity' and “obese as used herein, refers to class I obesity, class imer's disease, atherosclerosis, bronchitis, bursitis, cancer, 15 II obesity, class III obesity or pre-obesity (e.g., being “over chronic prostatitis, conjunctivitis, Chagas disease, chronic weight') as defined by the World Health Organization. obstructive pulmonary disease, cermatomyositis, diverticuli In some embodiments, obesity-related disorder include, tis, diabetes (e.g., type I diabetes mellitus, type 2 diabetes but are not limited to, Type II diabetes mellitus, elevated mellitus), dermatitis, eosinophilic gastrointestinal disorders blood pressure, elevated cholesterol levels, ischemic heart (e.g., eosinophilic esophagitis, eosinophilic gastritis, eosino disease, arterial vascular disease, angina, myocardial infarc philic gastroenteritis, eosinophilic colitis), eczema, tion, stroke, migraines, congestive heart failure, deep vein endometriosis, gastrointestinal bleeding, gastritis, gastroe thrombosis, pulmonary embolism, gall stones, gastroesoph sophageal reflux disease (GORD, or its synonym GERD), agael reflux disease, obstructive sleep apnea, obesity Guillain-Barre Syndrome, infection, ischaemic heart disease, hypoVentilation syndrome, asthma, gout, poor mobility, back Kawasaki disease, glomerulonephritis, gingivitis, hypersen 25 pain, erectile dysfunction, urinary incontinence, liver injury, sitivity, headaches (e.g., migraine headaches, tension head fatty liver, and chronic renal failure. aches), ileus (e.g., postoperative ileus and ileus during sep In some embodiments, treatment of an obesity-related dis sis), idiopathic thrombocytopenic purpura, interstitial order or complication thereof involves reduction of body cystitis, inflammatory bowel disease (IBD) (e.g., Crohn's weight in the Subject. In some embodiments, treatment of an disease, ulcerative colitis, collagenous colitis, lymphocytic 30 obesity-related disorder or complication thereof involves colitis, ischaemic colitis, diversion colitis, Behcet’s Syn appetite control in the Subject. drome, indeterminate colitis), inflammatory bowel syndrome In other embodiments, provided herein are methods for (IBS), lupus, multiple Sclerosis, morphea, myeasthenia treating, preventing and/or managing a microbial infection gravis, myocardial ischemia, nephrotic syndrome, pemphi (e.g., Such as a bacterial infection, viral infection, fungal gus Vulgaris, pernicious aneaemia, peptic ulcers, psoriasis, 35 infection, or parasitic or protozoal infection) comprising polymyositis, primary biliary cirrhosis, Parkinson's disease, administering to a subject a therapeutically or prophylacti pelvic inflammatory disease, reperfusion injury, regional cally effective amount of at least one compound of formula (I) enteritis, rheumatic fever, systemic lupus erythematosus, or a pharmaceutically acceptable form thereof, or a pharma Schleroderma, Scierodoma, sarcoidosis, spondyloarthop ceutical composition comprising at least one compound of athies, Sjogren's syndrome, thyroiditis, transplantation rejec 40 formula (I) or a pharmaceutically acceptable form thereof. tion, tendonitis, trauma or injury (e.g., frostbite, chemical Also provided herein is the use of at least one compound of irritants, toxins, Scarring, burns, physical injury), Vasculitis, formula (I), or a pharmaceutically acceptable form thereof, or vitiligo and Wegener's granulomatosis. a pharmaceutical composition comprising at least one com In some embodiment, the inflammatory disorder is pound of formula (I) or a pharmaceutically acceptable form selected from anemia, asthma, arteritis, arthritis, chronic 45 thereof, for the treatment, prevention and/or management of a obstructive pulmonary disease, dermatitis, gastroesophageal microbial infection in a Subject. reflux disease, Crohn's disease, inflammatory bowel syn Also provided herein is the use of at least one compound of drome, multiple Sclerosis, psoriasis and an autoimmune dis formula (I), or a pharmaceutically acceptable form thereof, or CaSC. a pharmaceutical composition comprising at least one com Inhibition of FASN activity has also been observed to 50 pound of formula (I) or a pharmaceutically acceptable form reduce body weight (e.g., by blocking the body’s ability to thereof, in the manufacture of a medicament useful for treat convert carbohydrates to fat) and to suppress appetite (see ing, preventing and/or managing a microbial infection. Loftus et al., Science (2000) 288:2379-2381). Reduction of FASN has been identified as a target for treatment of micro storage fat is expected to provide various primary and/or bial infections, e.g., Such as a viral infection, for example, secondary benefits in a subject (e.g., in a Subject diagnosed 55 infection with an enveloped virus such as the herpes virus with a complication associated with obesity) Such as, for (e.g., human cytomegalomous virus (HCMV), herpes sim example, an increased insulin responsiveness (e.g., in a Sub plex virus 1 (HSV-1), herpes simplex virus 2 (HSV-2), vari ject diagnosed with Type II diabetes mellitus); a reduction in cella Zoster virus (VZV), Epstein-Barr virus), influenza A elevated blood pressure; a reduction in elevated cholesterol virus and Heptatitis C virus (HCV) (see Munger et al., Nature levels; and/or a reduction (or a reduced risk or progression) of 60 Biotechnology (2008) 26: 1179-1186: Syed et al., Trends in ischemic heart disease, arterial vascular disease, angina, Endocrinology and Metabolism (2009) 21:33-40; Sakamoto myocardial infarction, stroke, migraines, congestive heart et al., Nature Chemical Biology (2005) 1:333-337:Yang et al., failure, deep vein thrombosis, pulmonary embolism, gall Hepatology (2008) 48:1396-1403) or a picornavirus such as stones, gastroesophagael reflux disease, obstructive sleep Coxsackievirus B3(CVB3) (see Rassmann et al., Anti-viral apnea, obesity hypoVentilation syndrome, asthma, gout, poor 65 Research (2007) 76:150-158). Other exemplary viruses mobility, back pain, erectile dysfunction, urinary inconti include, but are not limited to, the hepatitis B virus, HIV. nence, liver injury (e.g., fatty liver disease, liver cirrhosis, poxvirus, hepadavirus, retrovirus, and RNA viruses such as US 8,546,432 B2 105 106 flavivirus, togavirus, coronavirus, Hepatitis D virus, orth family, iridovirus family, reovirus family, birnavirus family, omyxovirus, paramyxovirus, rhabdovirus, bunyavirus, and calicivirus family, and picornavirus family. Specific filovirus. examples include, but are not limited to, canine parvovirus, In some embodiments, the virus infects humans. In other parvovirus B19, porcine circovirus type 1 and 2. BFDV (Beak embodiments, the virus infects non-human animals. In 5 and Feather Disease virus, chicken anaemia virus, Polyoma another embodiment, the virus infects primates (e.g., cyno virus, simian virus 40 (SV40), JC virus, BK virus, Budgerigar molgus monkeys, rhesus monkeys); mammals, including fledgling disease virus, human papillomavirus, bovine papil commercially relevant mammals such as cattle, pigs, horses, lomavirus (BPV) type 1, cotton tail rabbit papillomavirus, sheep, goats, cats, and/or dogs; and/or birds, including com human adenovirus (HAdV-A, HAdV-B, HAdV-C, HAdV-D, mercially relevant birds such as chickens, ducks, geese, and/ 10 or turkeys. HAdV-E, and HAdV-F), fowl adenovirus A. bovine adenovi In certain embodiments, the virus is an enveloped virus. rus D, frog adenovirus, Reovirus, human orbivirus, human Examples include, but are not limited to, viruses that are coltivirus, mammalian orthoreovirus, bluetongue virus, members of the hepadnavirus family, herpesvirus family, iri rotavirus A, rotaviruses (groups B to G), Colorado tick fever dovirus family, poxvirus family, flavivirus family, togavirus 15 virus, aquareovirus A, cypovirus 1, Fiji disease virus, rice family, retrovirus family, coronavirus family, filovirus family, dwarf virus, rice ragged stunt virus, idnoreovirus 1, mycore rhabdovirus family, bunyavirus family, orthomyxovirus fam ovirus 1, Birnavirus, bursal disease virus, pancreatic necrosis ily, paramyxovirus family, and arenavirus family. Other virus, Calicivirus, Swine vesicular exanthema virus, rabbit examples include, but are not limited to, Hepadnavirus hepa hemorrhagic disease virus, Norwalk virus, Sapporo virus, titis B virus (HBV), woodchuck hepatitis virus, ground squir Picornavirus, human polioviruses (1-3), human coxsackievi rel (Hepadnaviridae) hepatitis virus, duck hepatitis B virus, ruses A1-22, 24 (CA1-22 and CA24, CA23 (echovirus 9)), heron hepatitis B virus, Herpesvirus herpes simplex virus human coxsackieviruses (B1-6 (CB1-6)), human echoviruses (HSV) types 1 and 2, varicella-zoster virus, cytomegalovirus 1-7,9,11-27, 29-33, vilyuish virus, simian enteroviruses 1-18 (CMV), human cytomegalovirus (HCMV), mouse cytome (SEV1-18), porcine enteroviruses 1-11 (PEV1-11), bovine galovirus (MCMV), guinea pig cytomegalovirus (GPCMV), 25 enteroviruses 1-2 (BEV1-2), hepatitis A virus, rhinoviruses, Epstein-Barr virus (EBV), human herpes virus 6 (HHV vari hepatoviruses, cardioviruses, aphthoviruses and echoviruses. ants A and B), human herpes virus 7 (HHV-7), human herpes In certain embodiments, the virus is a herpes virus, e.g., virus 8 (HHV-8), Kaposi's sarcoma-associated herpes virus HSV-I, HSV-2, and CMV. In another embodiment, the virus is (KSHV), B virus Poxvirus vaccinia virus, variola virus, HCMV. In another embodiment, the virus is a liver trophic Smallpox virus, monkeypox virus, cowpox virus, camelpox 30 virus. In another embodiment, the virus is an influenza virus. virus, ectromelia virus, mousepox virus, rabbitpox viruses, In some embodiments, the virus is HIV. In certain embodi raccoonpox viruses, molluscum contagiosum virus, orf virus, ments, the virus is a hepatitis B virus. In a specific embodi milker's nodes virus, bovin papular stomatitis virus, sheep ment, the virus is EBV. In some embodiments, the virus is pox virus, goatpox virus, lumpy skin disease virus, fowlpox Kaposi's sarcoma-associated herpes virus (KSHV). In cer virus, canarypox virus, pigeonpox virus, sparrowpox virus, 35 tain embodiments the virus is a variola virus. In one embodi myxoma virus, hare fibroma virus, rabbit fibroma virus, ment, the virus is a Dengue virus. In other embodiments, the squirrel fibroma viruses, Swinepox virus, tanapox virus, virus is a SARS virus. In one embodiment, the virus is an Yabapox virus, Flavivirus dengue virus, hepatitis C virus Ebola virus. In some embodiments the virus is a Marburg (HCV), GB hepatitis viruses (GBV-A, GBV-B and GBV-C), virus. In certain embodiments, the virus is a measles virus. In West Nile virus, yellow fever virus, St. Louis encephalitis 40 particular embodiments, the virus is a vaccinia virus. In some virus, Japanese encephalitis virus, Powassan virus, tick-borne embodiments, the virus is varicella-Zoster virus (VZV). In encephalitis virus, Kyasanur Forest disease virus, Togavirus, Some embodiments, the virus is a picornavirus. In certain Venezuelan equine encephalitis (VEE) virus, chikungunya embodiments the virus is a rhinovirus. In certain embodi virus, Ross River virus, Mayaro virus, Sindbis virus, rubella ments the virus is not a rhinovirus. In some embodiments, the virus, Retrovirus human immunodeficiency virus (HIV) 45 virus is an adenovirus. In particular embodiments, the virus is types 1 and 2, human T cell leukemia virus (HTLV) types 1, a coxsackievirus (e.g., coxsackievirus B3). In some embodi 2, and 5, mouse mammary tumor virus (MMTV), Roussar ments, the virus is a rhinovirus. In certain embodiments, the coma virus (RSV), lentiviruses, Coronavirus, severe acute virus is a human papillomavirus (HPV). respiratory syndrome (SARS) virus, Filovirus Ebola virus, In certain embodiments, the virus is a DNA virus. In other Marburg virus, Metapneumoviruses (MPV) such as human 50 embodiments, the virus is an RNA virus. In one embodiment, metapneumovirus (HMPV), Rhabdovirus rabies virus, the virus is a DNA or a RNA virus with a single-stranded vesicular stomatitis virus, Bunyavirus, Crimean-Congo hem genome. In another embodiment, the virus is a DNA or a RNA orrhagic fever virus, Rift Valley fever virus, La Crosse virus, virus with a double-stranded genome. Hantaan virus, Orthomyxovirus, influenza virus (types A, B, In some embodiments, the virus has a linear genome. In and C), Paramyxovirus, parainfluenza virus (PIV types 1, 2 55 other embodiments, the virus has a circular genome. In some and 3), respiratory syncytial virus (types A and B), measles embodiments, the virus has a segmented genome. In other virus, mumps virus, Arenavirus, lymphocytic choriomenin embodiments, the virus has a non-segmented genome. gitis virus, Junin virus, Machupo virus, Guanarito virus, In some embodiments, the virus is a positive-stranded Lassa virus, Ampari virus, Flexal virus, Ippy virus, Mobala RNA virus. virus, Mopeia virus, Latino virus, Parana virus, Pichinde 60 In other embodiments, the virus is a negative-stranded virus, Punta toro virus (PTV), Tacaribe virus and Tamiami RNA virus. In one embodiment, the virus is a segmented, virus. negative-stranded RNA virus. In another embodiment, the In some embodiments, the virus is a non-enveloped virus, virus is a non-segmented negative-stranded RNA virus. i.e., the virus does not have an envelope and is naked. In some embodiments, the virus is an icosahedral virus. In Examples include, but are not limited to, viruses that are 65 other embodiments, the virus is a helical virus. In yet other members of the parvovirus family, circovirus family, embodiments, the virus is a complex virus. polyoma virus family, papillomavirus family, adenovirus In some embodiments, the virus is a hepatitis C virus. US 8,546,432 B2 107 108 In certain embodiments, the virus is selected from: a herpes can treat one or more infections caused by four or more types virus such as HSV-1, HSV-2, VZV, EBV, CMV (HCMV, of viruses at the same time. In other embodiments, at least one MCMV, GPCMV), HMCV, CVB3, HHV-6 and HHV-8; an compound of formula (I) or a pharmaceutically acceptable influenza virus Such as influenza type A and influenza type B; form thereof, or a pharmaceutical composition comprising at respiratory viruses such as RSV. PIV (types 1, 2 and 3), 5 least one compound of formula (I) or a pharmaceutically measles virus, rhinovirus, adenovirus, HMPV and SARS acceptable form thereof provided herein can treat one or more virus; orthopoxviruses such as vaccinia virus, cowpox virus, infections caused by five or more types of viruses at the same ectromelia virus, monkeypox virus and rabbitpox virus; a time. In other embodiments, at least one compound of for hepatitis virus such as HBV and HCV: a papova virus such as mula (I) or a pharmaceutically acceptable form thereof, or a papillomavirus (e.g., cotton tail rabbit papillomavirus and 10 pharmaceutical composition comprising at least one com human papillomavirus) and BK virus; or other viruses such as pound of formula (I) or a pharmaceutically acceptable form VEE virus, Rift Valley fever virus, Tacaribe virus, Yellow thereof provided herein can treat one or more infections fever virus, West Nile virus, dengue virus, PTV and Pichinde caused by six, seven, eight, nine, ten, fifteen, twenty or more virus. types of viruses at the same time. In one embodiment, the virus is HSV-1. In another embodi 15 In certain embodiments, the microbial infections can ment, the virus is HSV-2. In another embodiment, the virus is encompass the disease related to infection by prions, e.g., VZV. In another embodiment, the virus is EBV. In another scrapie, madcow disease, and any modified forms thereof. In embodiment, the virus is HCMV. In another embodiment, the certain embodiments, the microbial infections encompass virus is MCMV. In another embodiment, the virus is GPCMV. those prion diseases that affect humans. In another embodiment, the virus is HHV-6. In another It is envisioned that a compound of formula (I) or a phar embodiment, the virus is HHV-8. maceutically acceptable form thereof, or a pharmaceutical In one embodiment, the virus is influenza type A virus. In composition comprising at least one compound of formula (I) another embodiment, the virus is influenza type B virus. or a pharmaceutically acceptable form thereof provided In one embodiment, the virus is RSV. In another embodi herein will also be useful in the treatment of other microbial ment, the virus is PIV-3. In another embodiment, the virus is 25 infections. Such as bacterial infections, fungal infections, and measles virus. In another embodiment, the virus is rhinovirus. parasitic infections. In another embodiment, the virus is adenovirus. In another In certain embodiments, the microbial infection is a bacte embodiment, the virus is HMPV. In another embodiment, the rial infection. Examples of bacterial infections include, but virus is SARS virus. are not limited to, infections by mycobacteria (e.g., Mycobac In one embodiment, the virus is vaccinia virus. In another 30 teria tuberculosis, M. bovis, M. avium, M. leprae, and M. embodiment, the virus is cowpox virus. In another embodi africanum), rickettsia, mycoplasma, chlamydia, and ment, the virus is ectromelia virus. In another embodiment, legionella. Other examples ofbacterial infections include, but the virus is monkeypox virus. In another embodiment, the are not limited to, infections caused by Gram positive bacillus virus is rabbitpox virus. (e.g., Listeria, Bacillus Such as Bacillus anthracis, Erysipelo In one embodiment, the virus is HBV. In another embodi 35 thrix species), Gram negative bacillus (e.g., Bartonella, Bru ment, the virus is HCV. cella, Campylobacter, Enterobacter, Escherichia, Fran In one embodiment, the virus is cotton tail rabbit papillo cisella, Hemophilus, Klebsiella, Morganella, Proteus, mavirus. In another embodiment, the virus is human papillo Providencia, Pseudomonas, Salmonella, Serratia, Shigella, mavirus. In another embodiment, the virus is BK virus. Vibrio and Yersinia species), Spirochete bacteria (e.g., Borre In one embodiment, the virus is VEE virus. In another 40 lia species including Borrelia burgdorferi that causes Lyme embodiment, the virus is Rift Valley fever virus. In another disease), anaerobic bacteria (e.g., Actinomyces and embodiment, the virus is Tacaribe virus. In another embodi Clostridium species), Gram positive and negative coccal bac ment, the virus is Yellow fever virus. In another embodiment, teria, Enterococcus species, Streptococcus species, Pneumo the virus is West Nile virus. In another embodiment, the virus coccus species, Staphylococcus species, and Neisseria spe is dengue virus. In another embodiment, the virus is PTV. In 45 cies. another embodiment, the virus is Pichinde virus. Specific examples of infectious bacteria include, but are In certain embodiments, at least one compound of formula not limited to: Helicobacter pyloris, Borelia burgdorferi, (I) or a pharmaceutically acceptable form thereof, or a phar Legionella pneumophilia, Mycobacteria tuberculosis, M. maceutical composition comprising at least one compound of avium, M. intracellulare, M. kansai, M. gordonae, Staphy formula (I) or a pharmaceutically acceptable form thereof 50 lococcus aureus, Neisseria gonorrhoeae, Neisseria menin provided herein can treat an infection caused by one type of gitidis, Listeria monocytogenes, Streptococcus pyogenes virus. In other embodiments, at least one compound of for (Group A Streptococcus), Streptococcus agalactiae (Group B mula (I) or a pharmaceutically acceptable form thereof, or a Streptococcus), Streptococcus viridans, Streptococcus faeca pharmaceutical composition comprising at least one com lis, Streptococcus bovis, Streptococcus pneumoniae, Haemo pound of formula (I) or a pharmaceutically acceptable form 55 philus influenzae, Bacillus antracis, corynebacterium diph thereof provided herein can treat one or more infections theriae, Erysipelothrix rhusiopathiae, Clostridium caused by two or more types of viruses at the same time. In perfingens, Clostridium tetani, Enterobacter aerogenes, other embodiments, at least one compound of formula (I) or a Klebsiella pneumoniae, Pasturella multocida, Fusobacte pharmaceutically acceptable form thereof, or a pharmaceuti rium nucleatum, Streptobacillus moniliformis, Treponema cal composition comprising at least one compound of for 60 pallidium, Treponema pertenue, Leptospira, Rickettsia, and mula (I) or a pharmaceutically acceptable form thereof pro Actinomyces israeli. vided herein can treat one or more infections caused by three In one embodiment, the bacterial infection is an infection or more types of viruses at the same time. In other embodi caused by Mycobacteria tuberculosis. ments, at least one compound of formula (I) or a pharmaceu In certain embodiments, at least one compound of formula tically acceptable form thereof, or a pharmaceutical compo 65 (I) or a pharmaceutically acceptable form thereof, or a phar sition comprising at least one compound of formula (I) or a maceutical composition comprising at least one compound of pharmaceutically acceptable form thereof provided herein formula (I) or a pharmaceutically acceptable form thereof US 8,546,432 B2 109 110 provided herein can treat an infection caused by one type of pharmaceutical composition comprising at least one com bacteria. In other embodiments, at least one compound of pound of formula (I) or a pharmaceutically acceptable form formula (I) or a pharmaceutically acceptable form thereof, or thereof provided herein can treat one or more infections a pharmaceutical composition comprising at least one com caused by six, seven, eight, nine, ten, fifteen, twenty or more pound of formula (I) or a pharmaceutically acceptable form 5 types of fungi at the same time. thereof provided herein can treat one or more infections In certain embodiments, provided herein are methods of caused by two or more types of bacteria at the same time. In treating, preventing and/or managing diseases, disorders, or other embodiments, at least one compound of formula (I) or a conditions caused by parasitic or protozoal infection. pharmaceutically acceptable form thereof, or a pharmaceuti Examples of parasitic or protozoal diseases and disorders cal composition comprising at least one compound of for 10 include, but are not limited to, diseases, disorders and condi mula (I) or a pharmaceutically acceptable form thereof pro tions caused by parasites such as, but not limited to, Pfalci vided herein can treat one or more infections caused by three farium, P. ovale, P. vivax, P malariae, L. donovari, L. infan or more types of bacteria at the same time. In other embodi tum, L. aethiopica, L. major; L. tropica, L. mexicana, L. ments, at least one compound of formula (I) or a pharmaceu braziliensis, T. Gondii, B. microti, B. divergens, B. coli, B. tically acceptable form thereof, or a pharmaceutical compo 15 hominis, C. parvum, C. Cayetanensis, D. fragilis, E. his sition comprising at least one compound of formula (I) or a tolytica, I. belli, S. mansonii, S. haematobium, Trypanosoma pharmaceutically acceptable form thereof provided herein ssp., Toxoplasma ssp., and O. volvulus. Other diseases, dis can treat one or more infections caused by four or more types orders and conditions include, but are not limited to, those of bacteria at the same time. In other embodiments, at least caused by Babesia bovis, Babesia Canis, Banesia Gibsoni, one compound of formula (I) or a pharmaceutically accept 20 Besnoitia darlingi, Cytauxzoon felis, Eimeria ssp., Hammon able form thereof, or a pharmaceutical composition compris dia ssp., T. Canis, Cestoda (i.e., tapeworms) and Theileria ssp. ing at least one compound of formula (I) or a pharmaceuti Specific diseases, disorders and conditions include, but are cally acceptable form thereof provided herein can treat one or not limited to, malaria, babesiosis, trypanosomiasis, Ameri more infections caused by five or more types of bacteria at the can trypanosomiasis (i.e., Chagas disease), leishmaniasis, same time. In other embodiments, at least one compound of 25 toxoplasmosis, meningoencephalitis, keratitis, amebiasis, formula (I) or a pharmaceutically acceptable form thereof, or giardiasis, cryptosporidiosis, isosporiasis, cyclosporiasis, a pharmaceutical composition comprising at least one com microSporidiosis, ascariasis, trichuriasis, ancylostomiasis, pound of formula (I) or a pharmaceutically acceptable form strongyloidiasis, toxocariasis, trichinosis, lymphatic filari thereof provided herein can treat one or more infections asis, onchocerciasis, filariasis, Schistosomiasis, and dermati caused by six, seven, eight, nine, ten, fifteen, twenty or more 30 tis caused by animal Schistosomes. types of bacteria at the same time. In one embodiment, the parasitic or protozoal disease is In certain embodiments, provided herein are methods of malaria. In another embodiment, the parasitic or protozoal treating, preventing and/or managing diseases, disorders, or disease is leishmaniasis. In another embodiment, the parasitic conditions caused by fungal infection. Examples include, but or protozoal disease is babesiosis. In another embodiment, are not limited to, aspergilliosis, crytococcosis, sporotricho 35 the parasitic or protozoal disease is toxoplasmosis. In another sis, coccidioidomycosis, paracoccidioidomycosis, histoplas embodiment, the parasitic or protozoal disease is trypanoso mosis, blastomycosis, Zygomycosis, and candidiasis. miasis. In certain embodiments, at least one compound of formula In certain embodiments, at least one compound of formula (I) or a pharmaceutically acceptable form thereof, or a phar (I) or a pharmaceutically acceptable form thereof, or a phar maceutical composition comprising at least one compound of 40 maceutical composition comprising at least one compound of formula (I) or a pharmaceutically acceptable form thereof formula (I) or a pharmaceutically acceptable form thereof provided herein can treat an infection caused by one type of provided herein can treat an infection caused by one type of fungi. In other embodiments, at least one compound of for parasite. In other embodiments, at least one compound of mula (I) or a pharmaceutically acceptable form thereof, or a formula (I) or a pharmaceutically acceptable form thereof, or pharmaceutical composition comprising at least one com 45 a pharmaceutical composition comprising at least one com pound of formula (I) or a pharmaceutically acceptable form pound of formula (I) or a pharmaceutically acceptable form thereof provided herein can treat one or more infections thereof provided herein can treat one or more infections caused by two or more types of fungi at the same time. In caused by two or more types of parasite at the same time. In other embodiments, at least one compound of formula (I) or a other embodiments, at least one compound of formula (I) or a pharmaceutically acceptable form thereof, or a pharmaceuti 50 pharmaceutically acceptable form thereof, or a pharmaceuti cal composition comprising at least one compound of for cal composition comprising at least one compound of for mula (I) or a pharmaceutically acceptable form thereof pro mula (I) or a pharmaceutically acceptable form thereof pro vided herein can treat one or more infections caused by three vided herein can treat one or more infections caused by three or more types of fungi at the same time. In other embodi or more types of parasite at the same time. In other embodi ments, at least one compound of formula (I) or a pharmaceu 55 ments, at least one compound of formula (I) or a pharmaceu tically acceptable form thereof, or a pharmaceutical compo tically acceptable form thereof, or a pharmaceutical compo sition comprising at least one compound of formula (I) or a sition comprising at least one compound of formula (I) or a pharmaceutically acceptable form thereof provided herein pharmaceutically acceptable form thereof provided herein can treat one or more infections caused by four or more types can treat one or more infections caused by four or more types of fungi at the same time. In other embodiments, at least one 60 of parasite at the same time. In other embodiments, at least compound of formula (I) or a pharmaceutically acceptable one compound of formula (I) or a pharmaceutically accept form thereof, or a pharmaceutical composition comprising at able form thereof, or a pharmaceutical composition compris least one compound of formula (I) or a pharmaceutically ing at least one compound of formula (I) or a pharmaceuti acceptable form thereofprovided herein can treat one or more cally acceptable form thereof provided herein can treat one or infections caused by five or more types of fungi at the same 65 more infections caused by five or more types of parasite at the time. In other embodiments, at least one compound of for same time. In other embodiments, at least one compound of mula (I) or a pharmaceutically acceptable form thereof, or a formula (I) or a pharmaceutically acceptable form thereof, or US 8,546,432 B2 111 112 a pharmaceutical composition comprising at least one com another embodiment, the ELOVL-mediated disorder is obe pound of formula (I) or a pharmaceutically acceptable form sity. In another embodiment, the ELOVL-mediated disorder thereof provided herein can treat one or more infections is diabetes mellitus. In another embodiment, the ELOVL caused by six, seven, eight, nine, ten, fifteen, twenty or more mediated disorder is fatty liver. types of parasite at the same time. In one embodiment, the ELOVL-mediated disorder is In some embodiments, compounds provided herein can ELOVL6-mediated disorder. treat infection by any combination of viruses, bacteria, fungi and parasites at the same time. For example, in certain 5. Administration embodiments, compounds provided herein can treat the infection by one or more viruses and one or more fungi. In 10 The compound of formula (I) or a pharmaceutically other embodiments, compounds provided herein can treat the acceptable form thereof, or a pharmaceutical composition infection by one or more viruses and one or more bacteria. In comprising at least one compound of formula (I) or a phar other embodiments, compounds provided herein can treat the maceutically acceptable form thereof can be administered infection by one or more fungi and one or more bacteria. In using any amount and any route of administration effective other embodiments, compounds provided herein can treat the 15 for treatment. The compounds provided herein are typically infection by one or more viruses and one or more parasites. In formulated in dosage unit form for ease of administration and other embodiments, compounds provided herein can treat the uniformity of dosage. It will be understood, however, that the infection by one or more fungi and one or more parasites. In total daily usage of the compounds provided herein will be other embodiments, compounds provided herein can treat the decided by the attending physician within the scope of sound infection by one or more bacteria and one or more parasites. medical judgment. The specific therapeutically effective dose In other embodiments, compounds provided herein can treat level for any particular subject will depend upon a variety of the infection by one or more viruses, one or more fungi and factors including the disease, disorder, or condition being one or more bacteria. In other embodiments, compounds treated and its severity; the activity of the specific compound provided herein can treat the infection by one or more bacte employed; the specific composition employed; the species, ria, one or more fungi and one or more parasites. In other 25 age, body weight, general health, sex and diet of the Subject; embodiments, compounds provided herein can treat the the time of administration, route of administration, and rate of infection by one or more viruses, one or more fungi and one excretion of the specific compound employed; the duration of or more parasites. In other embodiments, compounds pro the treatment; drugs used in combination or coincidental with vided herein can treat the infection by one or more viruses, the specific compound employed; and like factors well known one or more bacteria and one or more parasites. 30 in the medical arts. Compounds provided herein are inhibitors of FASN. Thus, A therapeutically effective amount of at least one com in certain embodiments, the compounds provided herein can pound of formula (I) or a pharmaceutically acceptable form be used to treat and/or manage other FASN-related disorders, thereof, or a pharmaceutical composition comprising at least examples of which include, but are not limited to, diabetes one compound of formula (I) or a pharmaceutically accept and general wellness of liver Such as treatment, prevention 35 able form thereof disclosed herein can be measured by the and/or management of fatty liver. therapeutic effectiveness of the compound. Compounds of In certain embodiments, the compound is an inhibitor of formula (I) can be administered in a dose of about 1 ug/kg to palmitate synthesis. As used herein “inhibition”, “inhibiting, about 200 mg/kg daily. Such as from about 1 ug/kg to about “inhibit” and “inhibitor', and the like, refer to the ability of a 150 mg/kg, from about 1 mg/kg to about 200 mg/kg, from compound to reduce, halt or prevent activity of a particular 40 about 1 ug/kg to about 100 mg/kg, from about 1 lug/kg to biological process (e.g., FASN activity, palmitate synthesis) about 1 mg/kg, from about 50 g/kg to about 200 mg/kg, from in a cell relative to vehicle. about 10 g/kg to about 1 mg/kg, from about 10 ug/kg to In other embodiments, provided herein are methods for about 100 g/kg, from about 100 g to about 10 mg/kg, and inhibiting ELOVL in a subject comprising administering to a from about 500 ug/kg to about 50 mg/kg. subject in need thereof a therapeutically effective amount of 45 In certain embodiments, atherapeutically effective amount at least one compound of formula (I) or a pharmaceutically of at least one compound of formula (I) or a pharmaceutically acceptable form thereof. acceptable form thereof, or a pharmaceutical composition In another embodiment, provided herein is use of at least comprising at least one compound of formula (I) or a phar one compound of formula (I) for the treatment of a ELOVL maceutically acceptable form thereof for administration one mediated disorder in a Subject. 50 or more times a day to a 70 kg adult human can comprise In another embodiment, provided herein is use of at least about 0.0001 mg to about 1000 mg of an compound per unit one compound of formula (I) in the manufacture of a medi dosage form. It will be appreciated that dose ranges as cament. In certain embodiments, the medicament is useful for described herein provide guidance for the administration of treating a ELOVL-mediated disorder. pharmaceutical compositions to an adult. The amount to be “ELOVL-mediated disorder” as used herein, refers to a 55 administered to, for example, a child oran adolescent can be disease, disorder or condition which is treatable by inhibition determined by a medical practitioner or person skilled in the of ELOVL activity. Typically, ELOVL-mediated disorders art and can be lower or the same as that administered to an are substantially similar to those mediated by FASN. Thus, adult. ELOVL-mediated disorders include the FASN-mediated dis The desired dosage can be delivered three times a day, two orders described herein above. Examples include, but are not 60 times a day, once a day, every other day, every third day, every limited to, hyperproliferative disorders, inflammatory disor week, every two weeks, every three weeks, or every four ders, obesity-related disorders and complications thereof, weeks. In certain embodiments, the desired dosage can be diabetes and general wellness of liver Such as treatment, delivered using multiple administrations (e.g., two, three, prevention and/or management of fatty liver. four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, In one embodiment, the ELOVL-mediated disorder is a 65 fourteen, or more administrations). hyperproliferative disorder. In another embodiment, the In one embodiment, the therapeutically effective amount ELOVL-mediated disorder is an inflammatory disorder. In of a disclosed compound of formula (I) or a pharmaceutically US 8,546,432 B2 113 114 acceptable form thereof, or a pharmaceutical composition pound of formula (I) or a pharmaceutically acceptable form comprising at least one compound of formula (I) or a phar thereof, as described above and herein, can be administered in maceutically acceptable form thereof is sufficient to establish combination with one or more additional therapeutically a maximal plasma concentration ranging from about 0.001 active agents. uM to about 100 uM, e.g., from about 1 uM to about 20 uM. By “in combination with, it is not intended to imply that Preliminary doses as, for example, determined according to the agents must be administered at the same time and/or animal tests, and the Scaling of dosages for human adminis formulated for delivery together, although these methods of tration is performed according to art-accepted practices. delivery are certainly within the scope of this disclosure. The The therapeutically effective dose can be estimated ini compound of formula (I) or a pharmaceutically acceptable 10 form thereof, or a pharmaceutical composition comprising at tially from cell culture assays. A dose can be formulated in least one compound of formula (I) or a pharmaceutically animal models to achieve a circulating plasma concentration acceptable form thereof can be administered concurrently range that includes the ICs (i.e., the concentration of the with, prior to, or Subsequent to, one or more other additional therapeutic which achieves a half-maximal inhibition of therapeutically active agents. In general, each agent will be symptoms) as determined in cell culture assays or animal 15 administered at a dose and/or on a time schedule determined models. Levels in plasma can be measured, for example, by for that agent. In will further be appreciated that the additional high performance liquid chromatography. The effects of any therapeutically active agent utilized in this combination can particular dosage can be monitored by a suitable bioassay. be administered together in a single composition or adminis Examples of dosages are: about 0.1XICso about 0.5xICso tered separately in different compositions. The particular about 1XICso about 5xICs, 10xICso about 50xICs, and combination to employ in a regimen will take into account about 100xICso. compatibility of the compound of formula (I) with the addi Therapeutically effective dosages achieved in one animal tional therapeutically active agent and/or the desired thera model can be converted for use in another animal, including peutic effect to be achieved. humans, using conversion factors known in the art (see, e.g., In some embodiments, additional therapeutically active Freireich et al., Cancer Chemother. Reports 50(4): 219-244 25 agents utilized in combination with at least one compound of (1966) and Table A for Equivalent Surface Area Dosage Fac formula (I) or a pharmaceutically acceptable form thereof, or tors). a pharmaceutical composition comprising at least one com pound of formula (I) or a pharmaceutically acceptable form TABLE A thereof will be administered at levels that do not exceed the 30 levels at which they are utilized individually. In some To: embodiments, the levels utilized in combination will be lower Mouse Rat Monkey Dog Human than those utilized individually. From: (20g) (150 g) (3.5 kg) (8 kg) (60 kg) By a “therapeutically active agent”, “therapeutic agent'. Mouse 1 1.2 1.f4 16 1,12 “agent' or “active agent” refers to any substance that is useful Rat 2 1 1.2 1f4 1/7 35 for therapy, including prophylactic and therapeutic treatment. Monkey 4 2 1 3.5 1,3 Also encompassed herein is the delivery of the pharmaceu Dog 6 4 3.5 1 1.2 tical compositions in combination with agents that can Human 12 7 3 2 1 improve their bioavailability, reduce and/or modify their metabolism, inhibit their excretion, and/or modify their dis In some embodiments, the compound of formula (I) or a 40 tribution within the body. It will also be appreciated that the pharmaceutically acceptable form thereof, or a pharmaceuti therapy employed can achieve a desired effect for the same cal composition comprising at least one compound of for disorder (for example, at least one compound of formula (I) or mula (I) or a pharmaceutically acceptable form thereof is a pharmaceutically acceptable form thereof, or a pharmaceu administered via a variety of routes, including oral, intrave tical composition comprising at least one compound of for nous, intramuscular, intra-arterial, intramedullary, intrathe 45 mula (I) or a pharmaceutically acceptable form thereof can be cal, Subcutaneous, intraventricular, transdermal, interdermal, administered in combination with an anti-inflammatory, anti rectal, intravaginal, intraperitoneal, topical (as by powders, anxiety and/or anti-depressive agent, etc.), and/or they can ointments, creams, and/or drops), mucosal, nasal, bucal, achieve different effects (e.g., control of any adverse side enteral, Sublingual; by intratracheal instillation, bronchial effects). instillation, and/or inhalation; and/or as an oral spray, nasal 50 Exemplary therapeutically active agents include, but are spray, and/or aerosol. Specifically contemplated routes are not limited to, anti-cancer agents, antibiotics, anti-obesity systemic intravenous injection, regional administration via drugs, anti-viral agents, anesthetics, anti-coagulants, inhibi blood and/or lymph Supply, and/or direct administration to an tors of an enzyme, steroidal agents, anti-inflammatory agents, affected site. In general the most appropriate route of admin antihistamine, immunosuppressant agents, anti-neoplastic istration will depend upon a variety of factors including the 55 agents, antigens, vaccines, antibodies, decongestants, seda nature of the agent (e.g., its stability in the environment of the tives, opioids, pain-relieving agents, analgesics, anti-pyret gastrointestinal tract), the condition of the Subject (e.g., ics, enhancing agents, hormones, prostaglandins, progesta whether the subject is able to tolerate oral administration), tional agents, anti-glaucoma agents, ophthalmic agents, anti etc. At present the oral and/or nasal spray and/or aerosol route cholinergics, anti-depressants, anti-psychotics, hypnotics, is most commonly used to deliver therapeutic agents directly 60 tranquilizers, anti-convulsants, muscle relaxants, anti-spas to the lungs and/or respiratory system. However, the delivery modics, muscle contractants, channel blockers, miotic of the pharmaceutical composition by any appropriate route, agents, anti-Secretory agents, anti-thrombotic agents, antico taking into consideration likely advances in the Sciences of agulants, anti-cholinergics, B-adrenergic blocking agents, drug delivery, is also encompassed herein. diuretics, cardiovascular active agents, vasoactive agents, It will be also appreciated that at least one compound of 65 Vasodilating agents, anti-hypertensive agents, angiogenic formula (I) or a pharmaceutically acceptable form thereof, or agents, modulators of cell-extracellular matrix interactions a pharmaceutical composition comprising at least one com (e.g. cell growth inhibitors and anti-adhesion molecules), or US 8,546,432 B2 115 116 inhibitors/intercalators of DNA, RNA, protein-protein inter centis(R), nilotinib (TASIGNAR), Sorafenib (NEXAVARR), actions, protein-receptor interactions, etc. Active agents everolimus (AFINITORR), alemtuzumab (CAMPATHR), include Small organic molecules such as drug compounds gemtuzumab ozogamicin (MYLOTARG(R), temsirolimus (e.g., compounds approved by the Food and Drugs Adminis (TORISEL(R), ENMD-2076, PCI-32765, AC220, dovitinib tration as provided in the Code of Federal Regulations lactate (TKI258, CHIR-258), BIBW 2.992 (TOVOKTM), (CFR)), antibodies, peptides, proteins, carbohydrates, SGX523, PF-04217903, PF-02341066, PF-299804, BMS monosaccharides, oligosaccharides, polysaccharides, nucle 777607, ABT-869, MP470, BIBF 1120 (VARGATEF(R), oproteins, mucoproteins, lipoproteins, synthetic polypep AP24534, JNJ-26483327, MGCD265, DCC-2036, BMS tides or proteins, Small molecules linked to proteins, glyco 690154, CEP-11981, tivozanib (AV-951), OSI-930, proteins, steroids, nucleic acids, DNAs, RNAs, nucleotides, 10 MM-121, XL-184, XL-647, and/or XL228), proteasome nucleosides, oligonucleotides, antisense oligonucleotides, inhibitors (e.g., bortezomib (VELCADE)), mTOR inhibitors lipids, hormones, antibodies, vitamins and cells, and combi (e.g., rapamycin, temsirolimus (CCI-779), everolimus nations thereof. (RAD-001), ridaforolimus, AP23573 (Ariad), AZD8055 (As In certain embodiments, the therapeutically active agent is traZeneca), BEZ235 (Novartis), BGT226 (Norvartis), XL765 an anti-cancer agent. Exemplary anti-cancer agents, include, 15 (Sanofi Aventis), PF-4691502 (Pfizer), GDC0980 but are not limited to, radiation therapy, interferon (e.g., inter (Genetech), SF1126 (Semafoe) and OSI-027 (OSI)), feron C. interferon Y), antibodies (e.g., HERCEPTIN (trastu oblimersen, gemcitabine, caminomycin, leucovorin, pemetr Zumab), T-DM1, AVASTIN (bevacizumab), ERBITUX exed, cyclophosphamide, dacarbazine, procarbizine, pred (cetuximab), VECTIBIX (panitumumab), RITUXAN (ritux nisolone, dexamethasone, campathecin, plicamycin, aspara imab) BEXXAR (to situmomab)), anti-estrogens (e.g., ginase, aminopterin, methopterin, porfiromycin, melphalan, tamoxifen, raloxifene, and megestrol), LHRH agonists (e.g., leurosidine, leurosine, chlorambucil, trabectedin, procarba goScrclin and leuprolide), anti-androgens (e.g., flutamide and Zine, discodermolide, caminomycin, aminopterin, and hex bicalutamide), photodynamic therapies (e.g., vertoporfin amethyl melamine. (BPD-MA), phthalocyanine, photosensitizer Pc4, and Exemplary combinations of therapeutically active agents demethoxy-hypocrellin A (2BA-2-DMHA)), nitrogen mus 25 useful for the treatment of cancer (a.k.a. an “anti-cancer treat tards (e.g., cyclophosphamide, ifosfamide, trofosfamide, ment regimen’) which can be used in combination with at chlorambucil, estramustine, and melphalan), nitrosoureas least one compound of formula (I) or a pharmaceutically (e.g., carmustine (BCNU) and lomustine (CCNU)), alkylsul acceptable form thereof, or a pharmaceutical composition phonates (e.g., buSulfan and treosulfan), triaZenes (e.g., dac comprising at least one compound of formula (I) or a phar arbazine, temozolomide), platinum containing compounds 30 maceutically acceptable form thereof includes, but is not (e.g., cisplatin, carboplatin, oxaliplatin), Vinca alkaloids (e.g., limited to: Vincristine, vinblastine, vindesine, and vinorelbine), taxoids ABVD Adriamycin (doxorubicin), bleomycin, vinblastine, (e.g., paclitaxel, albumin-bound paclitaxel (ABRAXANE), dacarbazine nab-paclitaxel, docetaxel, taxol), epipodophyllins (e.g., eto AC Adriamycin (doxorubicin), cyclophosphamide poside, etoposide phosphate, teniposide, topotecan, 9-ami 35 BEACOPP Bleomycin, etoposide, Adriamycin (doxorubi nocamptothecin, camptoirinotecan, irinotecan, crisinatol, cin), cyclophosphamide. Oncovin (Vincristine), procarba mytomycin C), anti-metabolites, DHFR inhibitors (e.g., Zine, prednisone methotrexate, dichloromethotrexate, trimetrexate, edatrex BEP Bleomycin, etoposide, platinum agent (cisplatin) ate), IMP dehydrogenase Inhibitors (e.g., mycophenolic acid, CA Cyclophosphamide, Adriamycin (doxorubicin) (same as tiazofurin, ribavirin, and EICAR), ribonucleotide reductase 40 AC) inhibitors (e.g., hydroxyurea and deferoxamine), uracil ana CAF Cyclophosphamide, Adriamycin (doxorubicin), fluo logs (e.g., 5-fluorouracil (5-FU), floxuridine, doxifluridine, rouracil (5-FU) ratitrexed, tegafur-uracil, capecitabine), cytosine analogs CAV Cyclophosphamide, Adriamycin (doxorubicin), Vinc (e.g., cytarabine (ara C), cytosine arabinoside, and fludara ristine bine), purine analogs (e.g., mercaptopurine and Thiogua 45 CBV Cyclophosphamide, BCNU (carmustine), VP-16 (eto nine), Vitamin D3 analogs (e.g., EB 1089, CB 1093, and KH poside) 1060), isoprenylation inhibitors (e.g., lovastatin), dopamin ChIVPP/EVA Chlorambucil, Vincristine (Oncovin), procar ergic neurotoxins (e.g., 1-methyl-4-phenylpyridinium ion), bazine, prednisone, etoposide, vinblastine, Adriamycin cell cycle inhibitors (e.g., staurosporine), actinomycin (e.g., (doxorubicin) actinomycin D, dactinomycin), bleomycin (e.g., bleomycin 50 CHOP Cyclophosphamide, hydroxydoxorubicin (doxorubi A2, bleomycin B2, peplomycin), anthracycline (e.g., dauno cin), Vincristine (Oncovin), prednisone rubicin, doxorubicin, pegylated liposomal doxorubicin, ida CHOP-R or R-CHOP CHOP-rituximab rubicin, epirubicin, pirarubicin, Zorubicin, mitoxantrone), COP or CVP Cyclophosphamide. Oncovin (Vincristine), MDR inhibitors (e.g., Verapamil), Ca2+ ATPase inhibitors prednisone (e.g., thapsigargin), imatinib, thalidomide, lenalidomide, 55 CMF Cyclophosphamide, methotrexate, fluorouracil (5-FU) tyrosine kinase inhibitors tyrosine kinase inhibitors (e.g., axi COPP Cyclophosphamide. Oncovin (Vincristine), procarba tinib (AG013736), bosutinib (SKI-606), cediranib (RECEN Zine, prednisone TINTM, AZD2171), dasatinib (SPRYCELR, BMS-354825), EC Epirubicin, cyclophosphamide erlotinib (TARCEVAR), gefitinib (IRESSAR), imatinib ECF Epirubicin, cisplatin, fluorouracil (5-FU) (Gleevec(R), CGP57148B, STI-571), lapatinib (TYKERB(R), 60 EP Etoposide, platinum agent (cisplatin) TY VERB(R), lestaurtinib (CEP-701), neratinib (HKI-272), EPOCH Etoposide, prednisone. Oncovin, cyclophospha nilotinib (TASIGNAR), semaxanib (semaxinib, SU5416), mide, and hydroxydaunorubicin sunitinib (SUTENTR), SU11248), toceranib (PALLADIAR), FEC Fluorouracil (5-FU), epirubicin, cyclophosphamide vandetanib (ZACTIMAR, ZD6474), Vatalanib (PTK787, FL (Also known as Mayo) Fluorouracil (5-FU), leucovorin PTK/ZK), trastuzumab (HERCEPTINR), bevacizumab 65 (folinic acid) (AVASTINR), rituximab (RITUXANR), cetuximab (ER FOLFOX Fluorouracil (5-FU), leucovorin (folinic acid), BITUX(R), panitumumab (VECTIBIX(R), ranibizumab (Lu oxaliplatin US 8,546,432 B2 117 118 FOLFIRI Fluorouracil (5-FU), leucovorin (folinic acid), cell response, interfering RNA, anti-sense RNA, Imiqimod, irinotecan an inosine 5'-phosphate dehydrogenase inhibitor, amantadine ICE Ifosfamide, carboplatin, etoposide (VP-16) and rimantadine. ICE-R ICE--rituximab Other examples include, but are not limited to, those m-BACOD Methotrexate, bleomycin, Adriamycin (doxoru described in WO 2009/023059, the entirety of which is incor bicin), cyclophosphamide. Oncovin (Vincristine), dexam porated herein by reference. ethasone In one embodiment, the anti-viral agent is interferon. In MACOP-B Methotrexate, leucovorin (folinic acid), Adria another embodiment, the anti-viral agent is telaprivir. In one mycin (doxorubicin), cyclophosphamide, Oncovin (vinc embodiment, combinations of two or more anti-viral agents ristine), prednisone, bleomycin 10 are used in further combination with a compound provided MOPP Mechlorethamine. Oncovin (vincristine), procarba herein. Zine, prednisone In certain embodiments, the anti-viral agent is a protease PCV Procarbazine, CCNU (lomustine), Vincristine inhibitor. Exemplary protease inhibitors include, but are not ProMACE-MOPP Methotrexate, Adriamycin (doxorubicin), limited to, Saquinavir, Ritonavir, Indinavir, Nelfinavir, cyclophosphamide, etoposide--MOPP 15 Amprenavir, Lopinavir, Atazanavir, Fosamprenavir, ProMACE-CytaBOM Prednisone, doxorubicin (adriamy Tipranavir and Darunavir. cin), cyclophosphamide, etoposide, cytarabine, bleomy In certain embodiments, the anti-viral agent is an integrase cin, Oncovin (Vincristine), methotrexate, leucovorin inhibitor. Exemplary integrase inhibitors include, but are not R-FCM Rituximab, fludarabine, cyclophosphamide, mitox limited to, Raltegravir, Elvitegravir and MK-2048, GSK-572. antrOne In certain embodiments, the anti-viral agent is a reverse Stanford V Doxorubicin, mechlorethamine, bleomycin, vin transcriptase inhibitor (e.g., a nucleoside analog reverse tra blastine, Vincristine, etoposide, prednisone scriptase inhibitor (NRTI), a nucleotide analog reverse tras Thal/Dex Thalidomide, dexamethasone criptase inhibitor (NtRTI), a non-nucleoside reverse transcri TIP Paclitaxel, ifosfamide, platinum agent cisplatin 25 pase inhibitor (NNRTI)). VAC Vincristine, Actinomycin, Cyclophosphamide Exemplary nucleoside analog reverse traScriptase inhibi VAD Vincristine, Adriamycin (doxorubicin), dexamethasone tors (NRTIs) include, but are not limited to, Zidovudine, VAPEC-B Vincristine, Adriamycin (doxorubicin), pred Didanosine, Zalcitabine, Stavudine, Lamivudine, Abacavir, nisone, etoposide, cyclophosphamide, bleomycin Emtricitabine, Entecavir and Aciclovir (partial nucleoside VIP Etoposide, ifosfamide, platinum agent cisplatin 30 structure). Exemplary nucleotide analog reverse traScriptase inhibi In other embodiments, the therapeutically effective agent tors (NtRTIs) include, but are not limited to, Tenofovir and is an anti-vial agent. Exemplary anti-viral agents include, but Adefovir. are not limited to, Abacavir, Aciclovir, Acyclovir, Adefovir, Exemplary non-nucleoside reverse transcripase inhibitors Amantadine, Amprenavir, Ampligen, Arbidol, Atazanavir, 35 (NNRTIs) include, but are not limited to, Efavirenz, Nevirap Atripla, BI201335, Boceprevir, BMS-858 (see, e.g., Gao et ine, Delavirdine and Etravirine. In certain embodiments, the al., Nature, 465(6): 96-102 (2010)), BMS-790052 (see, e.g., compound of formula (I) or a pharmaceutically acceptable Gao et al., Nature, 465(6): 96-102 (2010)), Cidofovir, Com form thereof, or a pharmaceutical composition comprising at bivir, Danoprivir (ITMN-191; RG-7227), Darunavir, least one compound of formula (I) or a pharmaceutically Delavirdine, Didanosine, Docosanol, Edoxudine, EI-1 to 40 acceptable form thereof provided herein and/or the anti-viral EI-12 (see, e.g., Baldick et al., PLOS Pathogens, 6(9) agent is further used in combination with an enhancing agent. e1001086: 1-14 (2010)), Elvitegravir, Efavirenz, Emtricitab An 'enhancing agent, used in this context, is an agent which, ine, Enfluvirtide, Entecavir, Etravirine, Famciclovir, Fosam when used in combination with a compound provided herein prenavir, Foscarnet, Fosfonet, Ganciclovir, GSK-572, and/or an anti-viral agent, improves treatment, prevention or Ibacitabine, Immunovir, Idoxuridine, Imiquimod, Indinavir, 45 management of the microbial infection relative to treatment Inosine, Interferon (e.g., Interferon type III, Interferon type with the compound of formula (I) or a pharmaceutically II, Interferon type I, Peginterferon alfa-2a, Peginterferon acceptable form thereof, or a pharmaceutical composition alpha-2b, standard interferon alfa-2a, standard interferon comprising at least one compound of formula (I) or a phar alfa-2b, consensus interferon, interferon alfacon-1, ALBUF maceutically acceptable form thereof provided herein and/or ERON, omega interferon, interferon gamma-1b, lymphoblas 50 an anti-viral agent without the enhancing agent. Exemplary toid interferon tau), Lamivudine, Lopinavir, Loviride, enhancing agents include, but are not limited to, chloroquine, Maraviroc. Moroxydine, Methisazon, MK-2048, Nelfinavir, a quinoline antimalarial, grapefruit juice, hydroxyurea, Nevirapine, Nexavir, Oseltamivir (Tamiflu), Penciclovir, Per leflunomide, myucophenolic acid, resveratrol and Ritonavir. amivir, Pleconaril, Podophyllotoxin, Raltegravir, Ribavirin, In one embodiment, the anti-viral agent is an anti-viral Rimantadine, Ritonavir, Pyramidine, Saquinavir, Stavudine, 55 agent described in U.S. Pub. No. 2011/0064698, which is Tenofovir (e.g., Tenofovir disoproxil), Telaprivir, Tipranavir, incorporated herein by reference in its entirety. Exemplary Trifluridine, Trizivir, Tromantadine, Truvada, Valaciclovir anti-viral agents include, but are not limited to, IP-501, Mer (Valtrex), Valganciclovir, Vaccines (e.g., VZV vaccines Such imebodib VX-497, IDN-6556, XTL-002, HCV/MF59, as Varivax and Zostavax), Vicriviroc. Vidarabine, Viramidine, CIVACIR, ZADAXIN, CEPLENE, VX 950/LY 570310, Zalcitabine, Zanamivir (Relenza), Zidovudine, and other 60 ISIS14803, JTK 003, Tarvacin, HCV-796, CH-6, ANA971, Small molecule anti-viral agents described, for example, in ANA245, CPG 10101, Rituximab, NM 283, HepXTM-C, Herker et al., Nature Medicine, Advance Online Publication IC41, Medusa interferon, E-1, multiferon, BILN 2061, doi:10.1038/nm2238: 1-4 (Oct. 10, 2010), and combinations TMC435350, Telaprevir, Boceprevir, ACH-1625, ABT-450, thereof. BI-201335, PHX-1766, VX-500, MK-7009, R7227, Narla Examples of additional anti-viral agents include, but are 65 previr, Alinia, ABT-072, ABT-333, Filibuvir, VCH-916, not limited to, interleukin 2, interleukin 6, interleukin 12, a R7128, IDX 184, R7128, R1626, MK-3281, PSI-7851, ANA compound that enhances the development of a type 1 helper T 598, BI-207127, GS9190, VCH-759, Clemizole, A-832, US 8,546,432 B2 119 120 BMS-790052, ITX 5061, GS-9450, ANA773, CYT 107, In another embodiment, at least one compound of formula SPC3649, Debio 25, SCY-635 and a combination thereof. (I) or a pharmaceutically acceptable form thereof, or a phar Other examples include, but are not limited to, AZD-7295, maceutical composition comprising at least one compound of BI207127, BIT225, BM824383, BMS65032, BMS791325, formula (I) or a pharmaceutically acceptable form thereof GS-9256, IDX 375, INX-189, PPI-461, PSI-938, PSI-7977, provided hereincan be used in combination with ribavirin and TMC435, TMC649128, VX-222, VX-759, VX-916 and a an interferon. In one embodiment, the interferon is Interferon combination thereof. These agents are currently in various type III, Interferon type II, Interferon type I, Peginterferon stages of clinical trials and information is readily available to alfa-2a, Peginterferon alpha-2b, standard interferon alfa-2a, those in the art. standard interferon alfa-2b, consensus interferon, interferon In one embodiment, provided herein is a method of treat 10 ing, preventing and/or managing hepatitis C virus (HCV) alfacon-1, ALBUFERON, omega interferon, interferon infection comprising administering a therapeutically or pro gamma-1b, lymphoblastoid interferon tau or a combination phylactically effective amount of at least one compound of thereof. In another embodiment, the interferon is interferon formula (I) or a pharmaceutically acceptable form thereof, or alfa-2a, interferon alfa-2b, peginterferon alfa-2a, peginter a pharmaceutical composition comprising at least one com 15 feron alpha-2b, consensus interferon or lymphoblastoid inter pound of formula (I) or a pharmaceutically acceptable form feron tau. thereof provided herein in combination with one or more other therapeutic agents provided herein. 6. Anti-Viral Assays Examples of Such therapeutic agents include compounds having anti-HCV activity, for example, by inhibiting the Anti-viral assays used to screen compounds having effi function of a target such as, but not limited to, HCV metallo cacy for a specific virus are well-known in the art and protease, HCV serine protease, HCV polymerase, HCV heli described, for example, in WO 2009/023059, the entirety of case, SCV NS4B protein, HCV entry, HCV assembly, HCV which is incorporated herein by reference. Exemplary anti egress, HCV NS5A protein and IMPDH. viral assays are provided herein below. In other embodiments, at least one compound of formula 25 6.1 Herpes Simplex Virus (HSV) (I) or a pharmaceutically acceptable form thereof, or a phar Mouse models of herpes simplex virus type 1 or type 2 maceutical composition comprising at least one compound of (HSV-1 or HSV-2) can be employed to assess the anti-viral formula (I) or a pharmaceutically acceptable form thereof activity of test compounds in vivo. BALB/c mice are com provided herein can be used in combination with at least one monly used, but other Suitable mouse strains that are Suscep additional therapeutic agent having anti-HCV activity, 30 tible can also be used. Mice are inoculated by various routes including, but not limited to, Alinia (Nitazoxanide), Bavitux with an appropriate multiplicity of infection of HSV, fol imab, Belerofon, Chronvac-C, Civacir, Clemizole, Fluvasta lowed by administration of test compounds and placebo. For tin, Glycoferon, HepavaXX. C. HuMax-HepC, Lenocta (so i.p. inoculation, HSV-1 replicates in the gut, liver, and spleen dium Stibogluconate SSG), Locteron, peginterferon, and spreads to the CNS. For i.n. inoculation, HSV-1 replicates Ribavirin, Suvus, Telaprevir (VX-950), Zadaxin thymalfa 35 in the nasaopharynx and spreads to the CNS. Any appropriate sin, ZALBIN (Albuferon albinterferon alfa-2b), A-837093, route of administration (e.g., oral, topical, Systemic and ABT-072, ABT-333, ABT-450, ACH-1095, ACH-1625, nasal), frequency and dose of administration can be tested to ACH-2684, ACH-2928, AN 025-1, ANA598, ANA773, ATI determine the optimal dosages and treatment regimens using 08.10 (formerly PG301029), AVL-181, AVL-192, AZD7295, test compounds, optionally in combination with other thera BI 201335, BI 207127, BIT225, BMS-650032, BMS 40 p1es. 790052, BMS-791325, BMS-824393, CB5300, CB-183872 In a mouse model of HSV-2 genital disease, intravaginal (formerly IB657), CF102, CSL123, CTS-1027, CYT107, inoculation of female Swiss Webster mice with HSV-1 or Debio 025, ECH18, EDP-239, GEA007.1, GI 5005, GNI HSV-2 is carried out, and vaginal swabs are obtained to evalu 103, GNI-104, GS 91.90, GS 9256, GSK625433, IC41, ate the effect of therapy on viral replication. (See, e.g., Crute ID-12, IDX 184, IDX320, IDX375, IMO-2125, IMMU 105, 45 et al., Nature Medicine, 2002, 8:386-391). For example, viral ITMN-191 R7227 (RO5190591), ITX2155, ITX4520, titers by plaque assays are determined from the vaginal ITX5061NS5A inhibitors, JKB-122, KPE02001003, swabs. A mouse model of HSV-1 using SKH-1 mice, a strain KPE00001113, MBL-HCV1, MDX-1106 (ONO-4538), of immunocompetent hairless mice, to study cutaneous Mito-Q, MK-0608, MX3235 Celgosivir, NOV-205, lesions is also described in the art. (See, e.g., Crute et al., PF-868554, PF-4878691, PHX1766, PYN17, PYN18, PPI 50 Nature Medicine, 2002, 8:386-391 and Bolger et al., Antiviral 461, PPI-1301, PRO-206, PSI-7977, PSI-9381NX08189, Res., 1997, 35:157-165). Guinea pig models of HSV have R7128 (RO5024048), REP 9C, RG7348, SCV-07, SCY-635, also been described. (See, e.g., Chenet al., Virol.J., 2004 Nov. SD-101, SIRNA-034, SP-30, SPC3649, TG4040, TT033, 23, 1:11). Statistical analysis is typically carried out to cal VCH-759, VX-222, VX-500, VX-813, and VX-985. culate significance of the anti-viral activity. In one embodiment, the other therapeutic agent is an inter 55 6.2 Human Cytomegalovirus (HCMV) feron. In one embodiment, the interferon is Interferon type Since HCMV does not generally infect laboratory animals, III, Interferon type II, Interferon type I, Peginterferon alfa-2a, mouse models of infection with murine CMV (MCMV) can Peginterferon alpha-2b, standard interferon alfa-2a, standard be used to assay anti-viral activity of test compounds in vivo. interferon alfa-2b, consensus interferon, interferonalfacon-1, For example, a MCMV mouse model with BALB/c mice can ALBUFERON, omega interferon, interferon gamma-1b. 60 be used to assay the anti-viral activities of test compounds in lymphoblastoid interferon tau or a combination thereof. In vivo when administered to infected mice, which is described, another embodiment, the interferon is interferon alfa-2a, for example, in Kern et al., Antimicrob. Agents Chemother, interferon alfa-2b, peginterferon alfa-2a, peginterferon 2004, 48:4745-4753. Tissue homogenates isolated from alpha-2b, consensus interferon or lymphoblastoid interferon infected mice treated or untreated with test compounds are tall. 65 tested using standard plaque assays with mouse embryonic In another embodiment, the other therapeutic agent is rib fibroblasts (MEFs). Statistical analysis is then typically car avirin. ried out to calculate significance of the anti-viral activity. US 8,546,432 B2 121 122 Alternatively, human tissue (i.e., retinal tissue or fetal thy ters); 2+ (moderate numbers of infected cells, usually affect mus and liver tissue) is implanted into SCID mice, and the ing clusters of adjacent cells in portions of the epithelial layer mice are subsequently infected with HCMV, preferably at the lining bronchioles, or in small sublobular foci in alveoli); and site of the tissue graft. (See, e.g., Kern et al., Antimicrob, 3+ (numerous infected cells, affecting most of the epithelial Agents Chemother, 2004, 48:4745-4753). The pfu of HCMV layer in bronchioles, or widespread in large sublobular foci in used for inoculation can vary depending on the experiment alveoli). and virus strain. Any appropriate routes of administration 6.4. Hepatitis Type B Virus (HBV) (e.g., oral, topical, systemic and nasal), frequency and dose of A HBV transgenic mouse model, lineage 1.3.46 (official administration can be tested to determine the optimal dosages designation, Tg HBV 1.3 genome Chi46) has been and treatment regimens using test compounds, optionally in 10 described previously and can be used to test the in vivo combination with other therapies. Implant tissue homoge anti-viral activities of test compounds as well as the dosing nates isolated from infected mice treated or untreated with and administration regimen. (See, e.g., Cavanaugh et al., J. test compounds at various time points are tested using stan Virol., 1997,71:3236-3243; and Guidotti et al., J. Virol., 1995, dard plaque assays with human foreskin fibroblasts (HFFs). 69:6158-6169). In these HBV transgenic mice, a high level of Statistical analysis is then typically carried out to calculate 15 viral replication occurs in liver parenchymal cells and in the significance of the anti-viral activity. proximal convoluted tubules in the kidneys of these trans Guinea pig models of CMV to study anti-viral agents have genic mice at levels comparable to those observed in the also been described, for example, in Bourne et al., Antiviral infected liver of patients with chronic HBV hepatitis. HBV Res., 2000, 47:103-109; Bravo et al., Antiviral Res., 2003, transgenic mice that have been matched for age (i.e., 6-10 60:41-49; and Bravo et al., J. Infectious Diseases, 2006, 193: weeks), sex (i.e., male), and levels of hepatitis B Surface 591-597. antigen (HBSAg) in serum can be treated with test com 6.3 Influenza Virus pounds or placebo followed by anti-viral activity analysis to Animal models, such as ferret, mouse and chicken, devel assess the activity of test compounds. Non-limiting examples oped for use to test anti-viral agents against influenza virus of assays that can be performed on these mice treated and have been described, for example, in Sidwell et al., Antiviral 25 untreated with test compounds include Southern analysis to Res., 2000, 48: 1-16 and McCauley et al., Antiviral Res., measure HBV DNA in the liver, quantitative reverse tran 1995, 27: 179-186. For mouse models of influenza, non scriptase PCR (qRT-PCR) to measure HBV RNA in the liver, limiting examples of parameters that can be used to assay immunoassays to measure hepatitise antigen (HBeAg) and anti-viral activity of test compounds administered to the HBV surface antigen (HBSAg) in the serum, immunohis influenza-infected mice include pneumonia-associated 30 tochemistry to measure HBV antigens in the liver, and quan death, serum C.1-acid glycoprotein increase, animal weight, titative PCR (qPCR) to measure serum HBV DNA. Gross and lung virus assayed by hemagglutinin, lung virus assayed by microscopic pathological examinations can be performed as plaque assays, and histopathological change in the lung. Sta needed. tistical analysis is typically carried out to calculate signifi 6.5 Human Immunodeficiency Virus (HIV) cance of the anti-viral activity. 35 The safety and efficacy of test compounds against HIV can Nasal turbinates and trachea can be examined for epithelial be assessed in vivo with established animal models well changes and Subepithelial inflammation. The lungs can be known in the art. For example, a Trimera mouse model of examined for bronchiolar epithelial changes and peribronchi HIV-1 infection has been developed by reconstituting irradi olar inflammation in large, medium, and Small or terminal ated normal BALB/c mice with murine SCID bone marrow bronchioles. The alveoli are also evaluated for inflammatory 40 and engrafted human peripheral blood mononuclear cells. changes. The medium bronchioles are graded on a scale of 0 (See Ayash-Rashkovsky et al., FASEB J., 2005, 19:1149 to 3+ as follows: 0 (normal: lined by medium to tall columnar 1151). These mice are injected intraperitoneally with T- and epithelial cells with ciliated apical borders and basal pseu M-tropic HIV-1 laboratory strains. After HIV infection, rapid dostratified nuclei; minimal inflammation); 1 + (epithelial loss of human CD4.sup.--T cells, decrease in CD4/CD8 ratio, layer columnar and even in outline with only slightly 45 and increased T cell activation can be observed. A test com increased proliferation; cilia still visible on many cells); 2+ pound can be administered to these mice and Standard assays (prominent changes in the epithelial layer ranging from known in the art can be used to determine the viral replication attenuation to marked proliferation; cells disorganized and capacity in animals treated or untreated with the compound. layer outline irregular at the luminal border); and 3+ (epithe Non-limiting examples of such assays include the COBAS lial layer markedly disrupted and disorganized with necrotic 50 AMPLICORTM RT-PCR assay (Roche Diagnostics, Branch cells visible in the lumen; some bronchioles attenuated and berg, N.J.) to determine plasma viral load (HIV-1 RNA cop others in marked reactive proliferation). ies/ml); active HIV-1 virus replication assay where human The trachea is graded on a scale of 0 to 2.5+ as follows: 0 lymphocytes recovered from infected Trimera mice were coc (normal: Lined by medium to tall columnar epithelial cells ultured with target T cells (MT-2 cells) and HIV-dependent with ciliated apical border, nuclei basal and pseudostratified. 55 syncytia formation was examined; and human lymphocytes Cytoplasm evident between apical border and nucleus. Occa recovered from infected Trimera mice were cocultured with sional Small focus with squamous cells); 1 + (focal squamous cMAGI indicator cells, where HIV-1 LTR driven trans-acti metaplasia of the epithelial layer); 2+ (diffuse Squamous vation of B-galactosidase was measured. Levels of anti-HIV-1 metaplasia of much of the epithelial layer, cilia may be evi antibodies produced in these mice can also be measured by dent focally); and 2.5+ (diffuse squamous metaplasia with 60 ELISA. Other established mouse models described in the art very few cilia evident). can also be used to test the anti-viral activity of test com Virus immunohistochemistry is performed using a viral pounds in vivo. (See, e.g., Mosier et al., Semin. Immunol., specific monoclonal antibody (e.g., NP-, N- or HN-specific 1996, 8:255-262: Mosier et al., Hosp. Pract. (Off Ed)., 1996, monoclonal antibodies). Staining is graded 0 to 3+ as follows: 31:41-48, 53-55, 59-60; Bonyhadi et al., Mol. Med. Today, 0 (no infected cells); 0.5+ (few infected cells); 1 + (few 65 1997, 3:246-253; Jolicoeur et al., Leukemia, 1999, 13:S78 infected cells, as widely separated individual cells); 1.5+ (few S80; Browning et al., Proc. Natl. Acad. Sci. USA, 1997, infected cells, as widely separated singles and in Small clus 94: 14637-14641; and Sawada et al., J. Exp. Med., 1998, 187: US 8,546,432 B2 123 124 1439-1449). A simian immunodeficiency virus (SIV) nonhu In comparison with four human diploid cell lines and vero man primate model has also been described, for example, in cells, HFF cells are the most sensitive and predictive of tox Schito et al., Curr: HIV Res., 2006, 4:379-386. icity for bone marrow cells. 6.6 In Vitro Screening Assays To determine if each compound has sufficient anti-viral 6.6.1 General Procedures for Assays for Herpes Viruses activity that exceeds its level of toxicity, a selectivity index To quickly Screen out samples that do not have activity (SI) is calculated according to CCso/ECso. This index, also against any of the herpes viruses, or are too toxic to evaluate, referred to as a therapeutic index, is used to determine if a an inexpensive, rapid assay Such as a CPE-inhibition assay compound warrants further study. Typically, a compound that that is semi-automated is commonly used initially to Screen had an SI of 10 or greater is evaluated in additional assay out the negatives. Typically, all screening assays are con 10 systems. ducted in low passage human cells, and each assay system For HSV-1 and HSV-2, compounds that show activity in contains a positive control (ACV, GCV, CDV) and a negative the CPE-inhibition assay are confirmed using the plaque control (AZT). Efficacy is demonstrated by at least two dif reduction assay. Susceptibility of additional virus strains, ferent assay systems that detect functional biologic activity including both lab passaged and clinical isolates, is deter and should be confirmed using low passaged clinical isolates 15 mined for selected compounds. A battery of ACV resistant and drug resistant mutants whenever available. In the case of HSV strains can be also utilized. For CMV, compounds that EBV, efficacy against EBV is confirmed using a hybridization show activity in the CPE-inhibition assay are confirmed using assay that quantifies DNA synthesis. Toxicity is determined the plaque reduction assay in HFF cells. A variety of labora using both resting and proliferating human fibroblast cells tory, clinical, and GCV resistant isolates are also available for and proliferating lymphoblastic cells, and for selected com testing. For VZV, compounds with activity in a CPE assay are pounds, toxicity in human myeloid and erythroid progenitor evaluated further in a plaque reduction assay. cells is assessed. 6.6.1.2 Epstein-Barr Virus (EBV) 6.6.1.1 HSV-1, HSV-2, CMV and VZV The initial system to be used to determine anti-viral activity All the screening assay systems utilized are selected to against EBV can be VCA production in Daudi cells using an show specific inhibition of a biologic function, i.e., cytopathic 25 ELISA assay. Six concentrations of drug covering a range of effect (CPE) in susceptible human cells. In the CPE-inhibi e.g., 50 ug/ml to 0.03 g/ml are utilized. Using the results tion assay, a test compound is added 1 hour prior to infection obtained from untreated and drug treated cells, an ECso can be so the assay system will have maximum sensitivity and detect calculated. Selected compounds that have good activity inhibitors of early replicative steps, such as adsorption or against EBV VCA production without toxicity are tested for penetration, as well as later events. To rule out non-specific 30 their ability to inhibit EBV DNA synthesis. inhibition of virus binding to cells, all compounds that show In each assay system utilized, drug treatment of uninfected reasonable activity in the CPE assay are confirmed using a cells is incorporated to obtain as much toxicity data as pos classical plaque reduction assay in which the compound is sible. In some embodiments, for calculation of the SI, the data added 1 hour after infection. In the case where a compound on toxicity is at least as reliable as the results for efficacy. An blocks attachment, a positive result will appear in the CPE 35 example of a toxicity assay is a colormetric method using assay, but may be negative by plaque assay. In this case, the MTS. plaque assay is repeated with compound being added prior to All compounds that have an SI of for example, greater than viral infection. These assay systems also can be manipulated 10 in the screening assay are confirmed in a hybridization by increasing the pretreatment time in order to demonstrate assay that measures the amount of EBV DNA produced by anti-viral activity with oligodeoxynucleotides and/or pep 40 P3HR-1 infected cells. A wide range of compound concen tides and by delaying addition of drug after infection. Infor trations can be utilized so an accurate ECso can be calculated. mation regarding which step in the virus life cycle is inhibited Uninfected control cells treated with compound are also uti (i.e., early vs. late functions) can be gained. lized as another measure of drug toxicity. In some cases, it is In all the assays used for primary screening, a minimum of possible that results obtained using assays for VCA produc six compound concentrations is contemplated to cover a 45 tion and DNA synthesis may not correlate since the two range of, e.g., 100 ug/ml to 0.03ug/ml, in 5-fold increments events may be independent. to determine efficacy. Dose response curves are obtained 6.6.1.3 Human Herpes Virus HHV-6 and HHV-8 from these data. The dose that inhibits viral replication by Cord Blood Lymphocytes (CBL) and the Human T cell 50% (effective concentration 50; ECs) is typically calculated lymphoblastoid lines, HSB-2 and SupT-1, are used in screen using a computer Software program, for example, MacSyn 50 ing assays for HHV-6. CBL are isolated from fresh heparin ergy II by M. N. Prichard, K. R. Asaltine, and C. Shipman, Jr., ized umbilical cord blood and are infected with the Z29 strain University of Michigan, Ann Arbor, Mich. of HHV-6. The body cavity based B-cell lymphoma cell line, The same compound concentrations used to determine effi BCBL-1, are used for screening against HHV-8. cacy are also used on uninfected cells in each assay to deter There are two variants of HHV-6 known as type A variants mine toxicity of each experimental compound. The com 55 or type B variants. The HHV-6 type A variant is, for example, pound concentration that is cytotoxic to cells as determined the GS strain which is propagated in HSB-2 or SupT-1 cells. by their failure to take up a vital stain, neutral red (cytotoxic The HHV-6 type B variant is, for example, Z29 (ATCC, concentration 50; CCs), is determined as described above. Rockville, Md.) which is grown as a stock in CBL. The In some embodiments, compounds to treat herpes virus HHV-8 is propagated in a latent state in the BCBL-1 cell line. infections are for systemic diseases, such as neonatal herpes, 60 Lytic growth of the HHV-8 can be induced by the addition of CMV, and disseminated VZV, and may need to be given the phorbol ester, TPA. parenterally. Therefore, the toxicity of test compounds on Six concentrations of each drug ranging from, e.g., 100 dividing cells is determined at a very early stage of testing. In ug/ml to 0.03 g/ml drug are tested to obtain the ECs. EC this regard, a cell proliferation assay using HFF cells can be a CCs, and ICso values. The initial assay for HHV-6 is a flow very sensitive assay for detecting compound toxicity to divid 65 cytometric analysis of HHV-6 antigens in either HSB-2 cells ing cells, and the compound concentration that inhibits cell (HHV-6A), CBL (HHV-6B), or SupT-1 (6A or 6B). For HHV growth by 50% (ICs) can be calculated as described above. 8, yytic infection of virus in BCBL-1 cells will be carried out US 8,546,432 B2 125 126 as described above. The initial assay for HHV-8 is a flow rinsed using tap water until all excess stain is removed. The cytometric analysis of HHV-8 antigens in BCBL-1 cells. As plates are allowed to dry for 24 hours and the amount of CPE with the other herpes virus assays, these assays contain the in each row determined using a BioTek Multiplate Auto positive (infected and untreated cells) and negative (unin reader. ECso and ICso values are determined by comparing fected or uninduced and compound treated cells) controls compound treated and untreated cells using a computer pro needed for effective analysis and cytotoxicity determinations. gram. 6.6.2 InVitro Laboratory Procedures for Assays for Herpes Plaque Production Assay for HSV-1 and HSV-2: Viruses Two days prior to use, HFF cells are trypsinized, counted, 6.6.2.1 Efficacy Screening for HSV-1, HSV-2, CMV and and plated into six well plates and incubated at 37°C. with 5% VZV 10 Preparation of Human Foreskin Fibroblast Cells: CO and 90% humidity. On the date of assay, the compound Newborn human foreskins can be obtained from the Uni is made up at twice the desired concentration in 2xMEM and versity of Alabama School of Medicine (UAB) or Brookwood then serially diluted 1:5 in 2xMEM to give six concentrations Hospital, Birmingham, Ala., as soon as possible after circum of compound. The compound concentrations utilized are usu cisions are performed and placed in minimal essential 15 ally 200 g/ml down to 0.06 g/ml. The virus to be used is medium (MEM) containing Vancomycin, fungizone, penicil diluted in MEM containing 10% FBS to a desired concentra lin, and gentamicin, at the usual concentrations, for four hours tion which will give 20-30 plaques per well. The media is then at room temperature. The medium is then removed, the fore aspirated from the wells and 0.2 ml of virus is added to each skin minced into Small pieces and washed repeatedly until red well in triplicate with 0.2 ml of media being added to drug cells are no longer present. The tissue is then trypsinized toxicity wells. The plates are then incubated for 1 hour with using trypsin at 0.25% with continuous stirring for 15 minutes shaking every 15 minutes. After the incubation period, an at 37° C. in a CO incubator. At the end of each 15 minute equal amount of 1% agarose is added to an equal Volume of period, the tissue is allowed to settle to the bottom of the flask. each compound dilution. This provides final compound con The Supernatant containing cells is poured through sterile centrations beginning with 100 lug/ml and ending with 0.03 cheesecloth into a flask containing MEM and 10% fetal 25 ug/ml and a final agarose overlay concentration of 0.5%. The bovine serum (FBS). The flask containing the medium is kept compound agarose mixture is applied to each well in a 2 ml on ice throughout the trypsinizing procedure. After each volume and the plates are incubated for three days, after decanting of cells, the cheese cloth is washed with a small which the cells are stained with a 1.5% solution of neutral red. amount of MEM containing serum. Fresh trypsin is added At the end of the 4-6 hours incubation period, the stain is each time to the foreskin pieces and the procedure repeated 30 aspirated, and plaques counted using a stereomicroscope at until no more cells become available. The cell-containing 10x magnification. medium is then centrifuged at 1000 RPM at 4° C. for 10 minutes. The Supernatant liquid is discarded and the cells are Plaque Production Assay for CMV: resuspended in a small amount of MEM with 10% FBS. The The procedures are nearly identical to those provided for cells are counted using a Coulter Counter and then placed in 35 HSV with a few minor changes. The agarose used for both the an appropriate number of 25 cm tissue culture flasks. As cells initial overlay and the two subsequent overlays is 0.8% rather become confluent and need trypsinization, they are gradually than 1%. The assay is incubated for 14 days with the addi expanded into 175 cm flasks. The cells are maintained on tional 1 ml overlays being applied on days 4 and 8. Vancomycin and fungizone to passage three. Cell lines are Plaque Production Assay for VZV: tested periodically for the presence of mycoplasma contami 40 The procedures are essentially identical to those described nation using the Hoechst fluorescent stain for mycoplasma for the HSV plaque assay with the following possible excep DNA. Cells are utilized usually only until passage 10. tions: after addition of the compound, the plates are incubated Cytopathic Effect Inhibition Assay: for ten days; on days 3 and 6, an additional 1 ml overlay with Low passage (3-10) human foreskin fibroblast (HFF) cells equal amounts of 2xMEM and 1% agarose are added. are trypsinized, counted, and seeded into 96 well tissue cul 45 Plaque Reduction Assay: ture plates at a cell concentration of 2.5x10" cells in 0.1 ml of In certain cases, some large or highly charged molecules MEM supplemented with 10% FBS. The cells are then incu that are active in the CPE inhibition assay may be inactive in bated for 24 hours at 37° C. in a 5% CO-95% air, 90% the plaque assay because the compound failed to diffuse humidified atmosphere. The media is then removed and 100 through the agarose overlay. Therefore, a modified plaque ul of MEM containing 2% FBS is added to all but the first row. 50 assay can be used for confirmation, wherein the overlay In the first row, 125ul of media containing the experimental medium is liquid rather than semi-solid. The procedure for compound is added in triplicate wells. Media alone is added the liquid overlay plaque assay is similar to that using the to both cell and virus control wells. The compound in the first agarose overlay. The procedure for adding the virus is the row of wells is then diluted serially 1:5 throughout the same as for the regular plaque assay. The compounds are remaining wells by transferring 25ul using a Beckman Bio 55 made up at the desired concentrations in MEM with 2% FBS. Mek Liquid Handling Machine. The plates are then incubated For HSV-1 and HSV-2 assays, an antibody preparation for 60 minutes and 100 ul of an appropriate virus concentra obtained from, e.g., Baxter Health Care Corporation is tion added to each well, excluding cell control wells which diluted 1:500 and added to the media that the compound is received 100 ul of MEM. For HSV-1 and HSV-2 assays, the diluted in to limit extracellular spread of virus through the virus concentration utilized is 1000 Plaque Forming Units 60 media. For VZV and CMV, no antibody in the overlay is (PFU) per well. For CMV and VZV assays, the virus concen necessary. For the CMV and VZV assays, additional medium tration added is 2500 and 1000 PFU per well, respectively. without the new compound is added on day five and allowed The plates are then incubated at 37°C. in a CO, incubator for to incubate for a total of 8 and 10 days, respectively. At the end three days for HSV-1 and HSV-2, 10 days for VZV, or 14 days of the incubation period for all of the assays, 2 ml of a 6.0% for CMV. After the incubation period, the media is aspirated 65 neutral red solution is added to each well and incubated for 6 and the cells are stained with a 0.1% crystal violet informalin hours. The liquidis then aspirated off and plaques enumerated solution for 4 hours. The stain is then removed and the plates using a stereomicroscope. US 8,546,432 B2 127 128 6.6.2.2 Efficacy Screening for EBV labeled goat anti-mouse IgG (Southern Biotechnology Asso Cells: ciates, Birmingham, Ala.) is used as the second antibody. The The two lymphoid cell lines, Raji and Daudi derived from slides are counterstained with 0.1% Evan's blue for 5 minutes Burkitt's lymphoma, are used. The Raji cell line is a non and mounted with 10% glycerin in PBS. The number of producer of viral gene products associated with the produc FITC-positive cells on each Smear is determined using a tive viral cycle. The Daudi cell line is a low level producer, Nikon fluorescence microscope. Five hundred cells are i.e., fewer than 1% of the cells express EA spontaneously. counted in each spot. The number of cells expressing EBV These cells are equally susceptible to superinfection by the VCA is calculated by multiplying the fraction of antigen P3HR-1 virus as determined by EBV VCA expression. The positive cells by the number of cells/ml in the culture at the cells are maintained at 37°C. in a humidified atmosphere with 10 time of harvest. The compound concentration is plotted 5% CO, in culture with RPMI-1640 medium containing 10% against the number of antigen positive cells/ml using a com heat inactivated FBS, 100 u/ml Penicillin, 25ug/ml gentami puter program, and ECso and E.Coo values are calculated. cin and 2 mM L-glutamine. The cells are passaged twice ELISA: weekly and the cell concentration adjusted to 2x10°/ml for Daudi cells infected with P3HR-1 virus and treated with SC. 15 drug are harvested by centrifugation and washed three times Virus: with PBS. The cells are pelleted and suspended to a concen The following prototypes of infectious EBV can be used: tration of 4x10° cells/ml in PBS. One hundred ul of each (1) one derived from supernatant fluids of the P3HR-1 cell Suspension is dispensed in triplicate into a 96-well plate, line, which produces non-transforming virus that induces the air-dried and fixed with 95% ethanol and 5% acetic acid. production of VCA after primary infection or superinfection Uninfected cells are prepared in the same manner and used as of B cell lines; and (2) B95-8 virus, which immortalizes cord controls. After washing the plate, primary and secondary blood lymphocytes and induces tumors in marmosets, but antibodies diluted in 1% bovine serum albumin containing does not induce an abortive productive infection even in cell 0.05% Tween-20 are added sequentially to each well and lines harboring EBV genome copies. As an example, for virus incubated at room temperature. Antibody additions are sepa production, P3HR-1 cells are cultured at a concentration of 25 rated by 3 washes with PBS containing 0.005% Tween-20. 2x10/ml for two weeks in medium containing 2% FCS at 34° O-phenyldiamine (OPD) substrate is added and the reaction C. in a humidified atmosphere with 5% CO. Concentrated stopped with 3N HSO after about 10 minutes. The optical virus then is prepared from the supernatant of the culture by density is measured at 492 nm and the ECso extrapolated centrifugation at 12,000 g for 90 minutes in a Sorvall Centri using the computer software program described herein. fuge. The pellets are resuspended in RPMI-1640 medium at 30 Evaluation of Anti-viral Agents against EBV DNA Repli /100 of the original volume and stored at -70° C. cation: Antibodies: The Enzo Simply Sensitive Horseradish Peroxidase-AEC Murine monoclonal antibody to EBV VCA (Chemicon In Situ Detection System for EBV (Enzo Diagnostics, Farm International, Inc., Temecula, Calif.), is used in immunofluo ingdale, N.Y.) is used to determine anti-viral activity against rescence assays and ELISA. Optimal monoclonal antibody 35 DNA synthesis. Detection and staining are performed accord concentration is determined by antibody titration for each ing to the manufacturers instructions. Three days after Super assay system. For single fluorochrome analyses FITC-la infection and compound treatment, slides are prepared with belled goat anti-mouse total IgG (Southern Biotechnology 4x10" cells/spot for each cell suspension, and air-dried over Associates, Birmingham, Ala.) is used as the second anti night. The slides are fixed in acetone for 10 minutes. A biotin body. 40 labelled EBV probe is added to each spot offixed cells and the EBV Superinfection and Compound Treatment: slide is covered with a glass coverslip. The slide is then heated Superinfection is initiated by the incubation of 0.5 ml of an on a hot plate at 95°C. for 5 minutes. After heating, the slide appropriate concentration of EBV with 106 cells/tube in a is placed at 37°C. on a slide warmer for 30-60 minutes for the total of 1 ml/tube. In most cases, this amounts to a multiplicity DNAs to anneal. The coverslips are then removed and the of infection (MOI) of 0.1-0.2 based on VCA induction in 45 Post Hybridization Reagent is added to each spot. After incu Daudi cells. After adsorption at 37° C. for 1 hour, 3 ml of bation for 10 minutes and rinsing with washing buffer, Detec RPMI-1640 medium is added. The cells are pelleted by cen tion Reagent is applied. The Detection Reagent is left on the trifugation and Supernatants discarded. Compound concen slide for 30-60 minutes on a slide warmer and thenwashed off trations (0.08, 0.4, 2, 10, 50 g/ml) in 4 ml of RPMI-1640 are with washing buffer. Chromogen Substrate Solution is added added to the appropriate tubes. RPMI-1640 is added to posi 50 and incubated for 20 minutes on a slide warmer. The slides are tive and negative control tubes and each compound concen washed and counter stained with Blue Counterstain. The tration is added to Daudi cells without virus for toxicity slides are then rinsed with deionized water and mounted with controls. After incubation, the cells in each tube are counted water. The slides are viewed in a light microscope under a using a Coulter Counter and washed three times with phos magnification of 400x. Positive cells appear as red spots. All phate buffered saline solution (PBS) (without Ca and Mg). 55 the cells are counted in several fields. The fraction of red spots Each cell suspension is adjusted to a concentration of 4.0x10° in the total number of cells counted multiplied by 100 reflects cells/ml in PBS. For EBV IFA and DNA hybridization assays, the percent hybridization. two sets of slides are prepared with 4x10" cells/spot for each Primary Infection Assay: cell Suspension, and air-dried overnight. The primary infection of umbilical cord blood lympho Immunifluorescence Assay: 60 cytes with the transforming strain B95-8 of EBV induces the The infected and compound treated cells are counted and expression of the virus-associated nuclearantigen (EBNA) in washed three times with PBS. Cells, 4x10" in PBS, are spot the cell. It is also known that B95-8 virus induces cellular ted on multiwell slides and air dried. The cells are then fixed DNA synthesis after infection of CBL. The availability of for 10 minutes in acetone, washed in PBS and stained for EBNA virus-infected cells in culture allows for the identifi immunofluorescence with the mouse monoclonal antibodies 65 cation and quantitation of EBV-positive cell antigens by indi and FITC-labeled goat anti-mouse IgG. EBV VCA specific rect IFA staining and FACS. Cord blood lymphocytes sepa antibodies are used in the immunofluorescence assays. FITC rated by ficoll-hypaque gradient are cultured in complete US 8,546,432 B2 129 130 RPMI-1640. The EBV-B95-8 is produced by incubating the with other herpesviruses and fluorescence intensity as moni B95-8 cell line in RPMI-1640 plus 10% fetal calf serum for tored by FACS. Monoclonal antibodies selected for use in the 10-14 days. The supernatant is collected and stored at 0-4°C. HHV-6 assay systems are screened for variant specificity and One million CBL are infected by incubation with 1 ml of the demonstrated no A or B variant cross-reactivity in the assay B95-8 supernant for 1 hour. The virus is removed by centrifu 5 systems. Monoclonal antibody 8532 (Chemicon, Temecula, gation. After one wash with RPMI-1640, the infected cells are Calif.) targets HHV-6 induced early nuclear proteins and is treated with anti-viral compounds as described earlier for used as a primary antibody in the HHV-6GS assay systems at P3HR-1 superinfection. The cell cultures are incubated for a 5ug/ml concentration. Monoclonal antibody 8535 (Chemi 4-6 days. Cell harvesting and immunofluorescent staining is con, Temecula, Calif.) which targets a B variant 101 kDa the same as described above. 10 virion protein is used as a primary antibody in the HHV-6Z 6.6.2.3 Efficacy Screening for HHV-6 and HHV-8 29 assay system at a 5 g/ml concentration. The HHV-8 Cord Blood Lymphocytes (CBL) Cells: monoclonal antibody KS8.1 (Bala Chandran, University of Fresh heparinized umbilical cord blood can be obtained, Kansas Department of Microbiology, Molecular Genetics e.g., from the University of Alabama at Birmingham Hospi and Immunology) targets the HHV-8 viral envelope associ tal, and diluted 1:1 with Hank's balanced salt solution and 15 ated glycoprotein 8.1 expressed in the late lytic phase of layered on a Histopaque 1077 (Sigma Chemical Co., St. HHV-8 replication (Zoeteweij et al., 1999) and is used at Louis, Mo.) gradient. The tubes are centrifuged at 1600 rpm approximately 5 lug/ml. Monoclonal antibody to the EBV for 30 minutes at room temperature and serum is carefully VCA glycoprotein 125 (Chemicon, Temecula, Calif.) is used aspirated off. The lymphocytes are removed, washed with at a concentration of 2.5 ug/ml for ELISA and 5 lug/ml for Hank's balanced salt solution and centrifuged at 1200 rpm for IFA 10 minutes. The Supernatant is aspirated, and the cells are Efficacy Against HHV-6: resuspended in RPMI 1640 containing 10% heat-inactivated Serial 5-fold dilutions of drug starting at 50 lug/ml are FBS, 2 mM L-glutamine, 100 U/ml penicillin, 0.25 ug/ml prepared in media. CDV is used as a positive control. Samples fungizone, 25 ug/ml gentamicin, 0.1 U/ml Interleukin-2 for determining anti-viral efficacy are prepared by incubating (Sigma, St. Louis, Mo.) and 0.5ug/ml Phaseolus Vulagaris 25 1x10° cells for one hour with sufficient virus to infect agglutinin protein (PHAP). CBLs are used in the HHV-6, approximately 35% of the cells. After infection, the appropri Z-29 (Variant B) assays. ate dilution of compound is added and cells incubated for 4 to Human T Cell Lymphoblastoid Line HSB-2: 6 days at 37° C. Virus free controls are prepared by incubating The HSB-2 cells can be obtained through, e.g., the NIH 1x10° cells incompound-free media for the designated period AIDS Research and Reference Reagent Program (Rockville, 30 and virus controls are prepared by incubating 1x10° cells for Md.), and are propagated in RPMI 1640 containing 10% one hour with sufficient virus to infect 35% of the cells heat-inactivated FBS, 100U/ml penicillin, 25 g/ml gentami followed by incubation in compound-free media for the des cin and 2 mM L-glutamine. The cells are split 1:5 in a 175 cm ignated period. After incubation, the cells are rinsed with PBS flask every 3-4 days and used in the HHV-6, GS (Variant A) and permeabilized overnight in methanol at -80C for use in assayS. 35 FACS Body Cavity-Based Lymphoma (BCBL-1) Cells: FACS Assay: Cells are rinsed thoroughly with PBS and a BCBL-1 cells (NIH AIDS Research and Reference Pro blocking solution containing 5% FBS, 4% Normal goat gram, Rockville, Md.) propagated in RPMI 1640 media con serum (NGS) and 0.5% DMSO. Cells are then incubated with taining 10% FBS, 2 mM L-Glutamine, 10 uM B-Mercapto the appropriate monoclonal antibody (HHV-6 early nuclear ethanol 100 L/l penicillin, and 25 ug/ml gentamicin are 40 proteins (Chemicon, Temecula, Calif.) for HHV-6, GS variant utilized in the HHV-8 assay. A, a 101 kDa virion protein (Chemicon, Temecula, Calif.) for Viruses: HHV-6, Z-29 variant B, and KS8.1 for HHV-8 (Bala Chan There are two variants of HHV-6 known as type A variants dran, University of Kansas, Department of Microbiology, or type B variants. An example of HHV-6 type A variant is the Molecular Genetics and Immunology). GS strain which is propagated in the HSB-2 cells and can be 45 6.6.2.4 Toxicity Screening for Herpes Viruses obtained through, e.g., the AIDS Research and Reference Neutral Red Uptake Assay HFF Cells: Reagent Program, Division of AIDS, NIAID, NIH. These Twenty-four hours prior to assay, HFF cells are plated into cells, referred to as HSB-2/HHV-6GS, are maintained at 96 well plates at a concentration of 2.5x10" cells per well. 5x10 cells/ml under the same conditions and in the same After 24 hours, the media is aspirated and 125 ul of each media as the uninfected HSB-2 cells. The cells are split every 50 compound concentration is added to the first row of wells and 3-4 days by addition of uninfected cells at 9 parts to 1 part then diluted serially 1:5 using the automated Bio-Mek Liquid infected cells. Stock titers of this virus of 1x10 both in Handling System in a manner similar to that used in the CPE cell-associated and cell-free virus can be obtained by growth assay. The plates are then incubated in a CO incubator at 37° for 5 days. An example of HHV-6 type B variant is Z29 C. for seven days. At this time the media/compound is aspi (ATCC, Rockville, Md.) which is grown as a stock in CBL by 55 rated and 200ul?well of 0.01% neutral red in DPBS is added. incubation for 10 days followed by collection, centrifugation This mixture is incubated in the CO, incubator for 1 hour. The and freezing of the supernatant. HHV-8, latently expressed in compound is aspirated and the cells are washed using a Nunc the primary effusion lymphoma derived BCBL-1 cell line Plate Washer. After removing the DPBS wash, 200ul/well of (NIH AIDS Research and Reference Program, Rockville, 50% ETOH/1% glacial acetic acid (in HO) is added. The Md.) is induced into lytic HHV-8 expression by addition of 60 plates are rotated for 15 minutes and the optical densities are 100 ng/ml phorbol 12-myristate 13-acetate. BCBL-1 cells are read at 550 nm on a plate reader. CCs values are calculated cultured in RPMI 1640 media containing 10% FBS, 2 mM using a computer program. L-glutamine, 10 uM 3-mercaptoethanol 100 U/ml penicillin Cell Proliferation Assay HFF Cells: and 25 ug/ml gentamicin. Twenty-four hours prior to assay, HFF cells are seeded in Primary Antibodies: 65 6-well plates at a concentration of 2.5x10" cells per well in The primary antibodies used for the indirect IFA and FACS MEM containing 10% FBS. On the day of the assay, test are selected for their antigen specificity, low cross-reactivity compounds are diluted serially in MEM containing 10% FBS US 8,546,432 B2 131 132 at increments of 1:5 covering a range from 100 ug/ml to 0.03 derived from linear regression analysis of the logarithm of ug/ml. For compounds that have to be solubilized in DMSO, compound concentration versus CFU-GM or BFU-E survival control wells receive MEM containing 1.0% DMSO. The fraction. media from the wells is then aspirated and 2 ml of each 6.6.3 In Vitro Laboratory Procedures for Assays for Influ compound concentration is then added to each well. The cells 5 enza, Respiratory and Other Viruses are then incubated in a CO incubator at 37°C. for 72 hours. 6.6.3.1 Screening Efficacy for RSV. PIV and Flu, Measles, At the end of this time, the media-compound solution is Rhino, Adeno, SARS, VEE, Yellow Fever, West Nile, removed and the cells are washed. One ml of 0.25% trypsin is Pichinde, Punta Toro and Dengue Viruses added to each well and incubated until the cells start to come Rapid Screening Assay: 10 When relatively large numbers (10 or more) of test com off of the plate. The cell-media mixture is then pipetted up and pounds are available, the compounds are evaluated in a 2-con down vigorously to break up the cell Suspension, and 0.2 ml centration test. In this procedure, two concentrations (e.g., of the mixture is added to 9.8 ml of Isoton III and counted 200 and 20 ug/ml) are tested. Compounds are diluted 1:2 using a Coulter Counter. Each sample is counted 3 times with when virus is added, making final concentrations 100 and 10 2 replicate wells per sample. 15 ug/ml. The standard CPE test uses an 18 hour monolayer MTS Tetrazolium Cytotoxicity Assay: (80-100% confluent) of the appropriate cells, medium is Serial 5-fold dilutions of test compound starting at 50 drained and each of the concentrations of test compound or ug/ml are prepared in media and added to 1x10 cells. Con placebo are added, followed within 15 minutes by virus or trols are prepared by incubating 1x10° cells in compound virus diluent. Two wells are used for each concentration of free media. After an incubation period of 3-6 days depending compound for both anti-viral and cytotoxicity testing. The on the assay system, 200 ul is transferred to a 96 well plate in plate is sealed and incubated the standard time period duplicate. 20 ul of MTS is added, and the plate is wrapped in required to induce near-maximal viral CPE. The plate is then foil and incubated at 37° C. for 4 hours. MTS is bioreduced by stained with neutral red by the method described below, and dehydrogenase enzymes found in metabolically active cells the percentage of uptake indicating viable cells read on a into an aqueous soluble formazan. The quantity of formazan 25 microplate autoreader at dual wavelengths of 405 and 540 product as measured by the amount of 490 nm absorbance is nm, with the difference taken to eliminate background. An directly proportional to the number of living cells in culture. approximated virus-inhibitory concentration, 50% endpoint Compound concentration is plotted against the optical den (ECs) and cell-inhibitory concentration, 50% endpoint sity of each sample and CCso values were calculated using (ICs) are determined from which a general selectivity index MacSynergy II. 30 is calculated: SI=(ICs)/(ECs). An SI of 3 or greater typically Cell Proliferation Assay—HSB-2 and Daudi Cells: indicates confirmatory testing is needed. Serial 5-fold dilutions of compound starting at 50 ug/ml are Inhibition of Cytopathic Effect (CPE): prepared in media and added to 1x10 cells. Controls are This test, run in 96 well flat-bottomed microplates, is used prepared by incubating 1x10° cells in compound-free media. for the initial anti-viral evaluation of all new test compounds. After an incubation period of 3-4 days depending on the assay 35 In this CPE inhibition test, four logo dilutions of each test system, a Coulter Counter is used to determine the total compound (e.g., 1000, 100, 10, 1 lug/ml) are added to 3 cups number of cells for each sample (HSB-2 and Daudicell lines). containing the cell monolayer, within 5 minutes, the virus is Compound concentration is plotted against the total concen then added and the plate sealed, incubated at 37° C. and CPE tration of cells for each sample and ICso values are calculated read microscopically when untreated infected controls using MacSynergy II. 40 develop a 3 to 4+ CPE (approximately 72 to 120 hours). A Bone Marrow Assay: known positive control compound is evaluated in parallel In vitro toxicity can be determined by inhibition of myeloid with test compounds in each test. The positive control com colony-forming units granulocyte/macrophage (CFU-GM) pound, for example, is: ribavirin for dengue, influenza, and erythroid burst-forming unit-erythroid (BFU-E) colony measles, RSV, PIV, Pichinde, Punta Toro and VEE viruses: formation in Soft agar clonal assays. Using a 21-23 gauge 45 cidofovir for adenovirus; pirodovir for rhinovirus; 6-azauri needle attached to a syringe, rodent bone marrow cells are dine for West Nile and yellow fever viruses; and alferon collected from the leg bone of rats or mice by flushing with (interferon alfa-n3) for SARS virus. Follow-up testing with Isocoves Modified Dulbecco's medium (IMDM). A single compounds that are found active in initial Screening tests are cell Suspension is obtained by repeated aspiration through the run in the same manner except 8 one-half logo dilutions of needle. Nucleated cells are enumerated with a hemacytom 50 each compound are used in 4 cups containing the cell mono eter and adjusted to the desired cell concentration in IMDM. layer per dilution. The data are expressed as 50% effective Murine CFU-GM assays are prepared with 2.5x10 nucleated concentrations (ECso). cells/ml, 20% FBS, 10 ng/ml rmCM-CSF, and 0.2% agarose. Increase in Neutral Red (NR) Dye Uptake: BFU-E cultures include 30% FBS, 1% deionized BSA, 0.1 This test is run to validate the CPE inhibition seen in the mM 2-ME, 4 U/ml rhEpo, 10 ng/ml rm IL-3, 2.5x10 nucle 55 initial test, and utilizes the same 96-well micro plates after the ated cells/ml and 0.2% agarose (140). Triplicate wells (in 6 CPE has been read. Neutral red is added to the medium; cells well plates) containing 0.1 ml of compound (10x) receive 1 not damaged by virus take up a greater amount of dye, which ml of either culture mixture for each concentration group and is read on a computerized micro plate autoreader. For slowly mixed. The cultures are allowed to gel at 4°C. and then example, the method as described by McManus, Appl. Envi incubated for 7 (CFU-GM) or 9 (BFU-E) days at 37° C. in a 60 ronment. Microbiol. 1976, 31:35-38, can be used. An ECs is humidified atmosphere of 5% CO, in air. Colonies are determined from this dye uptake. counted using an inverted microscope. CFU-GM colonies are Decrease in Virus Yield: identified as cell clones containing at least 40 cells. BFU-E Compounds considered active by CPE inhibition and by cultures are stained with dianisidine, and aggregates of NR dye uptake are re-tested if additional, fresh material is greater than 60 hemoglobin-containing cells are counted as 65 available, using both CPE inhibition and, using the same erythroid colonies. The median inhibitory concentration plate, the effect on reduction of virus yield by assaying frozen (ICs) and the 90% inhibitory concentration (IC) are and thawed eluates from each cup for virus titer by serial US 8,546,432 B2 133 134 dilution onto monolayers of susceptible cells. Development both resting and proliferating human fibroblast cells and pro of CPE in these cells is the indication of presence of infectious liferating lymphoblastic cells, and for selected compounds, virus. As in the initial tests, a known active compound is run toxicity in rodent myeloid and erythroid progenitor cells is in parallel as a positive control. The 90% effective concen assessed. tration (ECoo), which is that test compound concentration that 6.6.4.1 Screening Assays for VV and CV inhibits virus yield by 1 log 10, is determined from these data. Compounds are initially screened for activity using the 6.6.32 Screening Toxocity for RSV. PIV and Flu, Measles, CPE assay in HFF cells. Further testing in two other cells Rhino, Adeno, SARS, VEE, Yellow Fever, West Nile, lines, Vero and MRC-5, and against other strains of virus is Pichinde, Punta Toro and Dengue Viruses possible for compounds that demonstrate activity in other Visual Observation: 10 assay systems. The screening assay systems utilized are In the CPE inhibition tests, two wells of uninfected cells selected to show specific inhibition of a biologic function, i.e., treated with each concentration of test compound are run in cytopathic effect (CPE) in susceptible human cells. In the parallel with the infected, treated wells. At the time CPE is CPE-inhibition assay, test compound is added 1 hour prior to determined microscopically, the toxicity control cells are also infection so the assay system will have maximum sensitivity examined microscopically for any changes in cell appearance 15 and detect inhibitors of early replicative steps such as adsorp compared to normal control cells run in the same plate. These tion or penetration as well as later events. To rule out non changes can be enlargement, granularity, cells with ragged specific inhibition of virus binding to cells, all compounds edges, a filmy appearance, rounding, detachment from the that show reasonable activity in the CPE assay are confirmed Surface of the well, or other changes. These changes are given using a classical plaque reduction assay in which the drug is a designation of T (100% toxic), PVH (partially toxic very added 1 hour after infection. These assay systems also can be heavy—80%), PH (partially toxic heavy—60%), P (par manipulated by increasing the pre-treatment time in order to tially toxic 40%), Ps (partially toxic—slight 20%), or 0 demonstrate anti-viral activity with oligodeoxynucleotides (no toxicity—0%), conforming to the degree of cytotoxicity and/or peptides. By delaying the time of addition of com seen. A 50% cell inhibitory (cytotoxic) concentration (ICs) pound after infection, information regarding which step in the is determined by regression analysis of these data. 25 virus life cycle is inhibited (i.e., early vs. late functions) can Neutral Red Uptake: be gained. A direct inactivation assay can be employed to In the neutral red dye uptake phase of the anti-viral test determine the virucidal activity of selected compounds. described above, the two toxicity control wells also receive Efficacy: neutral red and the degree of color intensity is determined In the assays used for primary screening, a minimum of six spectrophotometrically. A neutral red ICs (NRICs) is sub 30 compound concentrations is typically used, covering a range sequently determined. of, e.g., 100 mg/ml to 0.03 mg/ml, in 5-fold increments. Viable Cell Count: These data allow for creating dose response curves. From Compounds considered to have significant anti-viral activ these data, the dose that inhibited viral replication by 50% ity in the initial CPE and NR tests are re-tested for their effects (effective concentration 50; ECso) is usually calculated using on cell growth. In this test, 96-well tissue culture plates are 35 a computer software program, for example, MacSynergy II seeded with cells (sufficient to be approximately 20% con by M. N. Prichard, K. R. Asaltine, and C. Shipman, Jr., Uni fluent in the well) and exposed to varying concentrations of versity of Michigan, Ann Arbor, Mich. the test drug while the cells are dividing rapidly. The plates Toxicity: are then incubated in a CO incubator at 37°C. for 72 hours, The same compound concentrations used to determine effi at which time neutral red is added and the degree of color 40 cacy are also used on uninfected cells in each assay to deter intensity indicating viable cell number is determined spectro mine toxicity of each experimental compound. The com photometrically; an ICs is determined by regression analy pound concentration that is cytotoxic to cells as determined S1S. by their failure to take up a vital stain, neutral red (cytotoxic Data Analysis: concentration 50; CCs), is determined as described above. A Each test compounds anti-viral activity is expressed as a 45 neutral red uptake assay can be used. The assay is reproduc selectivity index (SI), which is the ICs or IC divided by the ible and allows quantitation of toxicity based on the number ECs. Generally, an SI of 10 or greateris indicative of positive of viable cells rather than cellular metabolic activity. In some anti-viral activity, although other factors, such as a low SI for cases, the toxicity of new compounds on dividing cells is the positive control, are also taken into consideration. Com determined at a very early stage of testing. A cell proliferation pounds having SI values of 10 or greater can be evaluated 50 assay using HFF cells is a sensitive assay for detecting com against additional strains of the original virus inhibited in pound toxicity to dividing cells. The compound concentration order to more fully determine the spectrum of anti-viral activ that inhibits cell growth by 50% (ICs) is calculated as ity of the compound. described above. In comparison with four human diploid cell 6.6.4 General Procedures for Assays for Orthopoxviruses lines and Vero cells, HFF cells are known to be very sensitive To quickly screen out compounds that do not have activity 55 and predictive of toxicity for bone marrow cells. against any of the herpes viruses, or are too toxic to evaluate, 6.6.4.2 Confirmation Assays for VV and CV an assay Such as a CPE-inhibition assay that is semi-auto Anti-viral Activity: mated is commonly used initially to screen out the negative Compounds that show activity in the CPE-inhibition assay compounds. Typically, all screening assays are conducted in are confirmed using the plaque reduction assay. Susceptibil low passage human cells, and each assay system contains a 60 ity of additional virus strains of VV, CV and activity in other positive control (CDV) and a negative control (ACV). Effi cell types can also be determined for selected compounds. cacy is demonstrated by at least two different assay systems Toxicity: that detect functional biologic activity and should be con In addition to the toxicity component incorporated into firmed using low passaged clinical isolates and drug resistant each assay system, a standardized cell cytotoxicity assay mutants whenever available. In the case of Vaccinia virus 65 using a vital stain uptake (Neutral Red) is performed using 7 (VV) and Cowpox virus (CV), efficacy against VV and CV is days of compound exposure to confluent non-dividing cells. confirmed using other isolates. Toxicity is determined using This assay measures direct cell cytotoxicity (CCs). In this US 8,546,432 B2 135 136 regard, a neutral red uptake assay is reproducible and allows desired concentration in 2xMEM and then serially diluted 1:5 for quantitation of toxicity based on the number of viable cells in 2xMEM using 6 concentrations of compound. The initial rather than cellular metabolic activity. In some cases, the starting concentration is usually 200 mg/ml down to 0.06 toxicity of new compounds on dividing cells is determined at mg/ml. The virus to be used is diluted in MEM containing a very early stage of testing. A cell proliferation assay using 10% FBS to a desired concentration which will give 20-30 HFF cells is a sensitive assay for detecting compound toxicity plaques per well. The media is then aspirated from the wells, to dividing cells, and the compound concentration that inhib and 0.2 ml of virus is added to each well in duplicate with 0.2 its cell growth by 50% (ICs) is calculated as described above. ml of media being added to drug toxicity wells. The plates are 6.6.5In Vitro Laboratory Procedures for Assays for Ortho then incubated for 1 hour with shaking every 15 minutes. poxviruses 10 After the incubation period, an equal amount of 1% agarose is 6.6.5.1 Efficacy Screening for VV and CV added to an equal Volume of each compound dilution. This Preparation of Human Foreskin Fibroblast (Hff) Cells: addition gives final compound concentrations beginning with Newborn Human foreskins are obtained as soon as possible 100 mg/ml and ending with 0.03 mg/ml and a final agarose after circumcision and placed in minimal essential medium overlay concentration of 0.5%. The compound/agarose mix (MEM) containing Vancomycin, fungizone, penicillin, and 15 ture is applied to each well in 2 ml volume and the plates are gentamicin at the usual concentrations, for 4 hours. The incubated for 3 days, after which the cells are stained with a medium is then removed, the foreskin minced into Small 0.01% solution of neutral red in phosphate buffered saline. pieces and washed repeatedly with phosphate buffered saline After a 5-6 hours incubation period, the stain is aspirated, and (PBS) deficient in calcium and magnesium (PD) until red plaques counted using a stereomicroscope at 10x magnifica cells are no longer present. The tissue is then trypsinized tion. using trypsin at 0.25% with continuous stirring for 15 minutes 6.6.5.2 Toxicity Screening for VV and CV at 37° C. in a CO incubator. At the end of each 15-minute Neutral Red Uptake Assay: period, the tissue is allowed to settle to the bottom of the flask. Twenty-four hours prior to assay, HFF cells are plated into The Supernatant containing cells is poured through sterile 96 well plates at a concentration of 2.5x10" cells per well. cheesecloth into a flask containing MEM and 10% fetal 25 After 24 hours, the media is aspirated and 125 ml of com bovine serum. The flask containing the medium is kept on ice pound is added to the first row of wells and then diluted throughout the trypsinizing procedure. After each addition of serially 1:5 using the BioMek 2000 Laboratory Automation cells, the cheeseclothis washed with a small amount of MEM Workstation in a manner similar to that used in the CPE assay. containing serum. Fresh trypsin is added each time to the After compound addition, the plates are incubated for 7 days foreskin pieces and the procedure repeated until all the tissue 30 in a CO, incubator at 37° C. At this time, the media/com is digested. The cell-containing medium is then centrifuged at pound mixture is aspirated and 200 ul/well of 0.01% neutral 1000 RPM at 4°C. for 10 minutes. The supernatant liquid is red in PBS is added. This mixture is incubated in the CO, discarded and the cells are resuspended in a small amount of incubator for 1 hour. The dye is aspirated and the cells are MEM with 10% FBS. The cells are then placed in an appro washed using a Nunc Plate Washer. After removing the PBS, priate number of 25 cm tissue culture flasks. As cells become 35 200 mg/well of 50% ETOH/1% glacial acetic acid (in HO) is confluent and need trypsinization, they are expanded into added. The plates are rotated for 15 minutes and the optical larger flasks. The cells are kept on Vancomycin and fungizone densities read at 540 nm on a plate reader. The ECso values are to passage four, and maintained on penicillin and gentamicin. determined by comparing compound treated and untreated Typically, cells are used only through passage 10. cells using a computer program. Cytopathic Effect Inhibition Assay: 40 Cell Proliferation Assay: Low passage HFF cells are seeded into 96 well tissue Twenty-four hours prior to assay, HFF cells are seeded in culture plates 24 hours prior to use at a cell concentration of 6-well plates at a concentration of 2.5x10" cells per well in 2.5x10 cells per ml in 0.1 ml of MEM supplemented with MEM containing 10% FBS. On the day of the assay, com 10% FBS. The cells are then incubated for 24 hours at 37° C. pounds are diluted serially in MEM containing 10% FBS at in a CO incubator. After incubation, the medium is removed 45 increments of 1:5 covering a range from 100 mg/ml to 0.03 and 125 ml of experimental compound is added to the first mg/ml. For drugs that have to be solubilized in DMSO, con row in triplicate wells, all other wells having 100 ml of MEM trol wells receive MEM containing 1% DMSO. The media containing 2% FBS. The compound in the first row of wells is from the wells is aspirated, and 2 ml of each drug concentra then diluted serially 1:5 throughout the remaining wells by tion is then added to each well. The cells are incubated in a transferring 25 ml using the BioMek 2000 Laboratory Auto 50 CO incubator at 37° C. for 72 hours. At the end of this time, mation Workstation. After dilution of the compound, 100 ml the media-compound solution is removed and the cells of the appropriate virus concentration is added to each well, washed. One ml of 0.25% trypsin is added to each well and excluding cell control wells, which received 100 ml of MEM. incubated until the cells start to come off of the plate. The The virus concentration utilized is 1000 PFU’s per well. The cell-media mixture is then pipetted up and down vigorously to plates are then incubated at 37°C. in a CO incubator for 7 55 break up the cell suspension and 0.2 ml of the mixture is days. After the incubation period, media is aspirated and the added to 9.8 ml of Isoton III and counted using a Coulter cells stained with a 0.1% crystal violet in 3% formalin solu Counter. Each sample is counted 3 times with 2 replicate tion for 4 hours. The stain is removed and the plates rinsed wells per sample. using tap water until all excess stain is removed. The plates Bone Marrow Clonogenic Assay: are allowed to dry for 24 hours and then read on a BioTek 60 In vitro toxicity to bone marrow progenitor cells can be Multiplate Autoreader at 620 nm. The ECs values are deter determined by inhibition of myeloid colony-forming units mined by comparing compound treated and untreated cells granulocyte/macrophage (CFU-GM) and erythroid burst using a computer program. forming unit-erythroid (BFU-E) colony formation in soft Plaque Reduction Assay: agar clonal assays. Using a 21-23 gauge needle attached to a Two days prior to use, HFF cells are plated into 6 well 65 Syringe, rodent bone marrow cells are collected from the leg plates and incubated at 37° C. with 5% CO, and 90% humid bone of rats or mice by flushing with Isocoves Modified ity. On the date of assay, the compound is made up at twice the Dulbecco's medium (IMDM). A single cell suspension is US 8,546,432 B2 137 138 obtained by repeated aspiration through the needle. Nucle icity is assessed by uptake of neutral red dye 24 hours follow ated cells are enumerated with a hemacytometer and adjusted ing the last treatment. Lamivudine (LMV) is used as the to the desired cell concentration in IMDM. Murine CFU-GM standard assay control, but other control compounds are also assays are prepared with 2.5x10 nucleated cells/ml, 20% available. FBS, 10 ng/ml rmCM-CSF, and 0.2% agarose. BFU-E cul- 5 ECso, ECoo and CCso values are calculated by linear tures include 30% FBS, 1% deionized BSA, 0.1 mM 2-ME, 4 regression analysis (MS EXCEL(R), QuattroPro(R) using data U/ml rhEpo, 10 ng/ml rmIL-3, 2.5x10 nucleated cells/ml combined from all treated cultures (Korba & Gerin, Antivir. and 0.2% agarose. Triplicate wells (in 6 well plates) contain Res. 1992, 19:55: Okuse et al., Antivir. Res. 2005, 65:23). ing 0.1 ml of compound (10x) receive 1 ml of either culture Standard deviations for ECs and EC values are calculated mixture for each concentration group and slowly mixed. The 10 from the standard errors generated by the regression analyses. cultures are allowed to gel at 4°C. and then incubated for 7 ECso and E.Coo are compound concentrations at which a (CFU-GM) or 9 (BFU-E) days at 37° C. in a humidified 2-fold, or a 10-fold depression of HBV DNA (relative to the atmosphere of 5% CO in air. Colonies are counted using an average levels in untreated cultures), respectively, is inverted microscope. CFU-GM colonies are identified as cell observed. CCso is the compound concentration at which a clones containing at least 40 cells. BFU-E cultures are stained 15 2-fold lower level of neutral red dye uptake (relative to the with dianisidine, and aggregates of greater than 60 hemoglo average levels in untreated cultures) is observed. The Selec bin-containing cells are counted as erythroid colonies. The tivity index (S.I.) is calculated as CCso/ECoo since at least a median inhibitory concentration (ICs) and the 90% inhibi 3-fold depression of HBV DNA levels is typically required to tory concentration (ICoo) are derived from linear regression achieve statistical significance in this assay system (Korba & analysis of the logarithm of compound concentration versus 20 Gerin, Antivir. Res. 1992, 19:55). CFU-GM or BFU-E Survival fraction. Secondary Assay: 6.6.6 Assays for Hepatitis Viruses Confluent cultures of 2.2.15 cells maintained on 48-well 6.6.6.1 Hepatitis B Virus (HBV) flat-bottomed tissue culture plates are treated as described for A variety of cell-culture based anti-HBV analyses are the primary assay. HBV virion DNA levels in the culture available. Candidate compounds are initially assayed in a 25 medium and cytotoxicity are assessed as described for the primary Screening assay. Compounds demonstrating reason primary assay. In addition, intracellular HBV DNA replica able anti-viral and cytotoxicity profiles are then candidates tion is measured by quantitative Southern blot hybridization for several additional follow-up analyses. For the primary analysis (Korba & Gerin, Antivir. Res. 1992, 19:55). ECs. screening assay, routinely 2-3 mg are required for compounds EC and S.I. values are calculated for both virion DNA and with molecular weights in the range of Standard nucleosides 30 intracellular HBV DNA replication intermediates. In certain (e.g., 300-500). Additional compound may be required for cases, additional assays (tertiary assays) can be conducted. follow-up analyses. Molecular weights and solubility infor Combination Studies: mation are provided if available. If no preferred solvent is Compounds are mixed at approximately equipotent con specified, 100% tissue culture DMSO will be used. Com centrations and this molar ratio is maintained during serial pounds are typically solubilized in aqueous solutions (normal 35 dilution(Korba, Antivir. Res. 1996, 29:49; Iyeretal. 2004). To pH range) at a minimum of a 10x final testing concentration compensate for potential unforeseen interactions (e.g., or in DMSO at a minimum 50x test concentration. EtOH is changes in uptake, metabolism, etc.), the concentration of one generally not well tolerated by the cell lines used for these compound is altered approximately 3-fold higher of lower studies, but final concentrations of EtOH of less than 0.03% relative to the second compound so that three different ratios are usually acceptable. Compounds which need to be tested in 40 are used in one experiment. Cultures are treated with 6-8 other solvents should be accompanied by a small amount of serial dilutions of the mixtures, as with the corresponding Solvent (under a separate accession number) to control for monotherapies, as described for the primary assay. Evalua cytotoxicity. For compounds in Solution, approximately 0.25 tion of compound interactions in the combination treatments ml of a 100x stock is minimally required. is conducted against the corresponding monotherapies in the Primary Assay: 45 same experiments using the Combostat(R) (Biosoft, Inc.) HBV anti-viral assays (Korba & Gerin, Antivir. Res. 1992, analysis Software. For combination treatments, ECso, ECoo 19:55) are conducted using confluent cultures of 2.2.15 cells CCs and S.I. (CCs/EC) are presented for the first com maintained on 96-well flat-bottomed tissue culture plates pound listed. The molar ratio of the compounds in each com (confluence in this culture system is required for active, high bination is also indicated. levels of HBV replication equivalent to that observed in 50 Alternatively, after the anti-viral activity oftest compounds chronically-infected individuals (Sells et al., J. Virol. 1988, against HBV is confirmed, the interactions of the compounds 62:2836: Korba and Gerin, Antivir. Res. 1992, 19:55). with 3TC, IFNC. and other compounds in terms of efficacy HepG2-2.2.15 is a stable cell line containing the hepatitis B (synergy, additivity, antagonism) and toxicity (combination virus (HBV) ayw strain genome. Anti-viral compounds toxicity) can be evaluated with the 2.2.15 cells using the blocking any late step of viral replication Such as transcrip- 55 quantitative HBV TaqMan PCR assay. tion, translation, pregenome encapsidation, reverse transcrip Drug Resistant HBV: tion, particle assembly and release can be identified and char Activity against recombinant HBV carrying clinically rel acterized using this cell line. evant mutations that confer resistance to licensed drugs is Cultures are treated with nine consecutive daily aliquots of performed using transient transfection of HBV DNA (Tatti et the test compounds. Typically, 4 doses (10-fold or 3-fold 60 al., Antivi. Res. 2002, 55:27: Iyer et al., AAC 2004, 48:2199). steps), in triplicate are used. HBV DNA levels in the culture Cultures are transfected in 48-well culture plates with Lipo medium (representing HBV virion production) are assessed fectamine 2000TM (Gibco, Inc) following the manufacturers by quantitative blot hybridization 24 hours after the last treat procedure. Beginning three days post-transfection, cultures ment. Alternatively, whether a compound reduces the produc are treated for 5 days with anti-viral compounds. Anti-viral tion of secreted HBV from cells can be initially assessed 65 activity is determined by quantitative Southern Blot hybrid utilizing real time quantitative PCR (TaqMan) assay to ization of intracellular HBV DNA replication intermediates. directly and accurately measure HBV DNA copies. Cytotox Currently, the following mutants are available: lamviudine US 8,546,432 B2 139 140 (LMV)-resistant, polM204V, polM2041, polL180M, genomes. Purified virions are used to assay the ability of polM204V/L180M (Allen et al., Hepatology 1998, 27:1670); anti-viral drugs to inhibit the endogenous polymerase adefovir dipovoxil (ADV)-resistant, polN236T (Angus et al., activity of HBV. Normally, this activity functions to Gastroenterology 2003, 125:292). Standardized nomencla complete (+) strand synthesis following the infection of ture is used for HBV polymerase assignment (Stuyver et al., new cells by HBV virions. Hepatology 2001, 33:751). Additional mutants (TNFR, 6.6.6.2 Hepatitis C Virus (HCV) Protocol I ETVR) are under construction. Cell line Huh7 ET (luc-ubi-neo/ET), which contains a new Other tests can be conducted in order to evaluate the ability HCV RNA replicon with a stable luciferase (LUC) reporter, is of compounds to inhibit the known 3TC- and penciclovir used. It is similar to the cell line 5-2 (Krieger et al., J. Virol. resistant mutants of HBV. Stable cell lines with control wild 10 2001, 75:4614-4624.), but contains additional modifications type HBV and the following mutations known to be associ that make the cell line more robust and provide stable LUC ated with resistance of HBV to these agents can be used: (1) expression for anti-viral screening. The composition of the L526M (rtL180M) of Domain B & YMDD M550V replicon is shown diagrammatically below: (rtM204V) of Domain C (the most common mutation pattern observed during HBV breakthrough viremia); (2) L526M 15 alone (the most common mutation associated with penciclo E-I Vir resistance; also associated with some resistance against 3TC); and control wild-type HBV. HBV Protein Production and RNA Transcription: Semi-quantitative ELISA-based analysis of HBV proteins The HCV RNA replicon ET contains the 5' NTR (IRES) of is performed (Korba & Gerin, Antivir. Res. 1995, 28:225; HCV 5" which drives the production of a firefly luciferase Korba et al., Antivir. Res. 2008, 77:56) using samples diluted (Luc), ubiquitin (Ubiq), and neomycin phosphotransferase (2 to 10-fold) to bring levels into the dynamic response ranges (Neo) fusion protein. Ubiquitin cleavage releases the LUC of the assays. Qualitative analysis of HBV proteins is also and Neogenes. The EMCV IRES element (E-I) controls the performed using standard Western blot techniques (Muller et 25 translation of the HCV structural proteins NS3-NS5. The al., J. Infect. Dis. 1992, 165:929). HBV surface (HBSAg) and NS3 protein cleaves the HCV polyprotein to release the HBV e (HBeAg) antigens are analyzed from culture medium mature NS3, NS4A, NS4B, NS5A and NS5B proteins that are samples, and HBV core (HBcAg) is analyzed from intracel required for HCV replication. At the 3' end of the replicon is lular lysates (normalized for total cell protein content in each the authentic 3' NTR of HCV. culture sample). Intracellular HBV RNA (normalized to the 30 The LUC reporter is used as an indirect measure of HCV level of cellular B-actin RNA in each culture sample) is replication. The activity of the LUC reporter is directly pro assessed by quantitative northern blot hybridization (Korba & portional to HCV RNA levels and positive control anti-viral Gerin, Antivir. Res. 1995, 28:225). compounds behave comparably using either LUC or RNA HBV Mechanism of Action Studies: endpoints. The use of the LUC endpoint is more economical A variety of assays can be used to pinpoint the mechanism 35 than HCV RNA and can be used for high-throughput appli of action of anti-viral compounds. Examples include the fol cations to Screen libraries of compounds. lowing: Primary HCV RNA Replicon Assay: Extracellular HBV virions: In addition to the quantitative First, the effect of compounds added in triplicate at a single PCR analysis, a Southern blot of the HBV particles high-test concentration of 20 mM on HCV RNA-derived Secreted from compound-treated cells can be performed; 40 LUC activity and cytotoxicity is examined. Human interferon Intracellular HBV particles: HBV particles can be isolated alpha-2b is included in each run as a positive control com from the treated 2.2.15 cells and the pregenomic RNA pound. Subconfluent cultures of the ET line are plated out into examined by Southern blot analysis. This can be helpful 96-well plates that are dedicated for the analysis of cell num in identifying the site of action of a late-acting com bers (cytotoxicity) or anti-viral activity, and test compounds pound; 45 are added to the appropriate wells the next day. Cells are Intracellular HBV replicative intermediates: Nucleic acids processed 72 hours later when the cells are still subconfluent. isolated from the cells can be examined by Southern Compounds that reduced the LUC signal by 50% or more blots to assess the distribution of circular partially relative to the untreated cell controls move forward to the next double-stranded HBV DNA, linear partially double screening steps. A compounds cytotoxicity is assessed as the stranded DNA and single stranded HBV DNA; 50 percent viable cells relative to the untreated cell controls. HBV transcription: Effects on HBV genomic and subge HCV RNA Replicon Confirmation Assay: nomic viral RNA synthesis are studied by Northern blot The HCV RNA replicon confirmatory assay is then used to and primer extension analysis; examine the effects of compounds at, for example, five half HBS Ag and HBeAg release assay: ELISAs are used to log concentrations each. Human interferon alpha-2b is quantify the amounts of the HBV envelope protein, Sur 55 included in each run as a positive control compound. Subcon face antigen (HBSAg), and of secreted e-antigen fluent cultures of the ET line are plated out into 96-well plates (HBeAg) released from cultures; that are dedicated for the analysis of cell numbers (cytotox Western blot analysis: Western blots are conducted to study icity) or anti-viral activity and the next day test compounds HBV core and envelope protein expression; are added to the appropriate wells. Cells are processed 72 Novel mechanism of action studies: Specific effects on 60 hours later when the cells are still subconfluent. Compound HBV transcription and replication may arise from alter ECs and EC values (anti-viral activity) are derived from ations in DNA-protein interactions, sometimes affected HCV RNA levels assessed as either HCV RNA replicon by cellular growth factors, at the HBV enhancers, pro derived LUC activity or as HCV RNA using TaqMan RT moters or through the transcriptional transactivator PCR. Compound ICso and ICoo values (cytotoxicity) are cal X-protein; and 65 culated using CytoToX-1 (Promega), a colorimetric assay Endogenous Polymerase Assay: Extracellular HBV viri used as an indicator of cell numbers and cytotoxicity when the ons contain partially double-stranded circular DNA LUC assay system is employed, while ribosomal (rRNA) US 8,546,432 B2 141 142 levels determined via TaqMan RT-PCR are used as an indi used in one experiment. Cultures are treated with 6-8 serial cation of cell numbers in the RNA-based assay. Compound dilutions of the mixtures, as with the corresponding mono selectivity indices SIs and SIoo values are also calculated. therapies, as described for the primary assay. Evaluation of 6.6.6.3 Hepatitis C Virus (HCV) Protocol II compound interactions in the combination treatments is con A variety of cell-culture based anti-HCV analyses are ducted against the corresponding monotherapies in the same available. Candidate compounds are initially assayed in a experiments using the Combostat(R) (Biosoft, Inc.) analysis primary Screening assay. Compounds demonstrating reason Software. For combination treatments, ECso, ECoo CCso and able anti-viral and cytotoxicity profiles are then candidates S.I. (CCs/EC) are presented for the first compound listed. for several additional follow-up analyses. For the primary The molar ratio of the compounds in each combination is also screening assay, routinely 2-3 mg are required for compounds 10 with molecular weights in the range of Standard nucleosides indicated. (e.g., 300-500). Additional compound may be required for Drug-Resistant HCV: follow-up analyses. Molecular weights and solubility infor Since no licensed anti-HCV drugs for which resistance mation are provided if available. If no preferred solvent is mutations have yet been identified, a panel of mutants con specified, 100% tissue culture DMSO is used. Compounds 15 ferring resistance to compounds in mid to late phase clinical are solubilized in aqueous solutions (normal pH range) at a trials is compiled. Some available stable replicon-containing minimum of a 10x final testing concentration or in DMSO at cell lines (Korba et al., Antivir. Res. 2008, 77:56) are genotype a minimum 50x test concentration. EtOH is generally not 1 B NS5B S282T (Perra et al., Nucleosides Nucleotides well tolerated by the cell lines used for these studies, but final Nucleic Acids 2005, 24:767), and NS3 A156S and NS3 concentrations of EtOH of less than 0.03% are acceptable. A156V (Courcambeck et al., Antivir. Ther: 2006, 11:847) Compounds which need to be tested in other solvents should drug-resistant mutants. The genetic background is the same be accompanied by a small amount of solvent (under a sepa as that in the BB7 replicon (AVA5 cells) used in the primary rate accession number) to control for cytotoxicity. For com assay. Activity against these mutants is assessed as described pounds in solution, approximately 0.25 ml of a 100x stock is in the primary assay, except that semi-quantitative real-time minimally required. 25 PCR is used for the analysis of HCV RNA due to reduced Primary Assay: replication levels. Anti-viral activity against HCV is assessed in a 3-day assay The genotype 1b mutants can also be assessed in this man (Okuse et al., Antivir. Res. 2005, 65:23: Korba et al., Antivir. ner. For this assay, Huh?.5 cells are transfected with HCV Res. 2008, 77:56) using the stably-expressing HCV replicon RNA using Liofectamine 2000TM (Gibco, Inc.) in 6-well cul cell line, AVA5 (sub-genomic (CON1), genotype 1b) (Blight 30 ture plates. Three days post-transfection, cultures are exposed et al., Science 2000, 290:1972) maintained as sub-confluent to 125 ug/mL G418 and test compounds. After 10-14 days, cultures on 96-well plates. Anti-viral activity is determined surviving colonies are fixed, stained, and counted. ECs and by blot hybridization analysis of intracellular HCV RNA EC values are calculated for each transfected RNA. (normalized to the level of cellular B-actin RNA in each 6.6.7. Assays for Papilloma Viruses culture sample). Cytotoxicity is assessed by neutral red dye 35 Assays for Human Papilloma Virus (HPV) 11 and 40: uptake in cultures maintained in parallel plates. A431 cells are plated 2x10 cells/well in 6-well cluster ECso, ECoo and CCso values are calculated by linear dishes. Replicate aliquots of HPV-11 (or HPV-40) are added regression analysis (MS EXCEL(R), QuattroPro(R) using data to each well representing an MOI of 150 particles per cell. combined from all treated cultures (Korba & Gerin, Antivir. Dilutions of compound are added to triplicate cultures. Con Res. 1992, 19:55: Okuse et al., Antivir. Res. 2005, 65:23). 40 trol wells without virus are included and receive media alone. Standard deviations for ECso and ECoo values are calculated Positive control compound can be, e.g., HPMPC (cidofovir) from the standard errors generated by the regression analyses. at 300 ug/ml. Cell cultures are harvested, lysed with Trizol ECso and E.Coo are compound concentrations at which a reagent (GIBCO/BRL) and RNA prepared. QRT-PCR is con 2-fold, or a 10-fold depression of intracellular HCV RNA ducted to quantitate the proportion of viral E1-E4 transcripts (relative to the average levels in untreated cultures), respec 45 and a cellular reference RNA for the TATA-binding protein tively, is observed. CCso is the compound concentration at (TBP). Anti-viral effects of compounds are assessed as an which a 2-fold lower level of neutral red dye uptake (relative ECs value representing a 50% reduction in the amount of to the average levels in untreated cultures) is observed. The El-E4 viral transcript when compared with cultures infected Selectivity index (S.I.) is calculated as CCs/EC. Recom with HPV-11 (or HPV-40) alone. CCs toxicity is calculated binant human interferon2b (PBL laboratories, Inc.) is used as 50 as the compound dose at which 50% of total cellular RNA is an assay control. Compounds currently in clinical trials that recovered. From these two values, the Selectivity Index (SI) is are directed against NS3 and NS5B can also be used. determined from CCs/ECs. Usually, SD5 would be signifi Secondary Assay: cant for the detection of an anti-viral activity. This assay assesses activity against additional genotypes The assay procedure can be modified to test compounds using the format described for the primary assay. Activity 55 with microbicidal activity if necessary. This modification is against the genotype 1b HCV is included for comparison. represented by drug addition to A431 cells at the same time as One exemplary replicon cell line contains H/FL-Neo (geno infectious virus. type 1a (H77), full length construct) (Blight et al., J. Virol. Assays for Bovine Papilloma Virus (BPV) 1: 2003, 77:3181). A genotype 2a construct (J6/JFH-1, full C127 cells are plated 3x10 cells/well into wells of a length) can be used to assess for future inclusion. ECso, ECoo 60 96-well flat-bottomed microculture plate. Replicate aliquots CCs and S.I. values are calculated for each replicon cell line. of BPV-1 are added to each well, representing approximately In some instances, additional assays (tertiary assays) may 100 focus-forming units. Control wells without virus are also be conducted. included. Dilutions of drug are added to triplicate cultures of Combination Studies: Compounds are mixed at approxi both BPV-1-infected and uninfected cultures. Control wells mately equipotent concentrations and this molar ratio is 65 receive media without compound. Positive control compound maintained during serial dilution (Korba, Antivir. Res. 1996, can be, e.g., cidofovir at 5ug/ml. Cell cultures are fed with 29:49; Iyer et al., 2004). Usually, three different ratios are medium and compound every 3-4 days. Cell numbers and US 8,546,432 B2 143 144 viability are assessed using the MTS assay. Anti-viral effects Each compound sensitivity experiment requires inocula of compounds are calculated using the following formula to tion of 50,000 log-phase WI-38 cells in six-well culture obtain% anti-viral activity: plates. After plating the cells, viral infection is achieved by the addition of 2x10 to 2x10 BKV particles to each culture B&A/B&C'x100%=% Anti-viral activity well in a volume of 0.5 mL. After a 2 hours of 37° C. incu bation, unbound virus is washed off with tissue culture A=O.D. of BPV-1-containing, compound-treated cultures medium. The cultures are maintained in DMEM medium, B=O.D. of BPV-1 containing cell cultures supplemented by 10% fetal bovine serum and L-Glutamine, C=O.D. of cultures of cells alone. at 37° C., under 5% CO, for 7 days. Cells are harvested by The ECso value represents a 50% reduction in the amount of 10 0.25% trypsin-1 mM Na-EDTA digestion at 37° C. for 10 O.D. values (MTS signal) of compound-treated virus-in minutes and viability assessed by Trypan blue dye exclusion fected cultures when compared with cultures containing test. DNA extraction on the cell lysates is performed with a BPV-1 alone and cultures containing cells alone. The Selec commercially available kit (QIAamp DNA Mini kit, Qiagen, tivity Index (SI) is determined from CCs/ECs. Usually, Valencia, Calif.). BKV VP-1 DNA is amplified from the total SD5 would be significant for the detection of an anti-viral 15 DNA by a TaqMan quantitative PCR reaction performed in an activity. ABI Prism 7700 Sequence Detector (ABI, Foster City, The assay procedure can be modified to test compounds Calif.). To keep track of variable input of cellular DNA in with microbicidal activity if necessary. This modification is different cell-culture experiments, each cell lysate is sub represented by drug addition to C127 cells at the same time as jected to simultaneous TaqMan PCR for ACY. infectious virus. 6.6.9Assays for Dengue Virus (DENV) Assays for Human Papilloma Virus (HPV)31b: In vitro assays for DENV can be conducted using proce Cultures of CIN612, clone 9E cells are prepared from dures substantially similar to those described, for example, in known protocols. Individual rafts are developed containing Heaton et al., Proc. Natl. Acad. Sci., 2010, 107(40): 17345 multi-layers of 9E cells growing on a collagen matrix impreg 17350. Huh-7.5 cells (a subline derived from the hepatocyte nated with mitomycin C-treated fibroblast cells. Rafts are 25 Huh7 cell line) are maintained in DMEM-high glucose treated with compound delivered into the cell culture media supplemented with 0.1 mM nonessential aminid acids, 5% that can diffuse into the 9E multilayer. Treatments are con V/v FBS, and penicillin-streptomycin. In certain cases, tinuous for the culture duration, which is typically a period of DENV-luciferase replicon RNAs are introduced in Huh-7.5 10 days. After 10 days culture, the 9E rafts are harvested and cells by electroporation. At 24 hour post-electroporation, the assayed for HPV-31b DNA (measure of viral DNA replica 30 cells are treated with varying concentrations of test com tion) and E1-E4 viral transcription (viral function) using the pounds, maintained for another 24 hours, and assayed for QRT-PCR assay described for the HPV-11 monolayer assay luciferase activity. system. Primers are prepared to quantitate HPV-31B DNA In other cases, Huh-7.5 cells are infected with DENV (mul and RNA (E1-E4) and the quantitation compared to TBP. tiplicity of infection=1) for 4 hours and then treated with test Anti-viral activity is measured quantitatively as either or both 35 compounds at varying concentrations. Twenty four hours a reduction in viral DNA and RNA when compared to pla post-infection, viral RNA levels or released virus are quanti cebo-treated 9E rafts. A portion of each raft is removed for fied along with cellular ATP levels. histology (H&E, immunostaining for specific marker ker Three types of in vitro assays for DENV include those atins keratin 10, involucrin). Viral DNA and RNA levels are described in Chen et al., Antimicrobial Agents and Chemo plotted against compound concentrations to determine ECso 40 therapy, 2010, 54(8):3255-3261. (50% reduction in viral DNA and/or RNA), CCs (50% Type 1: reduction in yield of total RNA/DNA). The Selectivity Index The assays measure viral titer reduction in the presence of (SI) is determined from CCs/ECs. Usually, SD5 would be test compounds. Vero cells are seeded in a 12-well plate significant for the detection of an anti-viral activity. (4x10 cells per well). At 24 hours post-seeding, the cells are 6.6.8 Assays for BK Virus (BKV) 45 infected with DENV at a multiplicity of infection of 0.1 and Assays for BK virus (BKV) can be conducted by following treated immediately with the test compounds. Culture the procedures described in, e.g., Farasati et al., Transplan medium are collected at an appropriate time, and viral titers tation, 2005, 79(1):116-118. Generally, the principle of the are determined using plaque assays. assay is to measure the effect of test compounds on the rate of Type 2: viral replication by quantitative real-time PCR for BKV viral 50 Cell-based flavivirus immunodetection (CFI) is used to capsid protein 1 DNA. Simultaneous quantification of a measure the amount of viral E protein in infected cells. A549 housekeeping gene Such as aspartoacylase (ACY) DNA per cells are seeded to a 96-well plate (2x10 cells per well). The mits monitoring of host-cell replication. Regression analysis cells are infected with DENV on the following day. During of dose-response curve allows for determination of ECso the infection, the cells are incubated with a test compound/ which is defined as the compound concentration that reduces 55 virus mixture for 1 hour, with shaking every 10 to 15 minutes. the yield of BKV DNA by 50%. The ratio of ICs to ECs The culture fluid is then replenished with fresh medium con (selectivity index) is used to compare the anti-viral effect of taining test compounds. On day 2 post-infection, the cells are different test compounds in relation to their safety. washed with PBS, fixed with 100% methanol at 4°C. for 10 For example, anti-viral testing can be performed using minutes, and detected for intracellular viral E protein by BKV, Gardner strain (available from ATCC). The cells are 60 ELISA. The ELISA uses mouse monoclonal antibody 4G2 expanded, e.g., in human embryonic lung fibroblasts (WI-38 against the DENV E protein and goat anti-mouse IgG conju cells), using DMEM medium supplemented by 10% fetal gated with hoseradish peroxidase as the primary and second bovine serum and L-glutamine, incubated at 37°C. under 5% ary antibodies, respectively. CO. Each test compound is typically tested at least three Type 3: times using a broad range of concentrations covering 4-5 65 The assay uses Huh-7 cells and luciferase-reporting repli orders of magnitude. Experiments usually include a negative con of DENV. The procedures are similar to those described control consisting of cells exposed only to the diluents. above. US 8,546,432 B2 145 146 6.7 In Vivo Assays days 4-21 and Swabs of the Snout area are taken on days 3-10. 6.7.1 In Vivo Assays for Herpes Viruses The samples are placed in 2.0 mls of media and frozen at -70° 6.7.1.1 HSV-1 and HSV-2 C. until titrated for HSV-1 on rabbit kidney fibroblast cells in Animal Models of Herpes Encephalitis: a CPE microtiter plate assay. All experimental drug efficacy studies are placebo or vehicle controlled and also the positive control, Zovirax, is administered topically. Mouse Model of Primary HSV-1/HSV-2 Challenge: Virus Species Route Disease The primary Screening model provides a rapid initial evalu HSV-1 BALBic Mice i.p. Encephalitis ation of anti-viral efficacy against HSV primary infection i.n. Encephalitis 10 with both clinical and virological endpoints. This model uti HSV-2 BALBic Mice i.p. Encephalitis lizes intravaginal inoculation of female Swiss Webster mice i.n. Disseminated infection of newborns and encephalitis (25 g) with HSV-1 or HSV-2 to evaluate potential anti-viral HSV-1 Rat i.n. Encephalitis therapies as well as Vaccine/adjuvant candidates. Animals are HSV-1 SKH-1 Mice i.cut. Herpes labialis followed daily for signs and systems of herpes disease and 15 vaginal swabs are obtained to evaluate the effect of therapy on viral replication. Single or combined anti-viral therapies can New compounds are screened initially for HSV activity in be administered topically, orally or systemically and can be BALB/c mice (Charles River Laboratories) inoculated i.p. given at varying intervals begun before or after viral chal with HSV-1 or HSV-2. Following i.p. inoculation with HSV-1 lenge. Dose range studies can also be performed. Dose and or HSV-2, virus replicates in the gut, liver, and spleen and route of administration are individualized for each experi spreads to the CNS by viremia and likely peripheral nerves as mental compound. Treatment group size is typically 12-16 well. Virus is detected first in the brain around day five, thus 1CC. allowing time for compounds to demonstrate anti-viral Microbicide Screening Model of Primary HSV-2 Chal effects. This model system one of the most sensitive for lenge in Mice: determining efficacy of a new anti-viral compound. Although 25 This model is designed to evaluate the protection afforded it does not simulate a natural route of infection, it allows for by a microbicide candidate against infection with HSV-2. The screening new compounds to determine optimal dosages and model utilizes intravaginal inoculation of female Swiss Web treatment regimens. This screening is followed by testing in ster mice for evaluation. The initial trial is usually performed mice inoculated in. which more closely simulates human by applying compound 5 minutes prior to challenge with infections. If the experimental compound exhibits activity in 30 HSV-2. Further evaluation of microbicides in this model mice inoculated i.p., it is next evaluated in mice inoculated by either extend the time between microbicide administration the in. route. and challenge or examine dose range. Compounds can be I.n. inoculation of three-week-old BALB/c mice with advanced to a secondary species evaluation in the guinea pig HSV-1 provides a model for herpes encephalitis of humans model of genital infection. Evaluation includes daily evalua utilizing a natural route of infection. After inoculation of 35 tion for signs and symptoms of genital herpes and viral approximately 105 pfu of HSV-1, strain E-377, virus repli examination of vaginal secretions. Treatment group size is cates in the nasopharynx and spreads to the CNS by way of typically 12-16 mice. olfactory and trigeminal nerves. Untreated animals generally Guinea Pig Model of Primary Genital HSV-2 Infection: die by days 6-10. The use of i.n. inoculation is known as a Because genital herpes disease in the guinea pig more natural route of infection for herpes encephalitis. I.n. inocu 40 closely resembles human disease, this animal is used as a lation of three-week-old BALB/c mice with about 4x10"pfu second species for therapies with demonstrated efficacy of HSV-2, strain MS, provides a model of disseminated neo against HSV in mice. As with humans, genital HSV infection natal herpes with CNS involvement. After viral inoculation, in guinea pigs is a self limited vesiculoulcerative disease virus replicates in nasopharyngeal and lung tissue, and dis which is followed by healing, the establishment of latency, seminates to the liver, spleen, and kidney. In addition, virus 45 and then both spontaneous and inducible symptomatic and spreads to the CNS via olfactory and trigeminal nerves. Acy asymptomatic recurrences. An exemplary model utilizes clovir, ACV. given parenterally or orally is effective in all the intravaginal inoculation of female Hartley guinea pigs and experimental infections mentioned above and is utilized as a provides both clinical and Virologic indices to asses both the positive control. effect of treatment on primary disease as well as on the The SKH-1 strain of immunocompetent hairless mice is 50 frequency or severity of Subsequent recurrent infections. used to facilitate scoring of cutaneous lesions. Orofacial Anti-viral therapy can be administered orally, topically or inoculation of HSV-1 in these mice provides an appropriate systemically and can be given at varying intervals beginning model for testing new anti-viral therapies. In this model, mice before or after virus challenge. Following intravaginal inocu are anesthetized with a ketamine/xylazine mixture and lation, animals are followed daily for the development of injected Subcutaneously with an electronic microchip for 55 genital herpes using a validated genital herpes scoring sys individual identification. Prior to inoculation, the snout, com tem. Vaginal swabs are also obtained to evaluate the effect posed of the triangular shaped area over the nasal bones from against viral replication. Because this is a non lethal model, the nose bridge to the eyes, is lightly abraded with a #113 animals can be sacrificed at the conclusion of the experiment tungsten-carbide engraving bit Dremmel tool. This procedure to evaluate the effects of treatment on latency. This model can is performed carefully to prevent bleeding. This area is then 60 be adapted to evaluate anti-viral activity against available swabbed for 10 seconds with a dacron swab soaked with drug resistant strains (ACV and Foscarnet). Dose, route of HSV-1. Following this procedure, animals are returned to administration and duration of treatment are individualized their cages and observed until recovery. for each experimental agent. Treatment group size is typically Animals infected with HSV-1 in the orofacial area exhibit 10-15 animals. lesions that begin to appear on days 4-6 and are usually 65 Guinea Pig Model of Recurrent Genital HSV-2 Infection: cleared by day 15. To determine the effect of treatment on The guinea pig model of genital herpes is unique in that cutaneous viral replication, severity of lesions is scored from after recovery from primary genital infection, animals expe US 8,546,432 B2 147 148 rience spontaneous recurrent genital lesions as well as viral lethal, chronic, generalized infection, which has many simi shedding in the absence of lesions. This allows a candidate larities to human CMV infections. At various times after compound to be evaluated for efficacy in controlling recurrent inoculation, virus can be readily isolated from blood, lung, disease. Female Hartley guinea pigs who have recovered liver, spleen, kidney, urine, intestine, and salivary gland. Virus from symptomatic primary genital infection are randomized replication persists in these target organs for 45-60 days and into treatment groups for anti-viral treatments beginning on in the salivary gland for months. The nature of the chronic day 21 PI and continued for 21 days after. Treatments can be infection allows for evaluating long term or maintenance given orally, topically or systemically. The indices for these therapy. studies include quantification and severity assessment of Severe combined immunodeficient (SCID) mice, which 10 lack functional T and B cells, are extremely sensitive to infec recurrent episodes during treatment and for 21 days following tion with MCMV and are utilized as models for CMV infec cessation of treatment. Additionally, vaginal Swabs are col tions in an immunocompromised host. SCID mice that are lected to evaluate any impact on shedding. Dose, route of inoculated with a range of 1.0-10 pfu of MCMV, and are left administration are individualized for each experimental untreated, eventually die in a dose dependent manner. Ani agent. Treatment group size is typically 10-15 animals, and 15 mals that receive 10 pfu have a mean day of death of about 14 duration of treatment is typically 21 days. days, whereas, those inoculated with 10 pful survive an aver Model of Neonatal HSV-2 Infection in Guinea Pigs: age of 25 days. With each log 10 increase in virus inoculum, An exemplary model of neonatal HSV infection mimics survival time is decreased by about three days. To determine the natural history of infection in the human newborn. This the pathogenesis of MCMV in SCID mice, mice are inocu model is available to evaluate candidate anti-viral compounds lated with 10 pfu. On each of various days post infection, and combined therapeutic approaches including combination three mice are euthanized, their tissues removed, homog of anti-virals or anti-virals and immune modulators. Addi enized, and assayed for MCMV. Virus is first detected in tionally, this model can be used to evaluate the efficacy of salivary gland by day six followed by lung, spleen, kidney, candidate vaccines by measuring the protection afforded by adrenals, and pancreas on days 9-12. Liver, which is one of transplacental antibody. In this model, newborn Hartley 25 the most permissive organs in normal mice, does not exhibit guinea pigs are inoculated intranasally with HSV-2 within 48 detectable virus until day 18. In addition, brain is infected by hours of delivery. Newborn animals are then randomized to day 18. These data indicate that inoculation of SCID mice receive experimental compound, placebo or ACV (control). A with low concentrations of MCMV results in a disseminated positive control of ACV (60 mg/kg/day) BID is typically infection with viral replication in the same target organs as used. Animals are evaluated daily for evidence of cutaneous 30 observed in immunodeficient humans. These animals dem herpetic disease and weight gain as well as pulmonary, CNS onstrate high levels of virus in their tissues for 2-3 weeks, thus symptoms and death. Surviving animal are followed for 45 allowing adequate time to document an anti-viral response in days to assess the effectiveness of therapy on the incidence treated animals compared with placebo animals. and frequency of cutaneous herpetic recurrences. Dose and Human CMV infections can cause a wide range of clinical route of administration are individualized for each experi 35 manifestations, especially in the immunocompromised host. mental agent. Duration of treatment is typically 10 days or Few models exist to study HCMV infection since the virus is O. host-specific, and infection and replication are limited to 6.7.1.2 Cytomegaloviruses human cells. In this regard, a model that involves HCMV Animal Models for Cytomegalovirus Infections: infection offetal human retinal tissue implanted in the eyes of 40 severe combined immunodeficient (SCID) mice can be uti lized. Small fragments of fetal human retinas are implanted into the anterior chamber, and four to six weeks after trans Virus Species Route Disease plantation are inoculated with 2,000 to 10,000 pfu of HCMV. MCMV BALBc mice i.p. Disseminated CMV Animals are euthanized and eyes enucleated at various time acute, chronic 45 points after infection. Eyes are prepared for microscopy by SCID-mice i.p. Disseminated CMV acute sectioning fixed tissue, or are homogenized for detection of HCMV SCID-hu-Ret i.oc. HCMV replication in infectious HCMV by plaque assay. The model has been vali Retinal tissue dated using GCV, CDV, and other anti-viral therapies. In SCID-hu-thy/liv i.im. HCMV replication in thymus, liver tissue addition, this model can also be utilized to study and identify 50 the virulence characteristics of HCMV by examining the growth of various HCMV mutants. Human CMV does not generally infect laboratory animals. The SCID-hu thy/liv implant model can also be used in For this reason, it is necessary to use Surrogate models, that is, compound efficacy studies. In this model, Small fragments of a similar but different virus in its natural host. While there are human fetal thymus and liver are implanted under the kidney cytomegalovirus strains in a number of animal species, two 55 capsule in the SCID mouse. Approximately 12-16 weeks that have been studied include the murine and guinea pig later, implants that are fully vascularized and quite large CMVs. The murine model is predictive of efficacy for anti (10-50% the size of the kidney) are inoculated with 10-10' viral drugs, such as Foscarnet (PFA), ganciclovir (GCV), and pful of HCMV. At various time points after infection, implants cidofovir (CDV) that have been evaluated in humans. are biopsied and homogenized, and HCMV replication is I.p. inoculation of three-week-old BALB/c mice with 60 quantified by plaque assay. As with the SCID-humouse ocu approximately 20x10 pful of MCMV results in an acute, lar model, this model can be useful in determining the efficacy lethal infection with rapid virus replication in the lung, liver, of various anti-viral therapies. spleen, kidney, intestine, salivary gland, and other visceral Immunocompromised GPCMV Model: and glandular tissue. Animals die on approximately days 5-7. This guinea pig model mimics CMV infection of the Since this is a lethal infection, the model can be used for rapid 65 immunocompromised host, a common target population of identification of potential anti-viral compounds. Reduction of cytomegalovirus infections. Young Hartley guinea pigs are the virus inoculum to 10 pful of MCMV results in a non immunoSSuppressed with cylcophosphamide administered 1 US 8,546,432 B2 149 150 and 7 days prior to viral inoculation with ~10 pfusalivary 6.7.2 In Vivo Assays for Influenza Viruses gland passaged guinea pig cytomegalovirus (GPCMV). In a Efficacy: typical experiment, two groups of 12 animals each receive the The influenza animal model consists of an infection of experimental compound or placebo beginning 24 hours after laboratory mice with various strains of influenza A (H1N1, infection. Animals are followed daily for evidence of disease H3N2, H5N1) and B viruses, with the employment of several and death which usually occurs by day 14 as described in parameters to measure disease severity. The parameters Bourne et al., Antiviral Research 2000, 47:103-09. Effects on which can be used include the following: (a) monitoring of viral replication are measured by sacrificing animals and blood oxygen saturation (SaO) levels in live animals at fre quantitating virus in specific organs and blood by Real-Time quent intervals utilizing pulse oximetry; (b) measuring of 10 infectious pulmonary virus titers using in vitro endpoint dilu PCR and/or culture. The amount of compound is typically tion assay of homogenates of lungs taken at designated inter based on an average guinea pig weight of 350-500 g. vals during the infection; (c) assay of the degree of pulmonary Neonatal GPCMV Model: consolidation using lungs taken in as determined both by CMV infection of premature newborns may be life-threat score of lung discoloration and by weight of the lung; (d) ening disease if untreated. The neonatal guinea pig model 15 death of the animal due to viral pneumonia; (e) mean Survival resembles perinatal CMV infection and allows systematic time of the animals; and (f) selected histopathological analy evaluation of anti-viral compounds in a relatively immature sis of lung sections. Where appropriate, studies are conducted host. In this model, newborn Hartley guinea pigs are infected to determine the development of viruses resistant to signifi with -10°pful of salivary gland derived GPCMV 24-48 hours cant anti-viral drugs. after birth. Anti-viral or placebo treatments, administered Toxicity: orally or by intraperitoneal injection are begun at 0-24 hours One or more toxicity determinations are performed on the after infection. Infection results in decreased weight gain and test compounds under evaluation. These determinations mortality as high as 70% due to dissemination to target organs include: (a) lethality; and (b) host weight loss or failure to such as the liver, spleen and brain by day 10 post-infection gain weight. As needed and where applicable, the following (Bravo et al., Antiviral Research 2003, 60:41-49). Animals 25 additional parameters can also be investigated: (a) increase in are followed daily for signs of disease and death. The effects circulating serum levels of glutamic oxalic acid transaminase on viral replication are assessed by sacrificing animals and (SGOT) and pyruvic acid transaminase (SGPT) in the serum comparing viral titers in various target organs and blood by as markers for possible liver damage; (b) increase in circulat Real-Time PCR and/or culture. Dosing is typically based on ing creatinine (CT) level as an indicator of possible renal an average newborn guinea pig weight of 100 g. 30 impairment; and (c) increase in circulating creatinine phos Congenital GPCMV Model: phokinase (CK) levels as indicator of general tissue damage. CMV is the most common congenital infection. The guinea 6.7.3. In Vivo Assays for Respiratory Viruses pig is a small mammal in which virus crosses the placenta to 6.7.3.1 RSV, PIV-3, MV and hMPV cause fetal infection and disease, thus allowing the study of Respiratory syncytial virus (RSV), parainfluenza virus new anti-virals and unique therapies that may target placental 35 type 3 (PIV-3), measles virus (MV) and human metapneu and congenital infection. In this model, Hartley pregnant movirus (hMPV) are human pathogens where there is a lack guinea pigs are infected with ~10 pfu GPCMV at approxi of licensed vaccines for preventing illnesses caused by RSV. mately 45 to 55 days of a 70 day gestation. Animals can be PIV-3, or hMPV, although efficacious MV vaccines are avail treated by Systemic or oral routes. Endpoints include preven able. Ribavirin, immune serum globulins and the humanized tion of premature delivery, survival of the offspring and PCR 40 monoclonal antibody have been approved for use against analysis of placenta, and other maternal tissues (blood, liver Some of these paramyxoviruses. However, all of these agents and spleen) and pup organs (liver and spleen) harvested 3, 5 or have limitations and can be expensive. Thus, the elucidation 10 days post infection (Bravo et al., Journal of Infectious and development of new compounds, reagents or vaccines Diseases 2006, 193:591-7). The dose is typically based on a with activity against these viruses are needed. Potential anti pregnant guinea pig weight of about 1200 g. 45 virals and vaccines that can be effective against RSV. PIV-3, CMV Model of Hearing Loss: MV orhMPV are evaluated in cotton rats. In addition, studies Hearing loss is the most common manifestation of con are performed to characterize, enhance or further develop the genital CMV infection. Using direct inoculation of GPCMV different paramyxovirus-cotton rat models. Evidence (~10 pfu) into the cochlea through the round window, hear obtained in numerous studies Support the usefulness of the ing loss can be induced in guinea pigs as measured by ABR. 50 different paramyxovirus-cotton rat models for preclinical Animals can then be treated to prevent the hearing loss. Test evaluation of potential paramyxovirus anti-virals and vac compounds can be administered either systemically, orally cines. and possibly direct intratympanic administration. Dose is 6.7.32 SARS Virus typically based on the weight of the animals, approximately Efficacy: 350 g. 55 The SARS virus animal model utilizes weanling mice Murine CMV Model: infected intranasally with the virus. A moderate lung infec The murine CMV model is used to study CMV pathogen tion is manifested by occasional lung hemorrhaging but pri esis and to evaluate new anti-CMV compounds. In this model, marily by infectious virus recovered from the lungs Inhibition 5-week old female mice are infected with 1x10 pfu of of development of virus in the lungs of the mice is used as MCMV by intraperitoneal injection. Treatment can begin 60 parameters for evaluation of test agents. before or following infection and lasts 3-5 days. Animals are Toxicity: sacrificed at 3 to 5 days after infection and viral titers of the One or more toxicity determinations are performed on the spleen and liver are determined by plaque assay. Other tissues test compounds under evaluation. These determinations are: Such as salivary gland and lungs can be analyzed as well. (a) lethality; and (b) host weight loss or failure to gain weight. Ganciclovir (50 mg/kg, twice daily) serves as a control drug 65 As needed and where applicable, the following additional and inhibits MCMV replication in this model. Dosing parameters can also be investigated: (a) increase in circulat depends on the weight of the animals, typically about 25 g. ing serum levels of glutamic oxalic acid transaminase US 8,546,432 B2 151 152 (SGOT) and pyruvic acid transaminase (SGPT) in the serum 6.7.4.2 Ectromelia (Mousepox Assay) as markers for possible liver damage; (b) increase in circulat Ectromelia virus is the causative agent of mousepox, an ing creatinine (CT) level as indicator of possible renal impair acute exanthematous disease of mouse colonies in Europe, ment; and (c) increase in circulating creatinine phosphoki Japan, China, and the USA. Laboratory studies have shown nase (CK) levels as indicator of general tissue damage. ECTV to have a very narrow host range, infecting only certain 6.7.4. In Vivo Assays for Orthopoxviruses mouse species. A number of different strains of ECTV have 6.7.4.1 Vaccinia and Cowpox Viruses (Smallpox Assay) been isolated which have been shown to differ in their viru The Smallpox animal model is an intranasal infection of lence for the mouse. The Moscow, Hampstead, and NIH79 laboratory mice by the cowpox and vaccinia viruses, which strains have been studied, with the Moscow strain being one induce an infection of the nose and lungs resulting in a small 10 of the most infectious and virulent for the mouse. Studies in pox-like toxemia-associated death. Parameters used in evalu the last five decades have resulted in a detailed description of ating test compounds in this model include: (a) death of the the Virologic and pathologic disease course in genetically animal; (b) mean Survival time of the animals; (c) lung and susceptible (A, BALB?c. DBA/2, and C3H/He; death -7 days nose virus titers; and (d) host weight loss. Other parameters 15 post-infection) and resistant (C57BL/6 and AKR) inbred and can include: (a) monitoring SaO levels; (b) assay of degree of out-bred mice; identification and characterization of the pulmonary consolidation both by lung score and lung weight important cell-mediated and innate responses for recovery increase; and (c) histopathological analysis of lungs and other from infection; and the discovery of rmp-1, rmp-2, rmp-3 and Organs. rmp-4 loci which govern resistance to severe mousepox. Also utilized is a cutaneous infection in immunocompro Varying mouse genotype, virus strain, and dose of virus result mised hairless mice that can be induced by vaccinia virus. in distinct disease patterns for a given route of infection. This infection is progressive and leads to the death of the Mousepox differs from smallpox in at least two features mice. It is now also being used in selected anti-viral experi following a respiratory tract infection. First, the disease ments. Parameters used in evaluating test agents in this cuta course in mousepox is shorter as compared to Smallpox. The neous infection model include: (a) death of the animal; (b) 25 eclipse period in mousepox and Smallpox are 6 and 10 days, severity score in initially induced lesions; (c) size of initially respectively. Fatal cases of mousepox usually occur 7 to 14 induced lesions; (d) number of spontaneous "satellite' days post-infection (p. i.), whereas deaths in ordinary Small lesions; and (e) virus titer in various organs in the animal. pox occur from ~18 to 22 days p.i. Second, the major lesions Animal Models for Vaccinia and Cowpox Virus Infections: in mousepox are observed in the liver and spleen, whereas 30 these organs are relatively uninvolved in Smallpox. A feature of mousepox that is similar to smallpox is the relatively small Virus Species Route Disease dose of virus that is required to initiate disease in the upper and lower respiratory tract. Another similarity is the detection Cowpox Virus (BR) BALBic Mice i.p. Death - Rapid of virus in respiratory gases during the preexanthem period. Liver-Visceral 35 nvolvement Additionally, both diseases present with a characteristic exan BALBic Mice in. Death - Slower thematous rash. In the case of mousepox, the development of Lung-respiratory rash is dependent on a number of parameters including mouse involvement Vaccinia Virus (WR) SKH-1 mice i.d. Skin lesions strain, virus strain, route of inoculation and virus dose. SCID Mice i.p. Disseminated disease 40 Efficacy: BALBic Mice in. Death An important use of the mousepox model is the evaluation Disseminated Disease Vaccinia Virus (IHD) BALBic Mice in. Death of orthopoxvirus compounds and vaccines. The ECTV aero Disseminated Disease Sol model provides a broad dynamic range for evaluating Vaccinia Virus (WR) SCID Mice i.p. Death compounds. An aerosol lethal dose of 100 PFU can be used, Disseminated Disease 45 which is ~3-fold greater that the LDso value of 32 PFU, and is Vaccinia Virus (NYC) SCID Mice i.p. Death likely in the range of the infectious dose for aerosolized smallpox. Alternatively, a dose 1000 to 10,000 times the LDso The causative agent of Smallpox, variola virus, cannot be can be used to fully examine the robustness of the test com utilized outside a BSL-4 containment area and does not cause pound. disease in adult mice. Various orthopoxviruses can be utilized 50 6.7.4.3 Monkeypox Virus (MPXV) as surrogate viruses for smallpox including VV and CV. They Animal assays for monkeypox virus (MPXV) can be per can be inoculated i.p. ori.n. into SCID mice with an endpoint formed by following the procedures described in, e.g., of death. In normal mice, CV, VV-WR, or VV-IHD, but not Americo et al., Journal of Virology, 2010, 84(16): 8172-8180. VV-Copenhagen Strain, will produce mortality when inocu Generally, the assay is based on an intranasal or intraperito lated by variety of routes. Intranasal inoculation of mice with 55 nial infection of CAST/Ei.J mice with MPXV, for example, an CV produced an infection with features similar to systemic or isolate of Congo Basin Glade of MPXV or West African disseminated Smallpox. Other routes of inoculation Such as Glade of MPXV. Upon infection, the animals exhibit loss of i.p. or i.v. with VV or CV result in less bronchial involvement weight, morbidity and death in a dose dependent manner. In and more skin lesions. The IHD strain of VV is less virulent in addition, MPX replication is observed in the lung, spleen and BALB/c mice than the WR Strain. The WR Strain of VV 60 liver of the tested animals. produces mortality in BALB/c mice by i.n. inoculation and Consequently, anti-viral efficacy of the test compounds can SCID mice by i.p. inoculation. SKH-1 hairless mice can also be assessed by following the criteria Such as weight loss, be inoculated with VV and CV by inoculation of abraded morbidity and death in the presence and absence of the test orofacial areas, similar to the HSV techniques. Mice can be compounds upon infection with MPXV. Further, replication treated Systemically or topically with anti-viral compounds 65 in organs such as lung, spleen and liver of the animals in the for evaluation of efficacy against disease (lesion scores) or presence and absence of the test compounds upon infection viral replication (viral titers). can also be examined to assess the anti-viral efficacy. US 8,546,432 B2 153 154 6.7.4.4 Rabbitpox Virus (RPV) visible papillomas to be assessed, and is a clinically relevant Animal assays for rabbitpox virus (RPV) can be performed situation. Treatment is once weekly (Group B), 3 times by following the procedures described in, e.g., Rice et al., weekly (group C MWF), and 5 times weekly (MTWTF), Viruses, 2011, 3:63-82, and Adams et al., J. Virol., 2007, for eight weeks with a dose of 0.1 ml per site. Alternatively, 81:11084-11095 (). Generally, the model is based on bilat treatments may begin 14 days after infection at a time when eral, intrademal infection of New Zealand White rabbits with there are no visible papillomas to maximize the effectiveness 100-1000 pfu of RPV. Upon infection, the animals exhibit of the treatments. Body weights are taken weekly, and sera weight loss, elevated body temperature (fever), severe respi collected at the end of the treatment period for blood chem ratory distress, Swelling of primary and secondary lesions, istries as needed. Papillomas are measured weekly in 3 axes eye and nasal discharge and inoculation site necrosis. In addi 10 (lengthxwidth:xheight) in mm. Data are entered into a spread tion, viral replication is observed in the respiratory tract. If sheet and calculations conducted of the geometric mean untreated, the animals are eventually subjected to death (eu diameter of each papilloma, meantSEM for each group, t-test thanasia) according to euthanasia guidelines. between each paired groups and plots made of papilloma size Consequently, anti-viral efficacy of the test compounds can VS time. Plots of weight changes are also conducted. Atter be assessed by following the criteria described above, includ 15 mination, kidney and liversamples are retrieved for histology ing length of Survival upon injection and viral replication. In and toxicity assessment. Skin/papilloma sites are monitored addition, an overall clinical score can be examined to assess photographically and biopsies assessed for histology at the anti-viral efficacy. experiment/treatment termination. Serum samples can be Alternatively, animal assays can be based on the proce collected to conduct blood chemistries to assess any toxicities dures described, e.g., in Roy et al., Viruses, 2010, 2:2096 of the compound under treatment. 2107, in which similar clinical criteria are examined follow 6.7.5.2 Mouse Xenograft Model ing an infection through aerosol containing RPV. In this Subcutanoues and cutaneous mouse Xenograft models are model, experimental infection with RPV initiates with expo schematically described in FIGS. 1 and 2. sure to aerosols with a particle size distribution that is pref. 6.7.6 In Vivo Assays for Other Viruses erential for penetration to the tracheobronchial and pulmo 25 6.7.6.1 Punta Toro Virus nary regions of the lung, with emphasis on the lower Efficacy: respiratory tract. The Punta Toro virus infection is achieved in C57BL/6 6.7.5 In Vivo Assays for Papillomaviruses mice and in Syrian golden hamsters, with a generalized dis 6.7.5.1 Cottontail Rabbit Papillomavirus (CRPV) Model ease resembling that induced by Rift Valley fever. Parameters The procedures substantially similar to those described, for 30 used for anti-viral testing include: (a) death of the animal; (b) example, in Christensen, Antiviral Chemistry & Chemo hepatic icterus, seen as yellowed liver; (c) elevated ALT levels therapy, 2005, 16:283-294 are followed in connection with in serum; (d) virus titers in liver and serum; and (e) host cottontail rabbit papillomavirus (CRPV) model. In short, weight loss. topical formulations of the test compound are tested at three Toxicity: doses in groups of 5 rabbits at 4 sites per rabbit. One addi 35 One or more toxicity determinations are performed on the tional rabbit group includes a placebo treatment. Alternative test compounds under evaluation. These determinations are: deliveries include interalesional and systemic treatments (a) lethality; and (b) host weight loss or failure to gain weight. depending on the nature of the compound to be tested (e.g., As needed and where applicable, the following additional anti-viral, immunomodulator). parameters can also be investigated: (a) increase in circulat Adult New Zealand White rabbits can be purchased from, 40 ing serum levels of glutamic oxalic acid transaminase for example, CoVance, Inc. Rabbits are of both genders. (SGOT) and pyruvic acid transaminase (SGPT) in the serum Rabbits are quarantined and cleared (14 days). Each rabbit is as markers for possible liver damage; (b) increase in circulat inoculated with 10°wtCRPV (4 sites: 2 on the left side of the ing creatinine (CT) level as indicator of possible renal impair back (L1 and L2) and 2 on the right side of the back (R1 and ment; and (c) increase in circulating creatinine phosphoki R2)) CRPV stock. Combinations of L1, R1, L2 and R2 sites 45 nase (CK) levels as indicator of general tissue damage. receive treatments. Exemplary treatment schemes are pro 6.7.6.2 Pichinde Virus vided below. Efficacy: Group A: all 4 sites placebo ointment; The Pichinde virus model utilizes Syrian goldenhamsters. Group B: L1 and L2=GS327422 (0.1%); R1 and Parameters used for anti-viral testing include: (a) death of the R2=GS327422 (0.03%) 50 animal; (b) Virus titers in brain, liver, spleen and serum; and Treatments are once/week (Monday) for 8 weeks; (c) elevated ALT levels in serum. Group C: L1 and L2=GS327422 (0.1%); R1 and Toxicity: R2=GS327422 (0.03%) One or more toxicity determinations are performed on the Treatments are three times/week (MWF) for 8 weeks; test Substances under evaluation. These determinations are: and 55 (a) lethality; and (b) host weight loss or failure to gain weight. Group D: L1 and L2=GS327422 (0.1%); R1 and As needed and where applicable, the following additional R2=GS327422 (0.03%) parameters can also be investigated: (a) increase in circulat Treatments are five times/week (MTWTF) for 8 weeks. ing serum levels of glutamic oxalic acid transaminase The experiment contains 20 rabbits. Most experiments (SGOT) and pyruvic acid transaminase (SGPT) in the serum include 4-5 groups of rabbits (Groups A-E). A placebo group 60 as markers for possible liver damage; (b) increase in circulat serves as controls to assess local effects of treatment in treated ing creatinine (CT) level as indicator of possible renal impair Groups B to D. Vehicle consists of placebo. Groups B-D ment; and (c) increase in circulating creatinine phosphoki represents test compound comparisons VS placebo negative nase (CK) levels as indicator of general tissue damage. control. Doses of compounds are chosen based on various 6.7.6.3 VEE Virus criteria including the past experience. Treatments (topical) 65 Efficacy: The VEE virus animal model utilizes the TC-83 beginata time when the papillomas are visible but not greater vaccine strain of virus administered intranasally to C3H/Hen than a GMD of 5.0 mm. This time point allows effects on mice; the virus progresses to the central nervous system caus US 8,546,432 B2 155 156 ing high virus titers in the brain and death of the animal. The and rate can be monitored in IFN deficient mice immunized Semliki Forest virus model is very similar to that for the Banzi with test vaccine to assess the efficacy of a test vaccine in virus, with the same disease parameters. The Semliki Forest vivo. virus is a BSL-3-rated pathogen which requires special han 6.7.7 In Vivo Assays for Prion Diseases dling. Parameters for evaluation include: (a) death of the Efficacy: animal; (b) prolongation in mean day to death; (c) virus titers The prion transgenic mouse model utilizes knockout mice in the brains; and (d) host weight loss. for endogenous mouse PrP-sen. These mice express high Toxicity: levels of hamster PrP-sen in a wide range of tissues, including One or more toxicity determinations are performed on the the brain. The animals infected with hamster Scrapie agent test Substances under evaluation. These determinations are: 10 replace the Syrian hamster model. The latter animals require approximately 120 days to die of the scrapie infection, (a) lethality; and (b) host weight loss or failure to gain weight. whereas the prion transgenic mice die in approximately 82 As needed and where applicable, the following additional days when infected with the same agent. Death is used as the parameters can also be investigated: (a) increase in circulat parameter for anti-prion evaluation. ing serum levels of glutamic oxalic acid transaminase 15 Toxicity: (SGOT) and pyruvic acid transaminase (SGPT) in the serum One or more toxicity determinations are performed on the as markers for possible liver damage; (b) increase in circulat test Substances under evaluation. These determinations are: ing creatinine (CT) level as indicator of possible renal impair (a) lethality; and (b) host weight loss or failure to gain weight. ment; and (c) increase in circulating creatinine phosphoki As needed and where applicable, the following additional nase (CK) levels as indicator of general tissue damage. parameters can also be investigated: (a) increase in circulat 6.7.6.4 West Nile Virus ing serum levels of glutamic oxalic acid transaminase Efficacy: (SGOT) and pyruvic acid transaminase (SGPT) in the serum The West Nile virus animal model currently utilizes both as markers for possible liver damage; (b) increase in circulat mice and hamsters. In each, neurological signs are produced, ing creatinine (CT) level as indicator of possible renal impair leading to eventual death of the animals. This virus is a 25 ment; and (c) increase in circulating creatinine phosphoki BSL-3-rated pathogen which is recovered from various tis nase (CK) levels as indicator of general tissue damage. Sues. Other parameters such as functional abilities are also 6.7.8 Other Follow-Up Tests reviewed. Disease parameters used for anti-viral evaluation Follow-up determinations of promising anti-virals seen in include: (a) death of the animal; (b) prolongation on mean day the original animal studies can include effect of the adminis to death; (c) virus titers in brain and other tissues; and (d) host 30 tered test compounds on key immunologic components in weight loss. infected and in uninfected (toxicity control) mice. The immu Toxicity: nologic effects studied include: (a) cytotoxic T lymphocyte One or more toxicity determinations are performed on the activity; (b) natural killer cell activity; (c) total T, T-helper, test Substances under evaluation. These determinations are: T-Suppressor/cytotoxic and B cellenymeration; (d) response (a) lethality; and (b) host weight loss or failure to gain weight. 35 to the T-cell mitogen phytohemagglutinin (PHA); (e) produc As needed and where applicable, the following additional tion of interferon; and (f) production of neutralizing antibody. parameters can also be investigated: (a) increase in circulat Where appropriate, studies are conducted to determine the ing serum levels of glutamic oxalic acid transaminase development of viruses resistant to significant anti-viral (SGOT) and pyruvic acid transaminase (SGPT) in the serum drugs. as markers for possible liver damage; (b) increase in circulat 40 ing creatinine (CT) level as indicator of possible renal impair 7. Assays for ELOVL ment; and (c) increase in circulating creatinine phosphoki nase (CK) levels as indicator of general tissue damage. ELOVL assays can be conducted in vitro using procedures 6.7.6.5 Dengue Virus substantially similar to those described in, for example, Shi In vivo assays for DENV can be conducted using proce 45 mamura et al., European Journal of Pharmacology, 2010, dures substantially similar to those described, for example, in 630: 34-41. Guabiraba et al., PLoS ONE, 2010, 5(12):e15680 and Souza 7.1 In Vitro Assays et al., Proc. Natl. Acad. Sci., 2009, 106(33):14138-14143. 7.1.1 Elongation Enzyme Assay DENV virus stock solutions are diluted in endotoxin-free Elongation is carried out using 30 ul of Substrate reaction PBS or DPBS to appropriate concentrations. The virus is 50 mixture containing 100 mM potassium phosphate buffer (pH injected i.p. into mice. Test compounds are given via appro 6.5), 200 uM BSA (fatty acid free), 500 uM NADPH, 1 uM priate routes at appropriate dosing frequency (e.g., twice a rotenone, 20 uM malonyl-CoA, 833 kBq/ml (''CImalonyl day oral administration). Lethality rates are evaluated every CoA (GH Healthcare Science, Little Chalfont, UK) and acyl 12 hours and other parameters (body weight loss, inflamma CoA. The following long chain acyl-CoAS are used as a tion, etc.) are checked as appropriate. For tests using knock 55 preferential substrate for each ELOVL: ELOVL1, 10 uM out mice, the control typically includes the same test on the stearoyl-CoA: ELOVL2, 10 uM arachidonoyl-CoA: wild-type mice. Negative control usually involves the admin ELOVL3, 10 uM stearoyl-CoA: ELOVL5, 40 uMarachi istration of vehicle instead of test compound. donoyl-CoA; and ELOVL6, 40 uM palmitoyl-CoA. To start In the case of evaluation of vaccines for DENV virus, the reaction, 20 ul of the ELOVL microsomal fraction is assays can be conducted using procedures similar to those 60 added to the substrate mixture, and then incubated for 1 hour described, for example, in Johnson et al., Journal of Virology, at 37° C. with gentle shaking in a 96-well plate. After 1 h 1999, 73(1):783-786. The assay uses IFN deficient mice (e.g., incubation, 100 ul of 5 M HCl is added to hydrolyze acyl A129 mice, which lack alpha/bea IFN and gamma IFN recep CoA, and then the reaction mixture is filtered through a Uni tor genes) and involves intraperitoneal administration of filter-96, GF/C plate (PerkinElmer, Waltham, Mass.) using a DENV into such mice. Typically, IFN deficient mice are 65 FilterMate cell harvester (PerkinElmer, Waltham, Mass.). universally lethal upon administration of DENV regardless of The 96-well GF/C filter plate is subsequently washed with age. Based on this, criteria Such as changes in Survival time distilled water to remove excess 'C malonyl-CoA and US 8,546,432 B2 157 158 dried, after which 25ul of MICROSCINT 0 is added to each chloroform phase is transferred to a new glass tube containing well and radioactivity determined. 1 ml of distilled water and then 3 ml chloroform is added. The 7.1.2 Fatty Acid Elongation Assay in Mouse Hepatocytes lower phase is collected after centrifugation and evaporated to Mouse hepatoma H2.35 cells are grown on 24-well plates dryness. Extracts are dissolved in 2-propanol and the triglyc in Dulbecco's modified Eagle's medium (DMEM) (Invitro eride concentration is measured enzymatically (Determiner gen, Carlsbad, Calif.) supplemented with 200 nM dexametha TGII, Kyowa Medex, Tokyo Japan). For hepatic fatty acid son and 4% heat-inactivated fetal bovine serum (FBS) at 33° composition, the liver samples are incubated in 100-fold vol C. under 5% CO in a humidified incubator. The test com ume (w/v) of 5 MNaOH/ethanol (1:1) at 60° C., pound is dissolved in medium and incubated with subconflu After 2 hour incubation, 500 ul of 5 MC17:0 (internal ent H2.35 cells for 1 hour at 33° C. 1-Cpalmitic acid 10 (PerkinElmer Japan, Kanagawa, Japan) is added to each well standard) are added to all hydrolysates. The fatty acid com to a final concentration of 0.8 uCi/ml to detect elongase positions are analyzed as following. The fatty acids in the activity. After 4 hours of incubation at 33° C., the culture tissue hydrolysate are derivatized with 2-nitrophenylhydra medium is removed, and the labeled cells are washed with zine (2-NPH), and these derivatives are purified using an chilled PBS (3x0.5 ml) and dissolved in 250 ul of 2M sodium 15 Oasis FILE column. An aliquot (10 ul) of the eluate is injected hydroxide. The cell lysate is incubated at 70° C. for 1 hour to into the HPLC apparatus for analysis. HPLC analysis is per hydrolyze radiolabeled cellular lipids. After acidification formed with a Shimazu 10AVp system (Kyoto, Japan), with 100 ul of 5M HCl, fatty acids are extracted with 300 ul of equipped with a UV detector (SPD-10AVp), two pumps (LC acetonitrile. Radiolabeled palmitic acid (16:0), palmitoleic 10ADVp), an auto-sampler (SIL-10ADVp), and a column acid (16:1), stearic acid (18:0), and vaccenic acid:oleic acid oven (CTO-10ACvp). The mobile phase consist of CHCN (18:1) are quantified by reversed-phase radio-HPLC (RI water (80:20, flow rate: 0.6 ml/min). The separation is per HPLC). The identity of the labeled fatty acids is determined formed with a CAPCELL PAK C18 MGII (2.0 mm i.d.x150 by comparing the retention times with known fatty acid stan mm, 5um) at 35° C. and the UV absorbance is subsequently dards. Elongation activity was monitored as the elongation measured at 400 nm. The elongation index represents the ratio index (EI) which was the ratio of radiolabeled C18 (C18:0+ 25 of C18 (C18:0+C18:1) to C16 (C16:0+C16:1) which is quan C18:1) to C16 (C16:0+C 16:1) estimated from each peak area tified from each fatty acid amount. measured by RI-HPLC. 7.2.3 In Vivo Efficacy in KKAy Mice 7.2 In Vivo Assays Male KKAy mice given a regular diet (CE2, CLEAJapan) are orally administered ELOVL6 inhibitor (dissolved in 0.5% 7.2.1 (''CPalmitate Assay in Mouse Liver methylcellulose) twice daily (09:30, 18:30) for 28 days at 30 The in vivo efficacy of ELOVL6 inhibitor is determined by 30 mg/kg dose. At day 21, an intraperitoneal glucose tolerance following the conversion of radiolabeled 116:0 to 16:1, 18:0, test (0.5 kg/gglucose) is performed. At day 28, body compo and 118:1 in mice. Male C57BL/6J mice are orally adminis sition is determined and mice are sacrificed. Plasma param trated with ELOVL6 inhibitor and 1 hour later, the radioactive eters, hepatic triglyceride contents, and fatty acid composi tracer, 1-C palmitic acid, is interperitoneally administered tion are measured as described above. at 10 uCi/body. For time-course study of the pharmacody 35 namic effect, 1-Cpalmitic acid is administered 1, 8 or 12 7.2.4 Pharmacokinetic Studies in Mice hours after administration of test compounds. At 1 hour post Single doses of test compound at 10 mg/kg body weight are dosing of the radioactive precursor, animals are anesthetized administered orally to C57BL/6J mice by gavage in a vehicle with isoflurane (4%) and sacrificed for blood collection from of 0.5% methylcellulose aqueous suspension. Blood samples the vena cava. Liver (50 mg) is harvested and incubated in 40 from the abdominal vein and liver samples are obtained 2 potassium hydroxide/ethanol (2 ml/1.4 ml) at 70° C. for 1 hours after administration. In the case of an in diet regimen, hour. The nonacid-lipid is extracted by 4 ml of petroleum mice are dosed with 100 mg/kg at 17:00 and fed a diet con ether and discarded. Fatty acids are extracted by 2 ml of taining 0.13% test compound overnight. Then mice are sac petroleum ether following saponification by 2 ml of 6 MHC1. rificed the next morning. Blood samples are centrifuged to The ether phase containing the fatty acid fraction is evapo 45 separate the plasma. Liver samples are homogenized with rated under nitrogen gas and reconstituted in methanol to phosphate-buffered saline (pH 7.4). Each sample is depro measure the radioactivity by RI-HPLC. The radioactivity cor teinized with ethanol containing an internal standard. Test responding to each fatty acid is quantified to calculate the EI. compound and the internal standard are detected by liquid 7.2.2 In Vivo Efficacy in Diet-Induced Obesity (DIO) Mice chromatography mass spectrometry/mass spectrometry Mate C57BL/6J mice are maintained on a high-fat diet with 50 (Quattro Ultima mass spectrometer, Waters, Milford, Mass.) ad libitum access to water (D12492, Research. Diets, Inc., in positive ionization mode using an electrospray ionization NJ) for 7 months. Mice are orally administered ELOVL6 probe, and their precursor to production combinations are inhibitor (dissolved in 0.5% methylcellulose) twice daily (09: monitored using the Multiple Reaction Monitoring mode. 30 and 18:30) for 14 days at 30 mg/kg dose. At day 13, body composition is determined and an intraperitoneal glucose 55 tolerance test (0.5 kg/gglucose) is performed. At day 14, mice EXEMPLIFICATION are sacrificed. At 4 hour post-final dosing of ELOVL6 inhibi tor, mice are anesthetized and the liver tissues are immedi The disclosed compounds can be prepared in a number of ately isolated, weighed, frozen in liquid nitrogen and stored at ways well known to one skilled in the art of organic synthesis. -80°C. until use. Plasma is prepared and glucose, insulin and 60 More specifically, disclosed compounds can be prepared leptin are measured using commercially available assay kits using the reactions and techniques described herein. In the (Glucose, KyowaMedex, Tokyo, Japan; leptin and insulin, description of the synthetic methods described below, it is to Morinaga, Tokyo, Japan). Liver tissues are isolated for the be understood that all proposed reaction conditions, includ measurement of triglyceride contents and fatty acid compo ing choice of solvent, reaction atmosphere, reaction tempera sition. For hepatic triglyceride contents, isolated tissues are 65 ture, duration of the experiment and workup procedures, can homogenized in 2 ml distilled water, followed by the addition be chosen to be the conditions standard for that reaction, of 6 ml chloroform/methanol (2:1). After centrifugation, the unless otherwise indicated. It is understood by one skilled in US 8,546,432 B2 159 160 the art of organic synthesis that the functionality present on the reaction is determined to be complete by LCMS or TLC. various portions of the molecule should be compatible with The excess of azide is removed in vacuo and the residue is the reagents and reactions proposed. Substituents not com crystallized from toluene or methanol to provide the desired patible with the reaction conditions will be apparent to one tetrazolone intermediate (C) as a white solid. skilled in the art, and alternate methods are therefore indi (ii) Synthesis of the Amine Intermediate

I NHRC NH2 NO R RI O R R1 O Ré RI O R RI O - SS S-5 S-4 R7 R9 R7 R9 R7 R9 R7 R9 R8 R8 R8 R8 D-1 E D-3 D-2 cated. The starting materials for the examples are either com mercially available or are readily prepared by standard meth Step S-3: ods from known materials. Copper iodide (1 equiv) and cesium carbonate (2.0 equiv) are added to a microwave vial, and the vial is evacuated and SYNTHETIC METHODS filled with argon three times. An aryl iodide (D-1) in dry General Synthesis dimethylformamide (0.6 M), an alkylamine (R'NH) (2.0 25 equiv) and 2-isobutyrylcyclohexanone (0.2 equiv) are then (i) Synthesis of Tetrazolone Intermediate added to the vial, the vial is sealed and the resulting mixture is heated to 100° C. under microwave irradiation for 2 hours, R2 or until the reaction is determined complete by LCMS or R1 TLC. At that time, the vial is cooled to room temperature, and 30 R3 S-1 the reaction mixture is diluted with ethyl acetate and filtered -e- through a pad of Celite with the aid of ethyl acetate. The NH2 filtrate is washed with brine (3x), dried over MgSO, filtered R4 and concentrated in vacuo. The residue is purified by flash R5 chromatography on silica gel (ethyl acetate/hexanes as elut 35 A ant) to provide the desired amine intermediate (E). R2 Step S-4: RI To a mixture of nitro compound (D-2) (1.0 equiv) and iron R 3 S-2 (15.0 equiv) in 1:1 absolute ethanol/dry tetrahydrofuran solu 40 NCO tion (0.8 mL/mmol of ester) is added water (10 ul/ml of solvent). The mixture is then cooled to 0°C. and a solution of R4 R5 concentrated sulfuric acid (4.0 equiv) in water (1.2 ml/mmol B of ester) is added dropwise to the mixture. The reaction is R2 warmed to room temperature and stirred for 1 hour, or until R1 45 the reaction is determined complete by LCMS or TLC. The O reaction is then filtered through a pad of Celite(R) with the aid R3 -l of ethyl acetate. The filtrate is diluted with brine, saturated N NH aqueous Sodium bicarbonate solution and additional ethyl 4 V / acetate, the organic and aqueous layers are separated, and the R NRN R5 organic layer is washed with Saturated Sodium bicarbonate C solution, brine, dried over MgSO, filtered and concentrated in vacuo. The residue is purified by flash chromatography on silica gel (ethyl acetate/hexanes as elutant) to provide D-3. Step S-1: A solution of aniline (A) (1.0 equiv) and triethylamine (1.0 55 Step S-5: equiv) in dry dichloroethane (DCE) (0.2 M) under argon is To a stirred solution of D-3 (1.0 equiv) in acetic acid (0.2 treated with triphosgene (0.4 equiv) and heated to reflux for 2 M) is added a carbonyl compound (10.0 equiv of a ketone or hours, or until the reaction is determined to be complete by aldehyde) and sodium borohydride (10.0 equiv), and the LCMS or TLC. The reaction is then cooled to room tempera 60 resulting mixture is stirred at room temperature for 1 hour, or ture, diluted with dichloromethane, washed with 1N HCl (aq) until the reaction is determined complete by LCMS or TLC. and brine, dried over MgSO, and then filtered and concen The reaction is then diluted with ethyl acetate, and washed trated in vacuo. The desired isocyanate (B) is used in the next with Saturated aqueous Sodium bicarbonate Solution (5x) and step without purification. brine (2x), and the organic layer is dried over MgSO filtered Step S-2: 65 and concentrated in vacuo. The residue is purified by flash A mixture of the isocyanate (B) (1.0 equiv) and trimethyl chromatography on silica gel (ethyl acetate/hexanes) to pro silyl azide (2.0 equiv) is heated to reflux for 24 hours, or until vide the desired amine intermediate (E). US 8,546,432 B2 161 162 (iii) Coupling of Tetrazolone Intermediate and Amine Step S-6: Intermediate To a solution of amine intermediate (E) (1.3 equiv) in dry tetrahydrofuran (0.5 M) under argon is added triethylamine (1.3 equiv) followed by triphosgene (0.7 equiv). The resulting NHRC 5 heterogeneous mixture is stirred at room temperature for 15 minutes, treated with dimethylaminopyridine (DMAP) (1.0 equiv) and the tetrazolone intermediate (C) (1.0 equiv), diluted with dry tetrahydrofuran (to 0.25M final concentra tion with respect to amine) and heated to reflux for 1 hour, or R7 R9 '' until the reaction is determined to be complete by LCMS or 8 TLC. The reaction mixture is cooled to room temperature and R diluted with ethyl acetate and brine, and the aqueous and E organic layers are separated. The organic layer is washed with R2 brine, 10% aqueous HCl, then brine, and dried over MgSO, RI O filtered and concentrated in vacuo. The residue is purified by R3 l S-6 flash chromatography on silica gel (ethyl acetate/hexanes) to -S-S-e- provide the desired tetrazolone product (F). N NH (iv) Scope of the General Synthetic Method R4 \= The general synthetic method is not intended to be limited R5 '' to the coupling of amine intermediates such as (E) with phe C nyl tetrazolone intermediates such as (C) in the formation of R2 compounds provided herein. For example, other amines and RI R7 tetraZolones have been coupled using the general methods 3 O O R6 described above to provide a wide variety of tetrazolone R -l R8 ' compounds, e.g., such as the tetrazolone compounds pro N - vided in Tables 1-4.

R4 \NFN 1 N R5 RC R9 R10 30 F Exemplary Syntheses of Compounds (i) Synthesis of Tetrazolone Compounds 7 and 8

NH2 NCO O

F F -e- r F --- N l NH W f NFN 1 2 F

O O

N.-l N. - OMe W f N US 8,546,432 B2 163 164 Compound 3: tered and concentrated in vacuo. The residue was purified by A mixture of commercially available 2,6-difluorophenyl flash chromatography on silica gel (ethyl acetate/hexanes) to isocyanate (2) (1.0 equiv) and trimethylsilyl azide (2.0 equiv) provide compound (6). was heated to reflux for 12 hours. The excess of azide was removed in vacuo and the residue was crystallized from tolu ene to provide compound (3) as white needles. Compound 7: To a solution of compound (6) (1.5 equiv) in dry dichlo romethane (0.5 M) under argon was added triethylamine (3.0 Compound 5: equiv) followed by triphosgene (1.5 equiv). The resulting To a solution of commercially available 5-iodo-2-meth 10 heterogeneous mixture was stirred at room temperature for 15 oxybenzoic acid (4) (1.0 equiv) in dry dimethylformamide minutes, concentrated in vacuo, and the residue was taken up (DMF) (0.5 M) under argon was added solid potassium car in dry toluene (0.25M with respect to amine) and treated with bonate (1.5 equiv) followed by benzyl bromide (1.1 equiv), dimethylaminopyridine (DMAP) (1.0 equiv) and the com and the resulting mixture was stirred at room temperature for pound (3) (1.0 equiv). The resulting mixture was heated to 4 hours, acidified with 10% HCl, and diluted with brine and 15 reflux for 1 hour, cooled to room temperature and diluted with ethyl acetate. The aqueous and organic layers were separated ethyl acetate and brine, and the aqueous and organic layers and the organic layer was washed with brine (5x), dried over were separated. The organic layer was washed with brine, MgSO filtered and concentrated in vacuo to afford an oil, 10% aqueous HCl, then brine, and dried over MgSO filtered compound (5), which was used without further purification. and concentrated in vacuo. The residue was purified by flash chromatography on silica gel (ethyl acetate/hexanes) to pro vide compound (7) as a white foam. Compound 6: Copper iodide (1 equiv) and cesium carbonate (2.0 equiv) were added to a microwave vial and the vial was evacuated 25 Compound 8: and filled with argon three times. Compound (5) in dry dim To a solution of compound 7 (1.0 equiv) in 2:1 methanol/ ethylformamide (0.6 M), isopropylamine (2.0 equiv) and THF (0.1M) under argon was added 5% Pd/C (0.1 equiv) and 2-isobutyrylcyclohexanone (0.2 equiv) were then added to the inert atmosphere was replaced with hydrogen. The result the Vial, the vial was sealed and the resulting mixture was ing mixture was stirred at room temperature for 30 minutes, heated to 100° C. under microwave irradiation for 2 hours. At 30 filtered through a pad of Celite with the aid of methanol and that time, the vial was cooled to room temperature and the the resulting filtrate was concentrated in vacuo. The residue reaction mixture was diluted with ethyl acetate and filtered was purified by preparative HPLC (0.1% formic acid inaceto through a pad of Celite(R) with the aid of ethyl acetate. The nitrile/water) to provide compound (8) as a white solid. filtrate was washed with brine (3x), dried over MgSO fil (ii) Synthesis of Tetrazolone Compounds 15 and 16

I I I HN MeO MeO

He- He- Her o MeO HO Clu Clu O OMe O OMe O OMe O OMe 9 10 11 12

S-N S-N 13 14 US 8,546,432 B2 165 166 -continued

ls-s-s-s ls-s-s-s VS-N \NRN i? re- \S-N l \-NRN

HO MeO-( )- O O OMe O OMe 16 15

Compound 10: cipitate was collected via vacuum filtration and washed with To a stirred solution of commercially available compound cold toluene to afford compound (14) as a yellow solid. (9) (1.0 equiv) in 1:1 THF:HO (0.6M) under nitrogen was Compound 15: added LiOH (5.0 equiv) at room temperature. The reaction 25 was heated to 80°C. for two hours. The resulting solution was To a stirred solution of compound (12) (1.2 equiv) in cooled to room temperature and the volume was reduced by dichloromethane (0.5M) at 0°C. was added triethylamine rotary evaporation. The resulting Solution was acidified with (3.0 equiv) and triphosgene (1.0 equiv). The ice bath was 2N HCl to pH=1, extracted with ethyl acetate, dried with removed and the solution was stirred at room temperature for Sodium Sulfate, and concentrated via rotary evaporation to 30 15 minutes followed by concentrated by rotary evaporation. provide compound (10) which was advanced without purifi The resulting foam was dissolved in toluene (0.4M) followed cation. by addition of compound (14) (1.0 equiv) and dimethylami Compound 11: nopyridine (1.0 equiv). The resulting Suspension was heated at 70° C. for sixteen hours. The solution was cooled to room To a stirred solution of (10) (1.0 equiv) in 20:1 acetone: 35 DMF (0.2M) under nitrogen was added potassium carbonate temperature, diluted with ethyl acetate, and washed with (1.5 equiv), p-methoxybenzyl chloride (1.0 equiv), and water. The organic layer was dried with sodium sulfate and concentrated via rotary evaporation. The resulting oil was Sodium iodide (catalytic) at room temperature. The resulting purified by silica gel column chromatography (ethyl acetate/ mixture was heated to 70° C. for six hours. The resulting hexanes) to afford compound (15) as a white solid. Solution was cooled to room temperature and the Volume was 40 reduced by rotary evaporation. The resulting solution was Compound 16: diluted with hexanes and ethyl acetate, washed with water, To a stirred solution of compound (15) (1.0 equiv) in dried with sodium sulfate and concentrated via rotary evapo CHCl (0.1M) under nitrogen was added trifluoroacetic acid ration. The resulting oil was purified by silica gel column (10.0 equiv) followed by anisole (1.0 equiv) at room tempera chromatography (hexanes to 25% ethyl acetate/hexanes) to 45 ture. The reaction was run at room temperature for ten min afford compound (11) as an oil. utes. The reaction was diluted with dichloromethane and Compound 12: neutralized with aqueous Sodium bicarbonate Solution. The To a stirred solution of compound (11) (1.0 equiv) in DMF organic layer was dried with sodium Sulfate and concentrated (0.12M) at room temperature in a microwave vial was added via rotary evaporation. The resulting oil was purified by pre cesium carbonate (2.00 equiv), copper iodide (0.1 equiv), 50 parative HPLC (0.1% formic acid in acetonitrile/water) and 2-isobutyrylcyclohexanone (0.2 equiv), and cyclohexy lyophilized to give compound (16) as a white powder. lamine (3.0 equiv). The resulting Suspension was heated in (iii) Synthesis of Tetrazolone Compounds 17 and 18 the microwave at 100° C. for 1 hr. The resulting suspension was cooled to room temperature, diluted with ethyl acetate, 55 washed with dilute aqueous lithium chloride, and washed with water. The organic layer was dried with sodium sulfate and concentrated via rotary evaporation to afford agreen oil which was purified by silica gel column chromatography -e- (hexanes to 1:2 ethyl acetate/hexanes) to afford compound 60 S-NO'C NRN (12) as an oil. Compound 14. HO To a stirred solution of commercially available compound (13) (1.00 equiv) was added azidotrimethylsilane (4.00 equiv). The resulting suspension was heated at 90° C. for six 65 O OMe hours. The hot reaction solution was poured directly into a 16 beaker containing toluene.ice (1:1). The resulting yellow pre US 8,546,432 B2 167 168 -continued Compound 17: O O To a stirred solution of compound (16) (1.0 equiv) in MeOH (0.02M) under nitrogen was added palladium hydrox HN ide on carbon (0.2 equiv) followed by vacuum evacuation and V---, / exposure to hydrogen gas (1 atm, balloon). The reaction was NH, NFN run at room temperature overnight. The resulting solution was filtered thru celite, washed with dichloromethane and con HO centrated via rotary evaporation. The resulting oil was puri fied by preparative HPLC (0.1% formic acid in acetonitrile/ water) and lyophilized to afford compound (17) as a white solid. Compound 18: To a stirred solution of compound (17) was added formic acid (500 equiv). The reaction was heated at 100° C. for 1 hour. After cooling to room temperature, the reaction was diluted with ethyl acetate and the organic layer was washed with water, dried with sodium sulfate, and concentrated via rotary evaporation. The resulting oil was purified by prepara HO tive HPLC (0.1% formic acid in acetonitrile/water) and lyo philized to afford compound (18) as a white solid. Compound OMe (18) is depicted above as one compound, but it exists in 18 equilibrium as a mixture of tautomers as shown below.

O O

=s HN ---, \- \=

HO HO

OMe O OMe 18-tautomer

(iv) Synthesis of Tetrazolone Compounds 24 and 25

NHEt

COOH COOBI 19 2O

Ck

24

C 22 US 8,546,432 B2 169 -continued

Compound 20: was removed in vacuo and the residue was crystallized from To a solution of commercially available 4-oxocyclohexan methanol to provide compound (23) as white needles. ecarboxylic acid (19) (1.0 equiv) in dry DMF (0.5 M) under Compound 24: argon was added solid potassium carbonate (1.5 equiv) fol 15 To a solution of compound (21) (1.3 equiv) in dry tetrahy drofuran (0.5 M) under argon was added triethylamine (1.3 lowed by benzyl bromide (1.1 equiv). The resulting mixture equiv) followed by triphosgene (1.3 equiv). The resulting was stirred at room temperature for 4 hours, acidified with heterogeneous mixture was stirred at room temperature for 15 10% HCl, diluted with brine and ethyl acetate, and the layers minutes diluted with dry tetrahydrofuran (0.25 M with were separated. The organic layer was washed with brine respect to amine) and treated with DMAP (1.0 equiv) and (5x), dried (MgSO), filtered and concentrated in vacuo to compound (23) (1.0 equiv). The resulting mixture was heated afford a solid which was used without further purification. to reflux for 1 hour, cooled to room temperature and diluted Compound 21: with ethyl acetate and brine, and the aqueous and organic A solution of benzyl 4-oxocyclohexanecarboxylate (20) layers were separated. The organic layer was washed with (1.0 equiv) and a 2-Methylamine solution in THF (2.0 equiv) 25 brine, 10% aqueous HCl, then brine, and dried over MgSO, in dry DCE (1.4 M) under argon was treated with sodium filtered and concentrated in vacuo. The residue was purified triacetoxyborohydride (1.5 equiv) and the resulting mixture by flash chromatography on silica gel (ethyl acetate/hexanes) was stirred at rt for 1 h, and quenched with Saturated aqueous to provide compound (24) as a white Solid. Sodium bicarbonate solution. The layers were separated and Compound 25: the aqueous one extracted with DCM (3x). The combined 30 To a solution of compound (24) (1.0 equiv) in methanol organic layers were washed with brine, dried (MgSO), fil (0.1 M) under argon was added 10% Pd(OH) on carbon (0.1 tered and concentrated in vacuo. The residue was purified by equiv) and the inert atmosphere was replaced with hydrogen. flash chromatography on silica gel (ethyl acetate/hexanes) to The resulting mixture was stirred at room temperature for 2 h, provide compound (21) as an oil. filtered through a pad of Celite with the aid of methanol, and Compound 23: 35 the resulting filtrate concentrated in vacuo. The residue was A mixture of commercially available 2,6-dichlorophenyl purified by preparative HPLC (0.1% formic acid in acetoni isocyanate (22) (1.0 equiv) and trimethylsilyl azide (2.0 trile/water) to provide compound (25) as a white solid. equiv) was heated to reflux for 12 hours. The excess of azide (v) Synthesis of Tetrazolone Compounds 27 and 28

NH

NHBoc 26

C C C O -e- NCO s 27 C C N sN NH 22 US 8,546,432 B2 171 172 -continued

C O N N l Y V / N NH NRN C

28

Compound 27: Compound 28: Commercially available tert-butyl piperidin-3-ylcarbam- 20 ate (26) (1.3 equiv) in dry tetrahydrofuran (0.5 M) under Compound (27) (1.0 equiv) was dissolved in dry dichlo argon was added triethylamine (1.3 equiv) followed by triph romethane (0.2 M) at 0° C. and was treated dropwise with osgene (1.3 equiv). The resulting heterogeneous mixture was TFA (3:1 DCM/TFA). The resulting solution was stirred at stirred at room temperature for 15 minutes, diluted with dry room temperature for 1 h and was concentrated in vacuo. The tetrahydrofuran (0.25 M with respect to amine) and treated amine salt was then taken up in dry dichloromethane, cooled with DMAP (1.0 equiv) and compound (23) (1.0 equiv). The 25 to 0°C. and treated with excess of acetylchloride (10.0 equiv) resulting mixture was heated to reflux for 1 hour, cooled to and triethylamine (1.0 equiv). The resulting mixture was room temperature and diluted with ethyl acetate and brine, stirred at 0° C. for 15 min, quenched with 10% HCl and and the aqueous and organic layers are separated. The organic extracted with DCM (2x). The combined organic extracts layer was washed with brine, 10% aqueous HCl, then brine, and dried over MgSO filtered and concentrated in vacuo. 30 were washed with brine, dried (MgSO), filtered and concen The residue was used without further purification. An ana trated in vacuo. The residue was purified by preparative lytical sample was obtained by preparative HPLC (0.1% for HPLC (0.1% formic acid in acetonitrile/water) to provide mic acid in acetonitrile/water) to provide compound (27) as a compound (28) as a white solid. white solid. (vi) Synthesis of Tetrazolone Compounds 32 and 33

F O F F F F l -e- --- N NH W f NFN 1 2 F 3

COBn NH2 HN HN s N rN Na2 N-2 N-2

O1 OH O1 OH O1 OB 29 30 31

CO2H

33 US 8,546,432 B2 173 174 Compound 30: hydroxide on carbon (-0.2 equiv). The reaction was evacu To a stirred solution of 5-amino-2-pyridinecarboxylic acid ated and flushed with nitrogen followed by charging with (29) (1.0 equiv) in AcOH (0.9M) under nitrogen was added hydrogen gas. After stirring for one hour, the reaction was cyclohexanone (1.1 equiv) and sodium triacetoxyborohy filtered through celite, washed with dichloromethane, and dride (1.1 equiv) at room temperature. The resulting mixture concentrated by rotary evaporation. The resulting oil was was stirred at room temperature for 72 hours. Methanol was purified by silica gel column chromatography (dichlo added and the resulting solution was concentrated by rotary romethane to 9:1 dichloromethane:methanol) to afford 33 as evaporation. The resulting oil was dissolved in CHCl:IPA a clear oil. (4:1) and water, and the organic layer was concentrated in (vii) Synthesis of Tetrazolone Compounds 40 and 41

NO NO NO F F OPh OPh

He- Hip

O OH O O O O 34 35 36

F O F -l COOH N N \ W f N

41 40 vacuo to provide compound (30) which was advanced with Compound 36: out purification. To a stirred Suspension of commercially available com Compound 31: 40 pound (34) (1.0 equiv) and potassium carbonate (1.5 equiv) in To a stirred solution of compound (30) (1.0 equiv) in 2:1 dry DMF (0.3 M) under nitrogen was added benzyl bromide CHCl:DMSO (0.13M) was added AgO (3.0 equiv) fol (1.2 equiv) at room temperature. The reaction was stirred for lowed by benzyl bromide (1.1 equiv). The reaction was run at 19 hours, additional potassium carbonate (1.5 equiv) was room temperature overnight. The resulting Solution was fil added followed by phenol (1.2 equiv) and stirring was con tered through a celite pad, diluted with CHCl, washed with 45 tinued for 24 h. The resulting suspension was filtered through water, dried with sodium sulfate, and concentrated by rotary a pad of Celite with aid of AcOEt, the filtrate was diluted with evaporation. The resulting oil was purified by silica gel chro additional AcOEt and was washed with brine and 10% HC1. matography (hexanes to 50% ethyl acetate/hexanes) to afford The aqueous layer was back-extracted with AcOEt and the compound (31) as an off-white foam. combined organic layers were washed with brine (3x), dried Compound 32: 50 (MgSO), filtered and concentrated in vacuo. An orange oil To a stirred solution of compound (31) (0.9 equiv) in was obtained, which was purified by flash chromatography dichloromethane (0.5M) at 0C was added triethylamine (3.0 (Hexane/AcOEt 9:1) to render compound 36 as a thick yellow equiv) and triphosgene (1.0 equiv). The ice bath was removed oil (90% yield). and the solution was stirred at room temperature for 15 min utes followed by concentrated by rotary evaporation. The 55 resulting foam was dissolved in toluene (0.4M) followed by Compound 37: addition of compound (3) (1.0 equiv) and dimethylaminopy To a stirred solution of (36) (1.0 equiv) and iron powder ridine (1.0 equiv). The resulting suspension was heated at 90° (15.3 equiv) in 1:6 absolute EtOH/THF (0.3 M) cooled to 0° C. for two hours. The solution was cooled to room tempera C., was dropwise added a solution of HSO (0.6 ml/mmol) in ture, diluted with ethyl acetate, and washed with water. The 60 HO(1.8 ml/mmol). The resulting mixture was stirred at rt for organic layer was dried with sodium Sulfate and concentrated 1 h, filtered through a pad of Celite with the aid of AcOEt, and via rotary evaporation. The resulting oil was purified by silica the filtrate diluted with brine and additional AcOEt. The lay gel column chromatography (hexanes to 50% ethyl acetate/ ers were separated, the organic one was washed with brine, hexanes) to afford compound (32). saturated sodium bicarbonate solution, brine, dried (MgSO), Compound 33: 65 filtered and concentrated in vacuum. Compound (37) was To a stirred solution of (32) (1.00 equiv) in methanol obtained as a pale yellow oil which crystallized upon stand (0.1M) at room temperature was added 20% palladium 1ng. US 8,546,432 B2 175 176 Compound 38: (1.0 equiv). The resulting suspension was heated to reflux for To an stirred suspension of aniline (37) in glacial AcOH 3 h, was then cooled to room temperature, diluted with ethyl (0.3M) was added acetone (5 equiv) followed by sodium acetate, and washed with water, 10% hydrochloric acid solu borohydride (2 equiv). The resulting mixture was stirred at rt tion and brine. The organic layer was dried (MgSO), filtered for 1 h, diluted with AcOEt and washed with brine (3x), and concentrated in vacuo. The resulting crude was purified saturated sodium bicarbonate solution (6x, until basic pH) by flash chromatography (Hexane/AcOEt 5:1 to 2:1) to afford and brine (2x). The organic layer was dried (MgSO), filtered compound (40) as a pale yellow foam (66% yield). and concentrated in vacuo. Compound (38) was obtained as Compound 41: an oil after purification by flash chromatography (Hexane/ To a solution of compound (40) (1.0 equiv) in 1:1 metha AcOEt 91 to 3:1) (80% yield over 2 steps). 10 nol/AcOEt (0.3 M) under argon was added 10% Pd/C (0.15 Compound 40: equiv) and the inert atmosphere was replaced with hydrogen. To a stirred solution of compound (38) (1.0 equiv) in The resulting mixture was stirred at room temperature for 30 dichloromethane (0.3 M) at 0°C. was added triethylamine minutes, filtered through a pad of Celite with the aid of (2.0 equiv) and triphosgene (0.7 equiv). The ice bath was methanol and the resulting filtrate was concentrated in vacuo. removed and the solution was stirred at room temperature for 15 The residue was purified by flash chromatography (Hexane/ 15 minutes and was concentrated in vacuo. The resulting AcOEt2:1 to 1:4 and DCM/MeOH 9:1) to provide compound foam was dissolved in toluene (0.3 M) followed by addition (41) as a white foam (58% yield) M+1'=495.1. of compound (39) (1.1 equiv) and dimethylaminopyridine (viii) Synthesis of TetarZolone Compounds 51 and 52

US 8,546,432 B2 179 180 Compound 44: tion and brine. The organic layer was dried (MgSO), filtered Compound (44) was prepared in 64% yield via methylation and concentrated in vacuo. The resulting product was purified of commercially available 4-fluorosalicilic acid (42) followed by flash chromatography (Hexane/AcOEt 9:1 to 2:1) to afford by nitration as described in the literature (Del Corona, L.; compound (51) as a crystalline solid (22% yield). Signorelli, G.; Pinzetta, A.; Coppi, G. Eur: J. Med. Chem. Compound 52: 1993, 28: 419-425). To a solution of compound (51) (1.0 equiv) in 1:1 metha Compound 45: nol/THF (0.1 M) under argon was added 10% Pd/C (0.15 To a solution of compound (44) (1.0 equiv) in dry DMF equiv) and the inert atmosphere was replaced with hydrogen. (0.9M) was added phenol (1.1 equiv) and potassium carbon The resulting mixture was stirred at room temperature for 1 h, ate (1.5 equiv) and the resulting mixture was stirred at rt for 1 10 h, the solid filtered and washed with AcOEt, the filtrate further filtered through a pad of Celite with the aid of methanol and diluted with AcOEtand washed with 10% HCl and brine (5x). the resulting filtrate was concentrated in vacuo. Compound The aqueous layer was back-extracted with AcOEt, which (52) was obtained as a crystalline solid (99% yield). was washed with brine. The combined organic layers were BIOLOGICAL ASSAYS dried (MgSO), filtered and concentrated in vacuo. Com 15 pound (45) was obtained as an oil, which was used directly. Compound 46: Preparation of Human FASN Protein A solution of methyl ester (45) (1.0 equiv) in 2:2:5 MeCH/ Human FASN protein (SEQID NO. 1) was purified from THF/HO (0.3 M) was added LiOH (1.5 equiv). The suspen SKBR3 cells using procedures modified from those in Jaya sion was stirred at 60° C. for 1.5 h. The reaction mixture was kumar et al., PNAS 1995, 92:8695-8699. SKBR3 cells were allowed to cool to rt, diluted with brine and extracted with obtained from ATCC and grown in DMEM high glucose diethyl ether. The aqueous layer was acidified with 10% HCl medium supplemented with 10% FBS, 1 ug/mL bovine pan and extracted with AcOEt (3x). The combined AcOEt layers creas insulin, 100 U/mL penicillin and 100 g/mL strepto were washed with brine, dried (MgSO), filtered and concen mycin. The confluent cells were trypsinized and washed three trated in vacuo. Compound 46 was obtained as a pale yellow 25 times with PBS buffer before frozen in liquid N and stored at foam, which was used directly. -80° C. Frozen cells were thawed on ice and resuspended in Compound 47: lysis buffer (25 mM Tris-HCl, pH 7.0, 15 mM NaCl, 1 mM To a stirred solution of acid (46) (1.0 equiv) and potassium EDTA, and 1 mM DTT) with protease inhibitors. The cells carbonate (1.5 equiv) in dry DMF (0.6M) under nitrogen was were lysed by sonication, and the cell debris was removed by added benzyl bromide (1.1 equiv) at room temperature. The 30 centrifugation at 20,000 rpm for 30 min. To the supernatant, reaction was stirred for 19 hours, filtered through a pad of neutralized saturated ammonium sulfate Solution was added Celite with aid of AcOEt, the filtrate was acidified with 10% to a final concentration of 35%. The solution was left on ice HCl, diluted with brine and extracted with AcOEt (3x). The for 1 hr., and the precipitated proteins were harvested by combined organic extracts were washed with brine, dried centrifugation at 20,000 rpm for 30 min. The proteins were (MgSO), filtered and concentrated in vacuo. The residue 35 (oil) was used directly. redissolved in lysis buffer without NaCl and loaded on a Compound 48: To a stirred solution of (47) (1.0 equiv) in mono Q column. Bound proteins were eluted with a linear 2:1 THF/AcOH (0.6 M) cooled to 0°C., was portionwise gradient of NaCl in lysis buffer. Each fraction was analyzed added zinc powder (10.0 equiv). The resulting mixture was by SDS-PAGE and FASN NADPH consumption assay. The stirred at 0°C. for 1 h, filtered through a pad of Celite with the 40 fractions containing FASN were pooled and concentrated to aid of AcOEt and the filtrate was diluted with brine and 2-3 mg/mL. Glycerol was added to 20%, and the protein was additional AcOEt. The layers were separated, the organic one frozen in liquid N and stored at -80° C. was washed with brine, saturated sodium bicarbonate solu FASN NADPH Consumption Assay tion, brine, dried (MgSO), filtered and concentrated in All chemicals were purchased from Sigma (St. Louis, vacuo. Compound (48) was obtained as a pale yellow oil 45 Mo.). The procedures of NADPH consumption assay were which crystallized upon standing. similar to those described in Cox et al., PNAS 1983, 80:4233 Compound 49: 4237. On a 96-well polypropylene microplate, dilution series To an stirred suspension of aniline (48) in glacial AcOH (typical concentrations 60 nM-1.0 mM) of test compounds (0.3 M) was added cyclohexanone (4.0 equiv) followed by were prepared in DMSO, of which 4.0 uL each was trans sodium borohydride (2.5 equiv). The resulting mixture was 50 ferred to a black polystyrene assay microplate and mixed with stirred at rt for 1 h, diluted with AcOEtand washed with brine 36 ul. FASN assay buffer (50 mM potassium phosphate, pH (3x), saturated sodium bicarbonate solution (6x, until basic 7.0, 1.0 mM EDTA, 0.01% NP-40) plus 5.0 mM fresh DTT. pH) and brine (2x). The organic layer was dried (MgSO), FASN protein (40 uL 150 nMFASN) was added per well, and filtered and concentrated in vacuo. The residue was purified the microplate was incubated at 37°C. for 30 min. Enzyme by flash chromatography (Hexane/AcOEt 9:1) to give com 55 pound (49) as an oil (46% yield over 5 steps). activity measurement was initiated by addition of 20 uL 5x Compound 51: substrate mixture to final concentrations of 60 nM FASN, 2.4 To a stirred solution of compound (49) (1.0 equiv) in nM-40 uM compound, 0.2 mM NADPH, 50 uM butyryl dichloromethane (0.2 M) at 0°C. was added triethylamine CoA, 0.5 mM malonyl-CoA in 100 uL assay buffer plus 5.0 (2.0 equiv) and triphosgene (0.7 equiv). The ice bath was 60 mMDTT and 4.0% DMSO. NADPH consumption was moni removed and the solution was stirred at room temperature for tored kinetically by fluorescence ( 340 nm, w, 460 nm) 15 minutes and was concentrated in vacuo. The resulting on an EnVision 2100 multilabel plate reader (Perkin Elmer, foam was dissolved in toluene (0.2 M) followed by addition Waltham, Mass.). FASN enzyme activity (slope) in the pres of compound (50) (1.1 equiv) and dimethylaminopyridine ence of 4% DMSO was used as maximum control, whereas (1.0 equiv). The resulting suspension was heated to reflux for 65 background (minimum control) was measured by omission of 2 h, was then cooled to room temperature, diluted with ethyl malonyl-CoA in the substrate mixture. Inhibition curves were acetate, and washed with water, 10% hydrochloric acid solu fitted by a logistic function to yield ICso values: US 8,546,432 B2 181 182 less than 15 nM; “B” refers to compounds having an ICs of (Slope-Min) %. Inhibition= 1 - x 100% 15 nM to 100 nM, inclusive: “C” refers to compounds having (Max-Min) an ICs of greater than 100 nM to 200 nM, inclusive: “D’ 100 refers to compounds having an ICso of greater than 200 nM to %. Inhibition= 1 + (ICsof Ilyhill coefficient 5000 nM, inclusive; and “E” refers to compounds having an ICs of greater than 5000 nM. FASN Cellular Flashplate Assay Compounds provided herein were found to inhibit FASN Compound was added to HCT 116 cells 24 hours post activity using this assay. plating (1x106 cells per well, 6-well plates) and incubated Activities obtained by the above-described FASN NADPH 10 for 24 hours (37 C, 5% CO). Cell pellets were harvested and Consumption Assay are designated in Tables 1,2,3 and 4 with washed with PBS. 30 uL FAS buffer minus BGG (50 mM a star (*), wherein “A*” refers to compounds having an ICso KPB, pH 7.0, 1.0 mM EDTA, 0.01% NP-40, 1 mM TCEP) of less than 60 nM; “B” refers to compounds having an ICso was added to the cell pellet, and the cells were lysed by of 60 nM to 250 nM, inclusive: “C” refers to compounds freeze/thaw (3x, liquid nitrogen/42C water bath) before pel having an ICs of greater than 250 nM to 1000 nM, inclusive; 15 “D’ refers to compounds having an ICs of greater than 1000 leting debris (20,000 rcf. 15 minutes, 4 C). Total protein nM to 10,000 nM, inclusive; and “E*” refers to compounds concentration of the lysates were determined (Pierce BCA having an ICs of greater than 10,000 nM. Total Protein assay, BSA standard) and samples were normal FASN Scintillation Proximity Flashplate Assay ized to 1 mg/mL total protein in assay buffer. Acetyl-coenzyme A, malonyl-coenzyme A, NADPH, Assay setup: 50 uL assay buffer was added to each well of bovine gamma globulin, and ORLISTAT were purchased the FlashPlate (PerkinElmer, FlashPlate Plus Phospholipid from Sigma (St. Louis, Mo.). Tris(2-carboxyethyl) phosphine 96-well scintillant coated microplate). To the wells 10 uI of hydrochloride (TCEP) was purchased from Pierce Biotech each of the following were added: 1 mM Acetyl-CoA, 3 mM nologies (Rockford, Ill.). H-acetyl-coenzyme A was pur Malonyl-CoA, 0.05 mCi/mL HAcetyl-CoA and 20 mM chased from Moravek Biochemicals (Brea, Calif.). Flash 25 NADPH for final concentrations of 100 uM Acetyl-CoA,300 PlateR PLUS phospholipid 96-well scintillant coated uM Malonyl-CoA, 0.5 uCi/well HAcetyl-CoA and 2000 microplates were purchased from PerkinElmer Life and Ana uMNADPH. Finally, 10 uL of FAS enzyme was added to the lytical Sciences (Shelton, Conn.). The method of the FASN wells either in the form of HCT116 cell lysate or purified FAS Scintillation Proximity FlashPlate assay is similar to that for a standard curve. The FlashPlate was incubated at 37° C. described in Weiss and Glickman Assay Drug Dev Technol 30 for 120 min then read on the MicroBeta instrument. 2003, 1:161-6. In a 96-well polypropylene microplate, a dilu Compounds provided herein were found to inhibit FASN tion series (typical concentrations 60 nM-1.0 mM) of test activity using this assay. compounds were prepared in DMSO followed by a 20-fold HCV-Replicon Luciferase Assay dilution into FASN assay buffer (50 mM potassium phos DMEM complete medium (Life Technologies) was phate, pH 7.0, 1.0 mM EDTA, 0.01% NP-40), of which 5.0L 35 supplemented with 10% FCS, 2 mM glutamin, penicillin and each was transferred to a FlashPlateR PLUS 96-well plate streptomycin, and 1x nonessential amino acids and pre and mixed with 35 LL FASN assay buffer plus 0.5 mg/mL warmed in a 37°C. thermostat water bath for use as a growth bovine gamma globulin and 1 mMTCEP. FASN protein (10 medium. uL. 10 nM) was added per well, and the microplate was 40 A dish containing HCV-replicon reporter cells, which were incubated at 37° C. for 30 min. 10 uL of 20 mM NADPH was kept in a 37° C. CO incubator, was removed from the incu added, and the reaction was initiated by addition of 40 uL bator. The medium was aspirated, and cells were rinsed with substrate mixture to final concentrations of 1 nM FASN, 100 1 ml PBS. The solution was discarded, and 1 ml of 1.25% uMacetyl-coenzyme A, 6 uCi H1-acetyl-coenzyme A, 300 trypsin/0.02% EDTA was added to additionally rinse the uM malonyl-coenzyme A, 2 mMNADPH, 0.5 mg/mL bovine 45 cells. The trypsin/EDTA solution was removed with a gamma globulin, and 1 mMTCEP in a volume of 100 uL per vacuum pump, and the cells were incubated at 37°C. for 3 to well. Assay plates were incubated for 2 hr at 37° C. and the 5 minutes. Cell morphology was examined under an inverted reaction was stopped with 2 LL of 2.5 mM stock solution of microscope until a single cell Suspension was clearly visible, ORLISTAT in DMSO to -50 uM. The plates were read in a then the cell was suspended with 3 ml of complete medium by Wallac 1450 Microbeta Plus liquid scintillation counter (Per 50 gentle pipetting. kin Elmer, Waltham, Mass.), and counts per minute (CPM) Upon Suspension, cell numbers were counted with a were collected over 2 min. Each inhibitor well CPM was hematometer, and cell density was adjusted to 100K/ml by compared to the maximum FASN enzyme activity (Max) adding appropriate Volume of the complete medium. One CPM and the background (Min) CPM, as measured by omis hundred microliters (100 ul) of cell suspension was added to sion of FASN enzyme in the background well. 96 Inhibition 55 each well of a 96-well white plate to provided cell density of values were calculated, and curves were fitted by a four each of the wells to be 10K/well. The 96-well assay plate was parameter logistic function to yield ICso values: placed in a 37°C. 5% CO incubator for 24 hours. At the end of the incubation, the plate was removed and test (Inhibitor-Min) compound was added at various desired concentrations using %. Inhibition= 1 - (Max-Min) x 100% 60 a serial dilution. The plate was placed back into the 37° C. CO, incubator for 48 hours. After the incubation, 30 ul of Stead-Glo Luciferase System(R) (Promega) reagent was added Compounds provided herein were found to inhibit FASN to each well and mixed by gentle shaking for 5 minutes to activity using this assay. allow thorough cell lysis. Luminescence was measured with Activities obtained by the above-described FASN Scintil 65 Envision(R) (Perkin-Elmer) with an integration time of 2 sec lation Proximity Flashplate Assay are provided in Tables 1, 2, onds. 3 and 4 wherein “A” refers to compounds having an ICs of The tests were conducted using the following compounds: