I 1111111111111111 1111111111 11111 lllll 111111111111111 111111111111111 IIII IIII US010065924B2 c12) United States Patent (IO) Patent No.: US 10,065,924 B2 Nicolaou et al. (45) Date of Patent: Sep.4,2018

(54) PREPARATION AND BIOLOGICAL FOREIGN PATENT DOCUMENTS EVALUATION OF VIRIDICATUMTOXIN ANALOGS WO WO 2009/008906 1/2009

(71) Applicant: WILLIAM MARSH RICE OTHER PUBLICATIONS UNIVERSITY, Houston, TX (US) Breinholt, et al., "Hypomycetin-an Antifungal, tetracyclic (72) Inventors: Kyriacos C Nicolaou, Houston, TX Metabolite from Hypomyces aurantius: Production, Structure and (US); Christopher R. H. Hale, Biosynthesis," Acta Chem. Scand., 51:855-860, 1997. Houston, TX (US); Christian Nilewski, Brubaker and Myers, "A practical, enantioselective synthetic route Houston, TX (US); Heraklidia to a key precursor to the tetracycline antibiotics." Org. Lett., Ioannidou, Houston, TX (US); 9:3523-3525, 2007. Abdellatif El Marrouni, Houston, TX Charest, et al., "Synthesis of(-)-Tetracycline," J Am. Chem. Soc., (US) 127:8292-8293, 2005. Charest, et al., "A Convergent Enantioselective Route to Structur­ (73) Assignee: WILLIAM MARSH RICE ally Diverse 6-Deoxytetracycline Antibiotics," Science, 308, 395­ UNIVERSITY, Houston, TX (US) 398, 2005. Chooi, et al., "Identification of the Viridicatumtoxin and Griseoful­ ( *) Notice: Subject to any disclaimer, the term ofthis vin Gene Clusters from Penicillium aethiopicum," Chem. Biol., patent is extended or adjusted under 35 17:483-494, 2010. U.S.C. 154(b) by O days. Chooi, et al., "Discovery and Characterization of a Group of Fungal Polycyclic Polyketide Prenyltransferases," J Am. Chem. Soc., (21) Appl. No.: 15/327,912 134:9428-9437, 2012. Chooi, et al., J. Am. Chem. Soc., 135:16805-16808, 2013. (22) PCT Filed: Jul. 22, 2015 Chopra and Roberts, "Tetracycline antibiotics: mode of action, applications, molecular biology, and epidemiology of bacterial (86) PCT No.: PCT/US2015/041494 resistance," Microbial. Mo!. Biol. R., 65:232-260, 2001. De Jesus, A.E., et al., "Biosynthesis of viridicatumtoxin, a § 371 (c)(l), mycotoxin from Penicillin expansum," J Chem. Soc., Chem. (2) Date: Jan.20, 2017 Comm., 902-904, 1982. Hutchison, et al., "Viridicatumtoxin, a new mycotoxin from Penicil­ (87) PCT Pub. No.: WO2016/014643 lium viridicatum Westling," Toxicol. Appl. Pharmacol., 24:507-509, 1973. PCT Pub. Date: Jan. 28, 2016 (Continued) (65) Prior Publication Data

US 2017/0210699 Al Jul. 27, 2017 Primary Examiner - Barbara P Badia (74) Attorney, Agent, or Firm - Parker Highlander PLLC Related U.S. Application Data (60) Provisional application No. 62/027,500, filed on Jul. 22, 2014. (57) ABSTRACT In one aspect, the present invention provides novel deriva­ (51) Int. Cl. A61K 31/65 (2006.01) tives of viridicatumtoxin of the formula wherein the vari­ C07C 237126 (2006.01) ables are as defined herein. The application also provides C07C 235182 (2006.01) compositions, methods of treatment, and methods of syn­ C07D 261/20 (2006.01) thesis thereof. A61K 45106 (2006.01) (52) U.S. Cl. CPC ...... C07C 235182 (2013.01); A61K 31/65 (I) (2013.01); A61K 45106 (2013.01); C07C 237126 (2013.01); C07D 261/20 (2013.01); C07C 2603/94 (2017 .05) R2 ( 58) Field of Classification Search /0 CPC . C07C 235/82; C07C 237/26; C07C 2603/94; R10 C07D 261/20; A61K 31/65 USPC ...... 514/152; 552/203 0 R., See application file for complete search history. /0 /0 0 0 N (56) References Cited Rs R1 Rs/...... _~

U.S. PATENT DOCUMENTS

2010/0291622 Al 11/2010 Tomoda et al. 2013/0338117 Al 12/2013 Ricciardi 20 Claims, 4 Drawing Sheets US 10,065,924 B2 Page 2

(56) References Cited PCT International Search Report and Written Opinion issued in International Application No. PCT/US2015/041494; dated Oct. 19, 2015. OTHER PUBLICATIONS Sun, et al., "A Robust Platform for the Synthesis of New Tetracy­ cline Antibiotics" J Am. Chem. Soc., 130:17913-17927, 2008. Inokoshi, et al., "Spirohexalines, new inhibitors of bacterial Sutcliffe, et al., "Antibacterial activity of eravacycline (TP-434), a undecaprenyl pyrophosphate synthase, produced by Penicilliurn novel fluorocycline, against hospital and community pathogens." brasilianurn FKI-3368," J Antibiot., 66:37-41, 2013. Antimicrob. Agents Chemother., 57:5548-5558, 2013. Koyama, et al., "Anti-infectious agents against MRSA." Molecules, 18:204-224, 2013. Tally, et al., "Glycylcyclines: a new generation of tetracyclines" J Kummer, et al., "A practical, convergent route to the key precursor Antimicrob. Chemother., 35:449-452, 1995. to the tetracycline antibiotics." Chem. Sci., 2:1710-1718, 2011. Wright and Myers, "Methodological Advances Permit the Nicolaou, et al., "Total synthesis and structural revision of Stereocontrolled Construction of Diverse Fully Synthetic Tetracy­ viridicatumtoxin B." Angew. Chem. Int. Ed., (52) 8736-8741, 2013. clines Containing an All-Carbon Quaternary Center at Position Nicolaou, et al., "Total synthesis of viridicatumtoxin B and ana­ C5a." Tetrahedron, 67:9853-9869, 2011. logues thereof: strategy evolution, structural revision, and biologi­ Wzorek, et al., "A macrocyclic approach to tetracycline natural cal evaluation." J Am. Chem. Soc., 136(34):12137-12160, 2014. products. Investigation of transannular alkylations and Michael National Center for Biotechnology Information. PubChem Com­ additions." Org. Lett., 14:5840-5843, 2012. pound Database; CID~72203888, https://pubchem.ncbi.nlm.nih. Zheng, et al., "Viridicatnmtoxin B, a new anti-MRSA agent from gov/compound/72203888. Penicilliurn sp. FRll." J Antibiot. 61:633-637, 2008. U.S. Patent Sep.4,2018 Sheet 1 of 4 US 10,065,924 B2

a)

4: chiortetracycline (Aureomycin-:I~,1 5: R = OH, oxytetracyciine {Terramycin@) 6: R = H, tetracycBrie {,A,chromycin(~°'.'.l

Nfv1e2 H H 7Me2

+=· 'H~i JlQ f~J,,..~---,,J~~/~,,_l~.,.,.--<.,,(,OH..., ,._,ti, n '- ).! ,/ ,.___ ,.. A ,.<._, -"'___ ·~ ~ 'y-- 'lf ,r·h'r( ---~r OH O HOH O NH2

7: minocycllne (t,11inocini®) 9: Ugscyc!ine {Tygact!@)

8: doxycycline (Vlbramycin:E·l 10: eravacycHne (TP-434)

11: hypomycetin

? --..,.--­ ____~ _

14: BMS-192548

FIGS. lA& B U.S. Patent Sep.4,2018 Sheet 2 of 4 US 10,065,924 B2

? -----­•----­

3: spirohexaline

FIG.2 U.S. Patent Sep.4,2018 Sheet 3 of 4 US 10,065,924 B2

[Friede!­ CraftsJ

21

FIG.3 U.S. Patent Sep.4,2018 Sheet 4 of 4 US 10,065,924 B2

0 0

OH OH OH OH (+)-1: viridicatumtoxin B (+)-2: viridicatumtoxin A

Me Me Meo Meo 0 0

OH OH OR OH (±)-V2 R = H: (±)-V3 R = Me: (±)-V4

Me Me Meo Meo 0 0

OH OH OH OH

(±)-VS (±)-V6

FIG.4 US 10,065,924 B2 1 2 PREPARATION AND BIOLOGICAL of the commercial importance of these molecules for the EVALUATION OF VIRIDICATUMTOXIN biological activity, many efforts have been undertaken to ANALOGS synthesize tetracycline derivatives including recent efforts by the Myers (Charest, et al., 2005; Charest, et al., 2005; This invention was made with government support under 5 Brubaker and Myers, 2007; Sun, et al., 2008; Kummer, et al., Grant Number AI055475 awarded by the National Institutes 2011; Wright and Myers, 2011) and Evans groups (Wzorek, of Health. The government has certain rights in the inven­ et al., 2012). tion. In 2008, Kim, et al., isolated viridicatumtoxin B (1) from This application claims the benefit of priority to U.S. Penicillium sp. FRl 1 along with viridicatumtoxinA (2). This Provisional Application Ser. No. 62/027,500, filed on Jul. 10 compound was investigated through NMR spectroscopy and 22, 2014, the entire contents of which are hereby incorpo­ assigned the structure 1'. These compounds have been rated by reference. shown to have potent antibacterial properties in a number of bacterial strains including both gram positive and gram BACKGROUND OF THE INVENTION negative bacteria (Kim, et al., 2008). Without being bound 15 by theory, further study and analysis suggests that the 1. Field of the Invention viridicatumtoxin's antibacterial properties arise not by bind­ ing to the 30S subunit ofthe ribosome like many tetracycline This disclosure relates to the fields of medicine, pharma­ compounds (e.g., 4-10, FIG. lA) but by inhibiting UPP cology, chemistry, antimicrobial activity, and oncology. In synthase, an enzyme associated with bacterial peptidoglycan particular, new compounds, compositions, methods of treat­ 20 biosynthesis (Inokoshi, et al., 2013; Koyama, et al., 2013). ment, and methods of synthesis relating to viridicatumtoxin Furthermore, viridicatumtoxin A (2) shows promising anti­ and derivatives thereof are disclosed. cancer activity against a selection of cancer cell lines (NIH Results of Viridicatumtoxin A NCI 60 Cell line assay) as 2. Related Art well as shows antiviral activity (WO 2009/008906). 25 As such, new analogs of viridicatumtoxin could provide Since the discovery of chlortetracycline ( 4; FIG. lA) in access to a more efficacious antimicrobial or cancer drug and the late 1940's, tetracycline antibiotics such as chlortetra­ new methods of synthesis could allow cost effective clinical cycline (4), oxytetracycline (5), and tetracycline (6) have access to these compound for use in the treatment of been commonly prescribed to treat bacterial infections microbial infections and as chemotherapeutic agents. (Duggar, 1948). Throughout the years, as bacterial resis­ 30 tance grew or improved therapeutic properties were needed, SUMMARY OF THE INVENTION additional therapeutic agents including second generation tetracycline derivatives such as minocycline (7) and doxy­ Thus, in accordance with the present invention, there is cycline (8) and third generation tetracycline derivatives such provided a compound of the formula: as tigecycline (9) and eravacycline (TP-434, 10) have been 35 developed (FIG. lA) (Tally, et al, 1995; Sutcliffe, et al., 2013; Chopra and Roberts, 2001). (I) Penicillium is a genus of ascomycetous fungi of major importance in the natural environment as well as food and drug production. Members of the genus may be best known 40 for producing penicillin, a molecule that is used as an antibiotic, which kills or stops the growth ofcertain kinds of bacteria inside the body. According to the Dictionary ofthe Fungi (l 0th edition, 2008), the widespread genus contains over 300 species. Additionally, the majority of the tetracy­ 45 cline antibiotics have bacterial origins but some tetracycline antibiotics have fungal origins. Tetracycline antibiotics which have fungal origins include viridicatumtoxin B (1), viridicatumtoxinA (2), spirohexaline (3), hypomycetin (11 ), anthrotainin (TAN-1652, 12), TAN-1612 (13), and BMS­ 50 wherein: X 1 is absent such that atoms 16 and 17 are only 192548 (14) (FIG. 1B & FIG. 2) (Zheng, et al., 2008; connected by the shown single bond, a covalent bond such Hutchinson, et al., 1973; Inokoshi, et al., 2013; Breinholt, et that a double bond is formed between atoms 16 and 17, al., 1997; Wong, et al., 1993; JP 06-40995; Kodukula, et al., ---0-, alkanediylccss), or substituted alkanediylccss); Y 1 , 1995; and Shu, et al., 1995). Several tetracycline antibiotics Y2 , and Y3 are each independently alkylccsi 2 ) or substituted with fungal origins including viridicatumtoxin B (1), viridi­ 55 alkylccsi 2 ); R 1 is hydrogen, hydroxy, or oxo, provided that catumtoxinA (2), and spirohexaline (3) are also structurally when R 1 is oxo, the bond between R 1 and atom number 5 is unique from the earlier tetracycline derivatives in the struc­ a double bond and when the bond between R 1 and atom ture also contains a spirobicyclic system (ring system EF) number 5 is a double bond then R 1 is oxo, alkoxyccss), or derived from a geranyl subunit (Zheng, et al., 2008; substituted alkoxyccss); R2 is hydrogen, amino, carboxy,

Hutchinson, et al., 1973; Inokoshi, et al., 2013) (FIG. 2). 60 cyano, halo, hydroxy, nitro, or sulfa; alkylccsi 2), Without being bound by theory, the proposed biosynthetic alkoxy(csi2 ), alkylaminoccsi 2 ), dialkylaminoccsis), or a sub­ pathway to produce the viridicatumtoxin A (2) has been stituted version of any of these groups; R3 , R6 , R7 , and R 10 reported and is shown in FIG. 3 (De Jesus, et al., 1982; are each independently selected from: hydrogen, alkylccss),

Chooi, et al., 2010; Chooi, et al., 2012; Chooi, et al., 2013). alkenylccss), arylccsi 2 ), aralkylccsi 2 ), acylccss), or a substi­ Due to the biological activity of these compounds and the 65 tuted version of any of these groups; R4 and R5 are each need for multi-gram quantities of the compounds, a com­ independently selected from: hydrogen, alkylccss), mercially scalable synthesis is needed. Since the discovery alkanediylccss)-heterocycloalkylccss), alkanediylccss)-het­ US 10,065,924 B2 3 4 eroarylccss), alkanediylccss)-alkylaminoccss), ecule wherein the biomolecule is a protein, a polypeptide, an alkanediylccssfdialkylaminoccss), or a substituted version amino acid, a cofactor, an imaging agent, an antibody, a fatty of any of these groups; Rs is hydrogen, alkylccss), acid, a nucleic acid, or a small molecule therapeutic agent; alkenylccss), arylccsi 2 ), aralkylccsi 2 ), acylccss), or a substi­ and R9 is hydrogen, amino, carboxy, cyano, halo, hydroxy, tuted version of any of these groups; or -X2-Ru, 5 nitro, or sulfa; alkylccsi 2 ), alkoxy(csi 2 ), alkylaminoccsi 2 ), ), wherein: X2 is alkanediylccsi 2 ) or substituted dialkylaminoccsis), amidoccsi 2 or a substituted version of any of these groups; or -NR C(O)R -NR R s; alkanediylccsi 2); and Ru is hydroxy, amino, azido, carboxy, 12 13 14 1 wherein: R is hydrogen, alkylccss), or substituted or cyano, alkenylccs 6 ), alkynylccs 6), heterocycloalkylccsi 2 ), 12 alkylaminoccss), dialkylaminoccss), alkoxyccss), or a substi­ alkylccss); R13 is alkanediylccss) or substituted tuted version of any of these groups; or a -linker-biomol­ 10 alkanediylccss); and R14 and R 1s are each independently ecule wherein the biomolecule is a protein, a polypeptide, an selected from: hydrogen, alkylccsi 2 ), arylccsi 2 ), amino acid, a cofactor, an imaging agent, an antibody, a fatty aralkylccsi 2 ), acylccsi 2 ), or a substituted version of any of acid, a nucleic acid, or a small molecule therapeutic agent; these groups; or R14 and R 1s are taken together and are alkanediylccss), alkoxydiylccss), alkylaminodiylccss), or a and R9 is hydrogen, amino, carboxy, cyano, halo, hydroxy, 15 substituted version of any of these groups; or a pharmaceu­ nitro, or sulfa; alkyl(csi2 ), alkoxy(csi 2 ), alkylamino(csi 2 ), tically acceptable salt or tautomer thereof. In some embodi­ dialkylaminoccsis), amidoccsi 2), or a substituted version of ments, the compound is further defined as: any of these groups; or -NR12C(O)R13-NR14R 1s; wherein: R12 is hydrogen, alkylccss), or substituted alkylccss); R13 is alkanediylccss) or substituted 20 (III) alkanediylccss); and R14 and R 1s are each independently selected from: hydrogen, alkylccsi 2 ), arylccsi 2 ), aralkylccsi 2 ), acylccsi 2 ), or a substituted version of any of these groups; or R14 and R 1s are taken together and are alkanediylccss), alkoxydiylccss), alkylaminodiylccss), or a substituted version of any of these groups; or a pharmaceu­ 25 tically acceptable salt or tautomer thereof. In some embodi­ ments, the compound is further defined as:

(II) 30

wherein: X 1 is a covalent bond such that a double bond is formed between atoms 16 and 17, ---0-, alkanediylccss), or 35 substituted alkanediylccss); R 1 is hydrogen, hydroxy, or oxo, provided that when R 1 is oxo, the bond between R 1 and atom number 5 is a double bond and when the bond between R 1 and atom number 5 is a double bond then R 1 is oxo, alkoxyccss), or substituted alkoxyccss); R2 is hydrogen, amino, carboxy, cyano, halo, hydroxy, nitro, or sulfa; 40 alkylccsi 2 ), alkoxy(csi2 ), alkylaminoccsi 2 ), dialkylaminoccsis), or a substituted version of any of these

groups; R4 and Rs are each independently selected from: wherein: X 1 is absent such that atoms 16 and 17 are only hydrogen, alkylccss), alkanediylccss)-heterocycloalkylccss), connected by the shown single bond, a covalent bond such alkanedi_Ylccss)-heteroaryl(css), . ~lkanediylccss)­ that a double bond is formed between atoms 16 and 17, 45 alkylammo(css), alkaned1yl(css)-dialkylammoccss), or a -0-, alkanediylccss), or substituted alkanediylccss); Y 1 , substituted version of any of these groups; Rs is hydrogen,

Y2 , and Y3 are each independently alkylccsi 2 ) or substituted alkyl(css), alkenyl(css), aryl(csi2y aralkyl(Csl 2 ), acyl(css), alkylccsi 2 ); R 1 is hydrogen, hydroxy, or oxo, provided that or a substituted version of any of these groups; or -X2 ­ when R 1 is oxo, the bond between R 1 and atom number 5 is Ru, wherein: X2 is alkanediylccsi 2 ) or substituted ); a double bond and when the bond between R 1 and atom 50 alkanediylccsi 2 and Ru is hydroxy, amino, azido, carboxy, ), ), ), number 5 is a double bond then R 1 is oxo, alkoxyccss), or or cyan~, alkenyl(rs6 alk)'.nylccs 6 heterocycloalkylccsi 2 alkylammoccss), dialkylammoccss), alkoxyccss), or a substi­ substituted alkoxyccss); R2 is hydrogen, amino, carboxy, tuted version of any of these groups; or a -linker-biomol­ cyano, halo, hydroxy, nitro, or sulfa; alkylccsi ), 2 ecule wherein the biomolecule is a protein, a polypeptide, an alkoxy(csi ), alkylaminoccsi ), dialkylaminoccsis), or a sub­ 2 2 amino acid, a cofactor, an imaging agent, an antibody, a fatty stituted version of any of these groups; R4 and Rs are each 55 independently selected from: hydrogen, alkylccss), acid, a nucleic acid, or a small molecule therapeutic agent; and R is hydrogen, amino, carboxy, cyano, halo, hydroxy, alkanediylccss)-heterocycloalkylccss), alkanediylccss)-het­ 9 nitro, or sulfa; alkylccsi ), alkoxy(csi ), alkylamino(Csl ), eroarylccss), alkanediylccss)-alkylaminoccss), 2 2 2 dialkylaminoccsis), amidoccsi ), or a substituted vers10n of alkanediylccss)-dialkylaminoccss), or a substituted version 2 any of these groups; or -NR12C(O)R13-NR14R 1s; of any of these groups; Rs is hydrogen, alkylccss), 60 wherein: R12 is hydrogen, alkylccss), or substituted alkenyl(css), aryl(csi 2 ), aralkyl(csi 2 ), acyl(css), or a substi­ alkylccss); R13 is alkanediylccss) or substituted tuted version of any of these groups; or -X2-Ru, alkanediylccss); and R14 and R 1s are each independently wherein: X is alkanediylccsi ) or substituted 2 2 selected from: hydrogen, alkylccsi 2 ), arylccsi 2 ), alkanediylccsi ); and Ru is hydroxy, amino, azido, carboxy, 2 aralkylccsi 2 ), acylccsi 2 ), or a substituted version of any of or cyano, alkenylccs 6 ), alkynylccs 6), heterocycloalkylccsi 2 ), 65 these groups; or R14 and R 1s are taken together and are alkylaminoccss), dialkylaminoccss), alkoxyccss), or a substi­ alkanediyl(css), alkoxydiyl(css), alkylaminodiyl(css), or a tuted version of any of these groups; or a -linker-biomol­ substituted version of any of these groups; or a pharmaceu­ US 10,065,924 B2 5 6 tically acceptable salt or tautomer thereof. In some embodi­ wherein: R1 is hydroxy, alkoxyccss), or substituted ments, the compound is further defined as: alkoxy(css); R2 is hydrogen, amino, carboxy, cyano, halo, hydroxy, nitro, or sulfa; alkylccsi 2), alkoxyccsi2), alkylam­ inoccsi2), dialkylaminoccsis), or a substituted version of any (IV) 5 of these groups; R4 is hydrogen, alkylccss), or substituted alkylccss), Rs is hydrogen, alkylccss), alkanediylccss)-het­ erocycloalkylccss), alkanediylccss)-heteroarylccss), alkanediyl(css)-alkylamino(css), alkanediyl(css)-dialky­ laminoccss), or a substituted version of any of these groups; lO Rs is hydrogen, alkyl(css), alkenyl(css), aryl(csi 2), aralkylccsi2), acylccss), or a substituted version of any of these groups; or -X2-Ru, wherein: X2 is alkanediylccsi2) or substituted alkanediylccsi2); and Ru is hydroxy, amino, azido, carboxy, or cyano, alkenylccs 6 ), alkynylccs 6), hetero­ 15 cycloalkylccsi2), alkylaminoccss), dialkylaminoccss), alkoxyccss), or a substituted version of any of these groups; or a -linker-biomolecule wherein the biomolecule is a pro­ wherein: R1 is hydroxy, or oxo, provided that when R1 is tein, a polypeptide, an amino acid, a cofactor, an imaging oxo, the bond between R1 and atom number 5 is a double bond and when the bond between R1 and atom number 5 is agent, an antibody, a fatty acid, a nucleic acid, or a small 20 a double bond then R1 is oxo, alkoxy(css), or substituted molecule therapeutic agent; and R9 is hydrogen, amino, alkoxyccss); R2 is hydrogen, amino, carboxy, cyano, halo, carboxy, cyano, halo, hydroxy, nitro, or sulfa; alkylccsi 2), hydroxy, nitro, or sulfa; alkylccsi 2), alkoxy(csi2), alkylam­ alkoxy(csi2), alkylamino(csi2), dialkylamino(CslS)' inoccsi2), dialkylaminoccsis), or a substituted version of any amidoccsi2), or a substituted version of any of these groups; of these groups; R and Rs are each independently 4 or -NR12C(O)R13-NR14R1s; wherein: R12 is hydrogen, selected from: hydrogen, alkylccss), alkanediylccss)-hetero­ 25 alkylccss), or substituted alkylccss); R13 is alkanediylccss) or cycloalkylccss), alkanediylccss)-heteroarylccss), substituted alkanediylccss); and R14 and R1s are each inde­ alkanediylccss)-alkylaminoccss), alkanediylccss)-dialky­ pendently selected from: hydrogen, alkylccsi 2), arylccsi 2), lamino(css), or a substituted version of any of these groups; aralkylccsi2), acylccsi2), or a substituted version of any of Rs is hydrogen, alkylccss), alkenylccss), arylccsi 2), these groups; or R14 and R1s are taken together and are aralkylccsi 2), acylccss), or a substituted version of any of 30 alkanediylccss), alkoxydiylccss), alkylaminodiylccss), or a these groups; or -X2-Ru, wherein: X2 is alkanediylccsi2) substituted version of any of these groups; or a pharmaceu­ or substituted alkanediylccsi2); and Ru is hydroxy, amino, tically acceptable salt or tautomer thereof. In some embodi­ azido, carboxy, or cyano, alkenylccs 6 ), alkynylccs 6 ), hetero­ ments, the compound is further defined as: cycloalkylccsi2), alkylaminoccss), dialkylaminoccss), alkoxy(css), or a substituted version of any of these groups; 35 or a -linker-biomolecule wherein the biomolecule is a pro­ (V) tein, a polypeptide, an amino acid, a cofactor, an imaging agent, an antibody, a fatty acid, a nucleic acid, or a small molecule therapeutic agent; and R9 is hydrogen, amino, carboxy, cyano, halo, hydroxy, nitro, or sulfa; alkylccsi 2), 40 alkoxy(csi 2), alkylaminoccsi2), dialkylaminoccsis), amidoccsi2), or a substituted version of any of these groups; or -NR12C(O)R13-NR14R1s; wherein: R12 is hydrogen, alkylccss), or substituted alkylccss); R13 is alkanediylccss) or substituted alkanediylccss); and R14 and R1s are each inde- 45 pendently selected from: hydrogen, alkylccsi 2), arylccsi 2), aralkylccsi 2), acylccsi 2), or a substituted version of any of these groups; or R14 and R1s are taken together and are alkanediylccss), alkoxydiylccss), alkylaminodiylccss), or a wherein: R1 is hydroxy, alkoxy(css), or substituted substituted version of any of these groups; or a pharmaceu­ 50 alkoxyccss); R2 is hydrogen, amino, carboxy, cyano, halo, tically acceptable salt or tautomer thereof. In some embodi­ hydroxy, nitro, or sulfa; alkylccsi 2), alkoxyccsi2), alkylam­ ments, the compound is further defined as: inoccsi2), dialkylaminoccsis), or a substituted version of any

of these groups; R4 is hydrogen, alkylccss), or substituted alkyl(css), Rs is hydrogen, alkyl(css), alkanediyl(css)-het­ (V) 55 erocycloalkylccss), alkanediylccss)-heteroarylccss), alkanediyl(css)-alkylamino(css), alkanediyl(css)-dialky­ laminoccss), or a substituted version of any of these groups; Rs is hydrogen, alkylccss), alkenylccss), arylccsi 2), aralkylccsi2), acylccss), or a substituted version of any of 60 these groups; or -X2-Ru, wherein: X2 is alkanediylccsi2) or substituted alkanediylccsi2); and Ru is hydroxy, amino, azido, carboxy, or cyano, alkenylccs 6 ), alkynylccs 6), hetero­ cycloalkylccsi2), alkylaminoccss), dialkylaminoccss), alkoxyccss), or a substituted version of any of these groups; 65 or a -linker-biomolecule wherein: the linker is alkanediylccsi2), alkenediylccsi2), arenediylccsi2), het­ eroarenediylccsi2), heterocycloalkanediylccsi2) or a substi­ US 10,065,924 B2 7 8 tuted version of any of these groups; and the biomolecule is a protein, a polypeptide, an antibody, an imaging agent, or (V) a small molecule therapeutic agent; and R9 is hydrogen, amino, carboxy, cyano, halo, hydroxy, nitro, or sulfa; alkyl(csi2), alkoxy(Csl2), alkylamino(Csl2), 5 dialkylaminoccsis), amidoccsi2), or a substituted version of any of these groups; or -NR12C(O)R13-NR14R1s; wherein: R12 is hydrogen, alkylccss), or substituted alkylccss); R13 is alkanediylccss) or substituted 10 alkanediylccss); and R14 and R1s are each independently selected from: hydrogen, alkylccsi2), arylccsi2), aralkylccsi 2), acylccsi 2), or a substituted version of any of these groups; or R14 and R1s are taken together and are alkanediylccss), alkoxydiylccss), alkylaminodiylccss), or a 15 wherein: R1 is hydroxy, alkoxyccss), or substituted substituted version of any of these groups; or a pharmaceu­ alkoxy(css); R2 is hydrogen, amino, carboxy, cyano, halo, tically acceptable salt or tautomer thereof. In some embodi­ hydroxy, nitro, or sulfa; alkylccsi 2), alkoxyccsi2), alkylam­ ments, the compound is further defined as: inoccsi2), dialkylaminoccsis), or a substituted version of any

of these groups; R4 is hydrogen, alkylccss), or substituted 20 alkylccss), Rs is hydrogen, alkylccss), alkanediylccss)-het­ (V) erocycloalkylccss), alkanediylccss)-heteroarylccss), alkanediyl(css)-alkylamino(css), alkanediyl(css)-dialky­ laminoccss), or a substituted version of any of these groups; Rs is hydrogen or a -linker-biomolecule; wherein: the linker 25 is alkanediylccsi2), alkenediylccsi2), arenediylccsi2), het­ eroarenediylccsi2), or heterocycloalkanediylccsi2); and the biomolecule is an antibody; and R9 is hydrogen, amino, carboxy, cyano, halo, hydroxy, nitro, or sulfa; alkylccsi 2), alkoxy(csi2), alkylamino(csi2), dialkylamino(CslS)' 30 amidoccsi2), or a substituted version of any of these groups; or -NR12C(O)R13-NR14R1s; wherein: R12 is hydrogen, alkylccss), or substituted alkylccss); R13 is alkanediylccss) or substituted alkanediylccss); and R and R s are each inde­ wherein: R1 is hydroxy, alkoxyccss), or substituted 14 1 pendently selected from: hydrogen, alkylccsi ), arylccsi ), alkoxyccss); R2 is hydrogen, amino, carboxy, cyano, halo, 2 2 35 aralkylccsi ), acylccsi ), or a substituted version of any of hydroxy, nitro, or sulfa; alkylccsi 2), alkoxy(csi2), alkylam­ 2 2 these groups; or R and R s are taken together and are inoccsi2), dialkylaminoccsis), or a substituted version of any 14 1 alkanediylccss), alkoxydiylccss), alkylaminodiylccss), or a of these groups; R is hydrogen, alkylccss), or substituted 4 substituted version of any of these groups; or a pharmaceu­ alkyl(css), Rs is hydrogen, alkyl(css), alkanediyl(css)-het­ tically acceptable salt or tautomer thereof. In some embodi­ erocycloalkylccss), alkanediylccss)-heteroarylccss), 40 ments, the compound is further defined as: alkanediylccss)-alkylaminoccss), alkanediylccss)-dialky­ lamino(css), or a substituted version of any of these groups; Rs is hydrogen, alkylccss), substituted alkylccss), or -X2­ (V) Ru, wherein: X2 is alkanediylccsi2) or substituted alkanediylccsi2); and Ru is hydroxy, amino, azido, carboxy, 45 or cyano, alkenylccs 6 ), alkynylccs 6), heterocycloalkylccsi2), alkylaminoccss), dialkylaminoccss), alkoxyccss), or a substi­ tuted version of any of these groups; or a -linker-biomol­ ecule wherein: the linker is alkanediylccsi2), 50 alkenediylccsi2), arenediylccsi2), heteroarenediylccsi2), het­ erocycloalkanediylccsi2) or a substituted version of any of these groups; and the biomolecule is an antibody, an imaging agent, a protein, or a small molecule therapeutic agent; R9 is hydrogen, amino, carboxy, cyano, halo, hydroxy, nitro, or 55 sulfa; alkylccsi2), alkoxy(csi2), alkylaminoccsi2), dialky­ wherein: R1 is hydroxy, alkoxy(css), or substituted lamino(CslS)' amidoccsi2), or a substituted version of any of alkoxyccss); R2 is hydrogen, amino, halo, or hydroxy; alky­ these groups; or -NR12C(O)R13-NR14R1s; wherein: R12 laminoccsi2), dialkylaminoccsis), or a substituted version of is hydrogen, alkylccss), or substituted alkylccss); R is 13 any of these groups; R4 is hydrogen, alkylccss), or substi­ alkanediylccss) or substituted alkanediylccss); and R14 and 60 tuted alkylccss), Rs is hydrogen, alkylccss), alkanediylccss)­ R1s are each independently selected from: hydrogen, heterocycloalkylccss), alkanediylccss)-heteroarylccss), alkylccsi2), arylccsi2), aralkylccsi2), acylccsi2), or a substi­ alkanediylccss)-alkylaminoccss), alkanediylccss)-dialky­ tuted version ofany ofthese groups; or R14 and R1s are taken laminoccss), or a substituted version of any of these groups; together and are alkanediylccss), alkoxydiylccss), alkylam­ Rs is hydrogen, alkylccss), alkenylccss), arylccsi 2), inodiylccss), or a substituted version of any of these groups; 65 aralkylccsi2), acylccss), or a substituted version of any of or a pharmaceutically acceptable salt or tautomer thereof. In these groups; or -X2-Ru, wherein: X2 is alkanediylccsi2) some embodiments, the compound is further defined as: or substituted alkanediylccsi2); and Ru is hydroxy, amino, US 10,065,924 B2 9 10 azido, carboxy, or cyano, alkenylecs 6 ), alkynylecs 6 ), hetero­ cycloalkylecsl2), alkylaminoecss), dialkylaminoecss), (V) alkoxyecss), or a substituted version of any of these groups; or a -linker-biomolecule; and R9 is hydrogen, amino, car­ boxy, cyano, halo, hydroxy, nitro, or sulfa; alkylecsl 2), 5 alkoxyecsl 2), alkylaminoecsl2), dialkylaminoecsls), amidoecsl2), or a substituted version of any of these groups; or -NR12C(O)R13-NR14R1s; wherein: R12 is hydrogen, alkylecss), or substituted alkylecss); R13 is alkanediylecss) or 10 substituted alkanediylecss); and R14 and R1s are each inde­ pendently selected from: hydrogen, alkylecsl 2), arylecs 12), aralkylecs 12), acylecsl 2), or a substituted version of any of these groups; or R14 and R1s are taken together and are alkanediylecss), alkoxydiylecss), alkylaminodiylecss), or a 15 wherein: R1 is hydroxy, alkoxyecss), or substituted substituted version of any of these groups; or a pharmaceu­ alkoxyecss); R2 is hydrogen, amino, halo, or hydroxy; alky­ tically acceptable salt or tautomer thereof. In some embodi­ laminoecs12), dialkylaminoecsls), or a substituted version of ments, the compound further defined as: any of these groups; R4 is hydrogen, alkylecss), or substi­ tuted alkylecss), Rs is hydrogen, alkylecss), alkanediylecss)­ 20 heterocycloalkylecss), alkanediylecss)-heteroarylecss), alkanediylecss)-alkylaminoecss), alkanediylecss)-dialky­ (V) laminoecss), or a substituted version of any of these groups;

R8 is hydrogen, alkylecss), alkenylecss), arylecsl 2), aralkylecs 12), acylecss), or a substituted version of any of R1 R2 25 these groups; or -X2-Ru, wherein: X2 is alkanediylecs12) or substituted alkanediylecsl ); and Ru is hydroxy, amino, MeO 2 azido, carboxy, or cyano, alkenylecs 6 ), alkynylecs 6), hetero­ cycloalkylecsl2), alkylaminoecss), dialkylaminoecss), 0 alkoxyecss), or a substituted version of any of these groups;

30 or a -linker-biomolecule; and R9 is -NR12C(O)R13­ NR14R1s; wherein: R12 is hydrogen, alkylecss), or substi­ /0 OH 0 N Rs R{'..- '-~ tuted alkylecss); R13 is alkanediylecss) or substituted alkanediylecss); and R14 and R1s are each independently selected from: hydrogen, alkylecs 12), arylecs 12), 35 aralkylecsl2), acylecsl2), or a substituted version of any of wherein: R1 is hydroxy, alkoxyecss), or substituted these groups; or a pharmaceutically acceptable salt or tau­ alkoxyecss); R2 is hydrogen, amino, halo, or hydroxy; alky­ tomer thereof. In some embodiments, R1 is alkoxyecss) or laminoecsl2), dialkylaminoecsls), or a substituted version of substituted alkoxyecss)· In some embodiments, R1 is alkoxyecs 6). In some embodiments, R1 is methoxy. In other any of these groups; R4 is hydrogen, alkylecss), or substi­ 40 embodiments, R is oxo. In some embodiments, R is hydro­ tuted alkylecss), Rs is hydrogen, alkylecss), alkanediylecss)­ 1 2 gen. In other embodiments, R2 is halo. In some embodi­ heterocycloalkylecss), alkanediylecss)-heteroarylecss), ments, R2 is fluoro, chloro, bromo, or iodo. In other embodi­ alkanediylecss)-alkylaminoecss), alkanediylecss)-dialky­ ments, R2 is hydroxy. In other embodiments, R2 is amino. In laminoecss), or a substituted version of any of these groups; other embodiments, R2 is dialkylaminoecsl2) or substituted 45 dialkylaminoecsl ). In some embodiments, R is dialkylam­ R8 is hydrogen, alkylecss), alkenylecss), arylecsl 2), 2 2 inoecsl2). In some embodiments, R is dimethylamino or aralkylecs 12), acylecss), or a substituted version of any of 2 (2-aminoethyl)methylamino. In some embodiments, R is these groups; or -X -Ru, wherein: X is alkanediylecs ) 4 2 2 12 hydrogen. In some embodiments, Rs is hydrogen. In other or substituted alkanediylecsl2); and Ru is hydroxy, amino, embodiments, Rs is alkanediylecss)-heterocycloalkylecss)· azido, carboxy, or cyano, alkenylecs 6 ), alkynylecs 6 ), hetero­ 50 In some embodiments, Rs is ----CH2CH2N(CH2)4. In other cycloalkylecsl2), alkylaminoecss), dialkylaminoecss), embodiments, Rs is alkanediylecss)-alkylaminoecss)· In alkoxyecss), or a substituted version of any of these groups; some embodiments, Rs is -(CH2)2NH(CH2)4CH(NH2) CO H. In some embodiments, R is hydrogen. In other or a -linker-biomolecule; and R9 is hydrogen, amino, cyano, 2 8 embodiments, R is -X -Ru. In some embodiments, X halo, hydroxy; alkoxyecsl 2), amidoecsl 2), or a substituted 8 2 2 55 is alkanediylecs 6 ) or substituted alkanediylecs 6). In some version of either of these groups; or -NR12C(O)R13­ embodiments, X2 is alkanediylecs 6). In some embodiments, NR R s; wherein: R is hydrogen, alkylecss), or substi­ 14 1 12 X2 is ----CH2CH2CH2- or -CH2CH2-. In some embodi­ tuted alkylecss); R13 is alkanediylecss) or substituted ments, Ru is amino. In other embodiments, Ru is hetero­ alkanediylecss); and R14 and R1s are each independently cycloalkylecsl2), or substituted heterocycloalkylecsl2). In selected from: hydrogen, alkylecs 12), arylecs 12), 60 some embodiments, Ru is 4-N-methyl-piperazinyl. In other embodiments, R is a -linker-biomolecule. In some embodi­ aralkylecs 12), acylecsl 2), or a substituted version of any of 8 these groups; or R and R s are taken together and are ments, the linker is alkanediylecsl2), alkenediylecs12), 14 1 arenediylecs ), heteroarenediylecs ), heterocycloalkanedi­ alkanediylecss), alkoxydiylecss), alkylaminodiylecss), or a 12 12 ylecsl2) or a substituted version of any of these groups. In substituted version of any of these groups; or a pharmaceu­ 65 some embodiments, the biomolecule is an antibody, a pro­ tically acceptable salt or tautomer thereof. In some embodi­ tein, or a small molecule therapeutic agent. In some embodi­ ments, the compound is further defined as: ments, the biomolecule is an antibody. In some embodi­ US 10,065,924 B2 11 12 ments, R9 is hydrogen. In other embodiments, R9 is halo. In described herein and an excipient. In some embodiments, some embodiments, ~ is fluoro or iodo. In other embodi­ the composition is formulated for administration: orally, ments, R9 is amidoccsi 2 ) or substituted amidoccsi 2 ). In some intraadiposally, intraarterially, intraarticularly, intracranially, embodiments, R9 is -NHC(O)CH2 NH2 . In other embodi­ intradermally, intralesionally, intramuscularly, intranasally, ments, R9 is -NR12C(O)R13-NR14R15 . In some embodi­ 5 intraocularly, intrapericardially, intraperitoneally, intrapleu­ ments, R12 is hydrogen. In other embodiments, R12 is rally, intraprostatically, intrarectally, intrathecally, intratra­ alkylccs 6 ) or substituted alkylccs 6). In some embodiments, cheally, intratumorally, intraumbilically, intravaginally, R13 is alkanediylccssy In some embodiments, R13 is intravenously, intravesicularlly, intravitreally, liposomally, -CH2-. In some embodiments, R14 is alkylccsi 2 ) or sub­ locally, mucosally, parenterally, rectally, subconjunctival, stituted alkylccsi ). In some embodiments, R is t-butyl. In 2 14 10 subcutaneously, sublingually, topically, transbuccally, trans­ some embodiments, R is hydrogen. In some embodiments, 15 dermally, vaginally, in cremes, in lipid compositions, via a the compound is further defined as: catheter, via a lavage, via continuous infusion, via infusion, via inhalation, via injection, via local delivery, or via local­ ized perfusion. In some embodiments, the composition is 15 formulated for administration: orally, intravenously, or topi­ cally. In yet another aspect, the present disclosure provides a MeO method of treating a disease or disorder comprising admin­ istering a pharmaceutically effective amount of a compound 20 or composition as described herein. In some embodiments, the disease or disorder is a microbial infection. In some embodiments, the microbial infection is a bacterial infection. In some embodiments, the infection is by a gram positive or gram negative bacteria. In some embodiments, the disease is 25 a bacteria infection by Enterococcus faecalis, Enterococcus faecium, Staphylococcus aureus, Acinetobacter baumannii, Escherichia coli, Acinetobacter calcoaceticus, Staphycococ­ MeO cus epidermidis, Pseudomonas aeruginosa, Klebsiella aero­ genes, Candida albicans, Salmonella typhinurium, Strepto­ 30 coccus pneumoniae, Micrococcus luteus, Bacillus cerues, or Bacillus subtilis. In some embodiments, the disease is a bacteria infection by Staphylococcus aureus 503, Staphylo­ coccus aureus 209, Staphylococcus aureus RN420, Methi­ cillin-resistant Staphylococcus aureus CCARM 3167, 35 Methicillin-resistant Staphylococcus aureus 371, Methicil­ lin-resistant Staphylococcus aureus CCARM 3506, qui­ nolone-resistant Staphylococcus aureus CCARM 3505, qui­ MeO nolone-resistant Staphylococcus aureus CCARM 3519, Bacillus subtilis KCTC 1021, Bacillus cerues KCTC 1661, 40 Micrococcus luteus KCTC 1056, Streptococcus pneumoniae KCTC 3932, Streptococcus pneumoniae KCTC 5412, Enterococcus faecium 501, Enterococcus faecium KCTC 3122, Enterococcus faecalis 5613, Enterococcus faecalis KCTC 5191, Enterococcusfaecalis KCTC 3511, Staphyco­ 45 coccus epidermidis KCTC 3958, Salmonella typhinurium OH H KCTC 1926, Acinetobacter calcoaceticus KCTC 2357, MeO Escherichia coli CCARM 1358, Escherichia coli KCTC 1682, Pseudomonas aeruginosa KCTC 2004, Pseudomonas aeruginosa KCTC 2742, Klebsiella aerogenes KCTC 2619, 0, or 50 Acinetobacter baumannii AB210, or Candida albicans KCTC 7535. In some embodiments, the bacteria is a drug­ OH OH 0 resistant bacteria. In some embodiments, the method further comprises administering a second therapeutic agent. In some embodiments, the second therapeutic agent is an antibiotic. 55 In some embodiments, the second therapeutic agent is a tetracycline antibiotic. In some embodiments, the second MeO therapeutic agent is viridicatumtoxin A, viridicatumtoxin B, vancomycin, tetracycline, spirohexaline, minocycline, tige­ cycline, doxycycline, a ~-lactam antibiotic, an aminoglyco­ 60 side antibiotic, a sulfonamide antibiotic, a macrolide anti­ biotic, a glycopeptide antibioitic, an ansamycin antibiotic, an oxazolidinone antibiotic, a quinolone antibiotic, a strep­ togramin antibiotic, or a lipopeptide antibiotic. In other or a pharmaceutically acceptable salt, tautomer, or optical embodiments, the microbial infection is a viral infection. In isomer thereof. 65 some embodiments, the virus is a poxvirus. In some embodi­ In another aspect, the present disclosure provides a phar­ ments, the poxvirus is variola virus, vaccinia virus, or maceutical composition comprising a compound as molluscum contagiosum. In some embodiments, the method US 10,065,924 B2 13 14 further comprises administering a second therapeutic agent. substituted alkanediylecss); and R14 and R15 are each inde­ In some embodiments, the second therapeutic agent is an pendently selected from: hydrogen, alkylecsl 2 ), arylecs 12), interferon or antiviral compound. In other embodiments, the aralkylecs 12), acylecsl 2 ), or a substituted version of any of disease or disorder is cancer. In some embodiments, the these groups; or R14 and R15 are taken together and are cancer is a carcinoma, sarcoma, lymphoma, leukemia, mela­ 5 alkanediylecss), alkoxydiylecss), alkylaminodiylecss), or a substituted version of any of these groups; and R and R noma, mesothelioma, multiple myeloma, or seminoma. In 20 21 are each independently hydrogen, alkylecss), alkenylecss), some embodiments, the cancer is of the bladder, blood, arylecsl ), aralkylecsl ), acylecss), or a substituted version of bone, brain, breast, central nervous system, cervix, colon, 2 2 any of these groups; or a salt, tautomer, or optical isomer endometrium, esophagus, gall bladder, gastrointestinal tract, thereof; comprising reacting a compound of the formula: genitalia, genitourinary tract, head, kidney, larynx, liver, 10 lung, muscle tissue, neck, oral or nasal mucosa, ovary, pancreas, prostate, skin, spleen, small intestine, large intes­ (VII) tine, stomach, testicle, or thyroid. In some embodiments, the method further comprises administering a second therapeu­ tic agent. In some embodiments, the second therapeutic 15 agent is a second chemotherapeutic agent, radiotherapy, immunotherapy, or surgery. In another aspect, the present disclosure provides a method of inhibiting the activity of a bacterial ribosome for the treatment of a disease or disorder comprising adminis­ 20 tering a compound or composition according to any one of the claims. In still another aspect, the present disclosure provides a method of inhibiting the activity of a bacterial UPP synthase for the treatment of a disease or disorder comprising administering a compound or composition as 25 wherein: Y , Y , Y , R , R ', X , R , R , and R are as described herein 4 5 6 16 16 5 18 19 20 defined above; and X is 0, NH, or S; with a compound of In yet another aspect, the present disclosure provides a 4 the formula: method of preparing a compound of the formula:

30 (VIII) (VI)

35

40 wherein: X3 and R17 are as defined above; R21 is hydrogen, alkylecss), alkenylecss), arylecs 122, aralkylecsl 2 ), acylecss), or a substituted version of any of these groups; and R22 is wherein: Y4 , Y5 , and Y 6 are each independently hydrogen, arylecsl 2 ), aralkylecsl 2 ), or a substituted version of either of alkylecss) or substituted alkyecss); R16 and R16' are each these groups; in the presence of a base. In some embodi­ independently hydrogen, alkoxyecss), aralkoxyecsl 2), sub­ 45 ments, Y4 , Y5 , and Y 6 are alkylecs 6). In some embodiments, stituted alkoxyecss), or substituted aralkoxyecs 12); or R16 Y4 , Y5 , and Y 6 are methyl. In some embodiments, R16 and and R16' are taken together and are alkoxydiylecsl 2 ); R17 is R16' are alkoxyecss) or substituted alkoxyecss)· In some hydrogen, amino, carboxy, cyano, halo, hydroxy, nitro, or embodiments, R16 and R16' are methoxy. In some embodi­ sulfa; alkylecsl 2 ), alkoxyecsl 2 ), alkylaminoecsl2 ), dialky­ ments, R 1 7 is hydrogen, amino, halo, hydroxy; laminoecsls), or a substituted version of any ofthese groups; 50 alkylaminoecsl 2 ), substituted alkylaminoecsl 2 ), dialkylam­ or -C(0)0CH2 CH2 Si(CH3 ) 3 ; X3 is 0, NH, S, or CH2 ; X4 inoecsls), substituted dialkylaminoecsls), or ----C(0) is hydroxy, amino, mercapto, 0, NH, or S provided that 0CH2 CH2 Si(CH3 ) 3 ; In some embodiments, R 1 7 is amino, when X4 is 0, NH, or S, the bond to which the atom is hydroxy, or halo. In other embodiments, R17 is dimethyl­ attached is a double bond and provide that when the bond to amino or 2-aminoethylmethylamino. In some embodiments, which the atom is attached is a double bond, then X4 is 0, 55 X3 and X5 are 0. In some embodiments, X4 is hydroxy. In NH, or S; X5 is 0, NH, or S; R18 is hydrogen, alkylecss), other embodiments, X4 is 0. In some embodiments, R18 is alkenylecss), aryltcsl 2 ), aralkylecsl 2 ), acylecss), or a substi­ hydrogen, alkylecss), substituted alkylecss), or -X2-Ru, tuted version of any of these groups; or -X2-Ru, wherein: X2 is alkanediylecsl 2 ) or substituted wherein: X2 is alkanediylecsl 2 ) or substituted alkanediylecsl 2 ) and Ru is hydroxy, amino, alkanediylecsl 2); and Ru is hydroxy, amino, azido, carboxy, heterocycloalkylecs12), substituted heterocycloalkylecs12), 60 or cyan~, alkenylcrs6 ), alk)'.nylecs 6), heterocycloalkylecsl 2), alkylaminoecss), or substituted alkylaminoecss), In some alkylammoecss), dialkylammoecss), alkoxyecss), or a substi­ embodiments, R18 is hydrogen. In some embodiments, R19 tuted version ofany ofthese groups; R19 is hydrogen, amino, is hydrogen, amino, halo, hydroxy, alkylaminoecsl 2 ), sub­ carboxy, cyano, halo, hydroxy, nitro, or sulfa; alkylecsl 2), stituted alkylaminoecsl2 ), amidoecsl 2 ), substituted alkoxyecsl 2 ), alkylaminoecsl 2 ), dialkylaminoecslS)' amidoecsl 2 ), or-NR12C(0)R13-NR14R15 ; wherein: R12 is amidoecsl 2 ), or a substituted version of any of these groups; 65 hydrogen, R13 is alkanediylecss) or substituted or -NR12C(0)R13-NR14R15 ; wherein: R12 is hydrogen, alkanediylecss); and R14 and R15 are each independently alkylecss), or substituted alkylecss); R13 is alkanediylecss) or hydrogen, alkylecsl 2 ), substituted alkylecsl 2 ), arylecsl 2 ), US 10,065,924 B2 15 16 substituted arylccsi 2 ), aralkylccsi 2 ), substituted stituted alkoxyccss), or substituted aralkoxyccsi 2); or R16 aralkylccsi 2 ), acylccsi 2 ), or substituted acylccsi 2 ). In some and R16' are taken together and are alkoxydiylccsi 2 ); R17 is embodiments, R19 is hydrogen, halo, amidoccsi 2 ), substi­ hydrogen, amino, carboxy, cyano, halo, hydroxy, nitro, or ), tuted amidoccsi2 or -NHC(O)CH2 -NHR15 wherein R15 sulfa; alkylccsi 2 ), alkoxy(csi 2 ), alkylaminoccsi 2 ), dialky­ is hydrogen, alkylccsi 2 ), or substituted alkylccsi 2 ). In some 5 laminoccsis), or a substituted version ofany ofthese groups; embodiments, R is hydrogen, alkylccs ), or substituted 20 6 or -C(O)OCH2 CH2 Si(CH3 ) 3 ; X3 is 0, NH, S, or CH2 ; X4 alkylccs 6 ). In some embodiments, R20 is methyl. In some is 0, NH, or S; X5 is 0, NH, or S; R18 is hydrogen, embodiments, R21 is aralkylccsi 2 ) or substituted alkyl(css), alkenyl(css), aryl(Csl 2 ), aralkyl(Csl 2 ), acyl(css), aralkylccsi 2). In some embodiments, R21 is benzyl. In some or a substituted version of any of these groups; or -X2­ embodiments, R22 is arylccsi 2 ) or substituted arylccsi 2 ). In 10 Ru, wherein: X2 is alkanediylccsi2 ) or substituted some embodiments, R22 is phenyl. In other embodiments, alkanediylccsi ); and Ru is hydroxy, amino, azido, carboxy, R17 is not hydrogen or ----C(O)OCH2 CH2 Si(CH3 ) 3 , R18 is 2 not benzyl, R19 are not hydrogen, and R20 is not methyl. In or cyano, alkenylccs 6 ), alkynylccs 6 ), heterocycloalkylccsi 2 ), some embodiments, the base is an alkoxideccsis)· In some alkylaminoccss), dialkylaminoccss), alkoxyccss), or a substi­ embodiments, the alkoxideccsis) is t-butoxide. In some 15 tuted version ofany ofthese groups; R19 is hydrogen, amino, embodiments, the method comprises adding from about 1.0 carboxy, cyano, halo, hydroxy, nitro, or sulfa; alkylccsi 2), to about 2.0 equivalents of base relative to the compound of alkoxy(csi ), alkylaminoccsi ), dialkylaminoccsis), formula VII. In some embodiments, the method comprises 2 2 amidoccsi ), or a substituted version of any of these groups; adding from about 1.0 to about 1.5 equivalents of base. In 2 or -NR C(O)R -NR R ; wherein: R is hydrogen, some embodiments, the method comprises adding about 1.2 12 13 14 15 12 equivalents of base. In some embodiments, the method 20 alkylccss)), or substituted alkylccss); R13 is alkanediylccss) or substituted alkanediylccss); and R and R are each inde­ comprises adding from about 0.9 to about 2.0 equivalents of 14 15 the compound of formula VIII relative to the compound of pendently selected from: hydrogen, alkylccsi 2 ), arylccsi 2 ), formula VII. In some embodiments, the method comprises aralkylccsi 2 ), acylccsi 2 ), or a substituted version of any of adding from about 1.0 to about 1.5 equivalents of the these groups; or R14 and R15 are taken together and are compound of formula VIII. In some embodiments, the 25 alkanediylccss), alkoxydiylccss), alkylaminodiylccss), or a method comprises adding about 1.1 equivalents of the substituted version of any of these groups; and R20 and R21 compound of formula VIII. In some embodiments, the are each independently hydrogen, alkylccss), alkenylccss), method further comprises a solvent. In some embodiments, arylccsi 2 ), aralkylccsi 2 ), acylccss), or a substituted version of the solvent is an organic solvent. In some embodiments, the any of these groups; or a salt, tautomer, or optical isomer solvent is an areneccsi 2 ). In some embodiments, the solvent 30 thereof, comprising reacting a compound of the formula: is toluene. In some embodiments, the reaction comprises running the reaction at a temperature from about 0° C. to about 50° C. In some embodiments, the temperature is from (X) about 20° C. to about 35° C. In some embodiments, the temperature is about 25° C. In some embodiments, the temperature is about room temperature. In some embodi­ 35 ments, the reaction is run for a time period from about 5 minutes to about 2 hours. In some embodiments, the time period is from about 10 minutes to about 45 minutes. In some embodiments, the time period is about 15 minutes. In some embodiments, the reaction results in a yield of greater 40 than about 50%. In some embodiments, the yield is greater than about 75%. In some embodiments, the yield is greater than about 90%. In some embodiments, the reaction pro­ duces a diastereomeric ratio of greater than about 1: 1. In some embodiments, the diastereomeric ratio is greater than 45 about 1.75:1. In some embodiments, the diastereomeric ratio wherein the variables are as defined above; with a metal is about 2: 1. catalyst and an oxidizing agent. In some embodiments, R 1 7 In some embodiments, the method further comprises is not hydrogen, R18 is not benzyl, R19 is not hydrogen, and forming a compound of the formula: R20 is not methyl. In some embodiments, the metal catalyst 50 is a nickel(II) salt. In some embodiments, the nickel(II) salt

is Ni(acac)2 . In some embodiments, the reaction comprises (IX) adding from about 0.01 to about 1.0 equivalents ofthe metal catalyst relative to the compound of formula X. In some embodiments, the reaction comprises adding from about 0.1 55 to about 0.5 equivalents of the metal catalyst. In some embodiments, the reaction comprises adding about 0.2 equivalents of the metal catalyst. In some embodiments, the oxidizing agent is a dioxirane compound. In some embodi­ ments, the oxidizing agent is dimethyldioxirane (DMDO). In 60 some embodiments, the reaction comprises adding from 0 about 1.5 equivalents to about 10.0 equivalents of dimeth­ I yldioxirane relative to the compound of formula X. In some R21 embodiments, the reaction comprises adding from about 4.0 equivalents to about 6.0 equivalents ofdimethyldioxirane. In wherein: Y4 , Y5 , and Y 6 are each independently hydrogen, 65 some embodiments, the reaction comprises adding about 5.1 alkylccss) or substituted alkyccss); R16 and R16' are each equivalents ofdimethyldioxirane. In some embodiments, the independently hydrogen, alkoxyccss), aralkoxyccsi 2), sub­ method further comprises adding additional dimethyldioxi­ US 10,065,924 B2 17 18 rane every two hours during the reaction. In some embodi­ any of these groups; or -NR12C(O)R13-NR14R15 ; ments, the additional dimethyldioxirane is about 1.5 equiva­ wherein: R12 is hydrogen, alkylccss), or substituted lents relative to the compound of formula X. In some alkylccss); R 13 is alkanediylccss) or substituted embodiments, the method further comprises a solvent. In alkanediylccss); and R14 and R15 are each independently some embodiments, the solvent is an organic solvent. In 5 selected from: hydrogen, alkylccsi 2 ), arylccsi 2 ), aralkylccsi ), acylccsi ), or a substituted version of any of some embodiments, the solvent is a haloalkaneccsi 2 ). In 2 2 some embodiments, the solvent is dichloromethane. In some these groups; or R14 and R15 are taken together and are embodiments, the reaction comprises rurming the reaction at alkanediylccss), alkoxydiylccss), alkylaminodiylccss), or a substituted version of any of these groups; R and R are a temperature from about -90° C. to about -40° C. In some 20 21 embodiments, the temperature is from about -80° C. to 10 each independently hydrogen, alkylccss), alkenylccss), about -60° C. In some embodiments, the temperature is arylccsi 2 ), aralkylccsi 2 ), acylccss), or a substituted version of any of these groups; and R is hydroxy, alkoxyccsi ), or about - 78° C. In some embodiments, the method further 23 2 substituted alkoxyccsi 2 ); or a salt, tautomer, or optical iso­ comprises allowing the reaction to warm to a temperature mer thereof. In some embodiments, the reducing agent is a from about -80° C. to about -30° C. In some embodiments, 15 soft hydride donor. In some embodiments, the reducing the temperature is about -60° C. In some embodiments, the agent is a metal borohydride reagent. In some embodiments, reaction is run for a time period from about 3 hours to about the reducing agent is sodium cyanoborohydride. In some 12 hours. In some embodiments, the time period is from embodiments, the reaction comprises adding from about 5.0 about 5 hours to about 8 hours. In some embodiments, the equivalents to about 25.0 equivalents of reducing agent time period is about 6.5 hours. In some embodiments, the 20 relative to the compound of formula IX. In some embodi­ starting material is recovered after the time period and ments, the reaction comprises adding from about 8.0 equiva­ subject to the reaction conditions again. In some embodi­ lents to about 12.0 equivalents of reducing agent. In some ments, the reaction results in a yield of greater than about embodiments, the reaction comprises adding about 10.0 35% based upon recovered starting material. In some equivalents of reducing agent. In some embodiments, the embodiments, the yield is greater than about 50%. In some 25 method further comprises a solvent. In some embodiments, embodiments, the yield is greater than about 60%. In some the solvent is an organic solvent. In some embodiments, the embodiments, the reaction produces a diastereomeric ratio solvent is a etherccsi 2). In some embodiments, the solvent is of greater than about 1: 1. In some embodiments, the diaste­ tetrahydrofuran. In some embodiments, the reaction com­ reomeric ratio is greater than about 1.75:1. In some embodi­ prises running the reaction at a temperature from about -90° ments, the diastereomeric ratio is about 2:1. 30 C. to about -40° C. In some embodiments, the temperature In some embodiments, the reaction further comprises is from about -80° C. to about -60° C. In some embodi­ reacting a compound offormula IX with a reducing agent to ments, the temperature is about -78° C. In some embodi­ form a compound of the formula: ments, the method further comprises allowing the reaction to warm to a temperature from about -80° C. to about -30° C. 35 In some embodiments, the temperature is about -60° C. In (XI) some embodiments, the reaction is run for a time period from about 30 minutes to about 6 hours. In some embodi­ ments, the time period is from about 1 hour to about 3 hours. In some embodiments, the time period is about 1.5 hours. In 40 some embodiments, the reaction results in a yield of greater than about 20%. In some embodiments, the yield is greater than about 25%. In some embodiments, the yield is greater than about 35%. In some embodiments, the reaction pro­ duces a diastereomeric ratio of greater than about 1: 1. In 45 some embodiments, the diastereomeric ratio is greater than about 1.75: 1. In some embodiments, the diastereomeric ratio is about 2: 1. In some embodiments, one or more steps of the reaction wherein: Y4 , Y5 , and Y 6 are each independently hydrogen, further comprises purifying the reaction in a purification alkylccss) or substituted alkyccss); R16 and R16' are each 50 step. In some embodiments, the purification method is independently hydrogen, alkoxyccss), aralkoxyccsi 2), sub­ chromatography. In some embodiments, the purification stituted alkoxyccss), or substituted aralkoxyccsi 2 ); or R16 method is column chromatography or high performance and R16' are taken together and are alkoxydiylccsi 2 ); R17 is liquid chromatography. hydrogen, amino, carboxy, cyano, halo, hydroxy, nitro, or It is contemplated that any method or compos1t10n sulfa; alkylccsi 2 ), alkoxy(csi 2 ), alkylaminoccsi2 ), dialky­ 55 described herein can be implemented with respect to any lamino(CslS)' or a substituted version ofany ofthese groups; other method or composition described herein. For example, or -C(O)OCH2 CH2 Si(CH3 ) 3 ; X3 is 0, NH, S, or CH2 ; X4 an aldehyde synthesized by one method may be used in the is 0, NH, or S; R18 is hydrogen, alkylccss), alkenylccss), preparation of a final compound according to a different arylccsi 2 ), aralkylccsi 2 ), acylccss), or a substituted version of method. any ofthese groups; or-X2 -Ru, wherein: X2 is alkanedi­ 60 The use ofthe word "a" or "an" when used in conjunction ylccsi 2 ) or substituted alkanediylccsi 2 ); and Ru is hydroxy, with the term "comprising" in the claims and/or the speci­ amino, azido, carboxy, or cyano, alkenylccs 6 ), alkynylccs 6), fication may mean "one," but it is also consistent with the heterocycloalkylccsi2 ), alkylaminoccss), dialkylaminoccss), meaning of "one or more," "at least one," and "one or more alkoxyccss), or a substituted version of any of these groups; than one." The word "about" means plus or minus 5% ofthe

R19 is hydrogen, amino, carboxy, cyano, halo, hydroxy, 65 stated number. nitro, or sulfa; alkylccsi 2 ), alkoxy(csi 2 ), alkylaminoccsi 2 ), Other objects, features and advantages of the present dialkylaminoccsis), amidoccsi 2), or a substituted version of invention will become apparent from the following detailed US 10,065,924 B2 19 20 description. It should be understood, however, that the number 5 is a double bond then R 1 is oxo, alkoxyccss), or detailed description and the specific examples, while indi­ substituted alkoxy(css); R2 is hydrogen, amino, carboxy, cating specific embodiments of the invention, are given by cyano, halo, hydroxy, nitro, or sulfa; alkylccsi 2), way of illustration only, since various changes and modifi­ alkoxyccsi 2 ), alkylaminoccsi 2 ), dialkylaminoccsis), or a sub­ cations within the spirit and scope of the invention will 5 stituted version of any of these groups; R3 , R6 , R7 , and R 10 become apparent to those skilled in the art from this detailed are each independently selected from: hydrogen, alkylccss), description. alkenyl(css), aryl(csi 2 ), aralkyl(csi 2 ), acyl(css), or a substi­ tuted version of any of these groups; R4 and R5 are each BRIEF DESCRIPTION OF THE FIGURES independently selected from: hydrogen, alkylccss), 10 alkanediylccss)-heterocycloalkylccss), alkanediylccss)-het­ The following drawings form part of the present specifi­ eroarylccss), alkanediylccss)-alkylaminoccss), cation and are included to further demonstrate certain alkanediylccss)-dialkylaminoccss), or a substituted version aspects ofthe present invention. The invention may be better of any of these groups; R8 is hydrogen, alkylccss), understood by reference to one or more ofthese drawings in alkenyl(css), aryl(csi 2 ), aralkyl(csi 2 ), acyl(css), or a substi­ combination with the detailed. 15 tuted version of any of these groups; or -X2-Ru, FIGS. lA & B-Structures of bacterial tetracyclines and wherein: X2 is alkanediylccsi2 ) or substituted designed analogs (IA) and fungal tetracyclines (18) alkanediylccsi2 ); and Ru is hydroxy, amino, azido, carboxy,

FIG. 2-Structures ofViridicatumtoxin B and A (1 and 2, or cyano, alkenylccs 6 ), alkynylccs 6 ), heterocycloalkylccsi 2 ), respectively), original proposed structure of Viridicatum­ alkylaminoccss), dialkylaminoccss), alkoxyccss), or a substi­ toxin B, and spirohexaline. 20 tuted version of any of these groups; or a -linker-biomol­ FIG. 3-Biosynthetic Pathway of Viridicatumtoxin A ecule wherein the biomolecule is a protein, a polypeptide, an production in vivo. amino acid, a cofactor, an imaging agent, an antibody, a fatty FIG. 4-Structures ofViridicatumtoxin B and A (1 and 2, acid, a nucleic acid, or a small molecule therapeutic agent; respectively) and analogs (V2-V6). and R9 is hydrogen, amino, carboxy, cyano, halo, hydroxy, 25 nitro, or sulfa; alkylccsi 2 ), alkoxy(csi 2 ), alkylaminoccsi 2 ),

DESCRIPTION OF ILLUSTRATIVE dialkylaminoccsis), amidoccsi2 ), or a substituted version of EMBODIMENTS any of these groups; or -NR12C(O)R13-NR14R15 ; wherein: R12 is hydrogen, alkylccss), or substituted The present disclosure relates to a series of novel analogs alkylccss); R13 is alkanediylccss) or substituted of viridicatumtoxin and an improved synthetic pathway to 30 alkanediylccss); and R14 and R15 are each independently obtain viridicatumtoxin and its analogs. In some aspects, the selected from: hydrogen, alkylccsi 2 ), arylccsi 2 ), analogs of the fungal secondary metabolites viridicatum­ aralkylccsi 2 ), acylccsi 2 ), or a substituted version of any of toxinA (2) and B (1) (FIG. 4) are useful as potent antibiotics these groups; or R14 and R15 are taken together and are against a variety of Gram-positive and certain Gram-nega­ alkanediylccss), alkoxydiylccss), alkylaminodiylccss), or a tive bacterial strains. In the present disclosure, a collection 35 substituted version of any of these groups; or a pharmaceu­ ofviridicatumtoxin analogs (V2-V6, FIG. 4) are synthesized tically acceptable salt or tautomer thereof. and their antibiotic profile is evaluated. These and other Additionally, the compounds provided by the present aspects of the disclosure are described in greater detail disclosure are shown, for example, above in the summary of below. the invention section and in the examples and claims below. 40 They may be made using the methods outlined in the I. Compounds and Formulations Thereof Examples section. Viridicatumtoxin and its derivatives can be synthesized according to the methods described, for In one aspect, the present invention provides compounds example, in the Examples section below. These methods can of the formula: be further modified and optimized using the principles and 45 techniques of organic chemistry as applied by a person skilled in the art. Such principles and techniques are taught, (I) for example, in March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure (2007), which is incorporated by reference herein. 50 Viridicatumtoxin and its derivatives ofthe disclosure may contain one or more asymmetrically-substituted carbon or nitrogen atoms, and may be isolated in optically active or racemic form. Thus, all chiral, diastereomeric, racemic form, epimeric form, and all geometric isomeric forms of a 55 chemical formula are intended, unless the specific stereo­ chemistry or isomeric form is specifically indicated. Com­ pounds may occur as racemates and racemic mixtures, single enantiomers, diastereomeric mixtures and individual diaste­ reomers. In some embodiments, a single diastereomer is wherein: X 1 is absent such that atoms 16 and 17 are only 60 obtained. The chiral centers ofthe compounds ofthe present connected by the shown single bond, a covalent bond such invention can have the S or the R configuration. that a double bond is formed between atoms 16 and 17, Chemical formulas used to represent viridicatumtoxin and

-0-, alkanediylccss), or substituted alkanediylccss); Y 1 , its derivatives of the disclosure will typically only show one Y2 , and Y3 are each independently alkylccsi 2 ) or substituted of possibly several different tautomers. For example, many alkylccsi 2 ); R 1 is hydrogen, hydroxy, or oxo, provided that 65 types of ketone groups are known to exist in equilibrium when R 1 is oxo, the bond between R 1 and atom number 5 is with corresponding enol groups. Similarly, many types of a double bond and when the bond between R 1 and atom imine groups exist in equilibrium with enamine groups. US 10,065,924 B2 21 22 Regardless of which tautomer is depicted for a given com­ trolled release parenteral formulation are carbohydrates pound, and regardless of which one is most prevalent, all (e.g., dextrans), proteins (e.g., albumin), lipoproteins, or tautomers of a given chemical formula are intended. antibodies. Materials for use in implants can be non-biode­ Viridicatumtoxin and its derivatives ofthe disclosure may gradable ( e.g., polydimethyl siloxane) or biodegradable also have the advantage that they may be more efficacious 5 (e.g., poly(caprolactone ), poly(lactic acid), poly(glycolic than, be less toxic than, be longer acting than, be more potent acid) or poly(ortho esters) or combinations thereof). than, produce fewer side effects than, be more easily Formulations for oral use include tablets containing the absorbed than, and/or have a better pharmacokinetic profile active ingredient(s) (e.g., viridicatumtoxin and its deriva­ (e.g., higher oral bioavailability and/or lower clearance) tives) in a mixture with non-toxic pharmaceutically accept­ than, and/or have other useful pharmacological, physical, or 10 able excipients. Such formulations are known to the skilled chemical properties over, compounds known in the prior art, artisan. Excipients may be, for example, inert diluents or whether for use in the indications stated herein or otherwise. fillers (e.g., sucrose, sorbitol, sugar, mannitol, microcrystal­ In addition, atoms making up viridicatumtoxin and its line cellulose, starches including potato starch, calcium derivatives of the present disclosure are intended to include carbonate, sodium chloride, lactose, calcium phosphate, all isotopic forms of such atoms. Isotopes, as used herein, 15 calcium sulfate, or sodium phosphate); granulating and include those atoms having the same atomic number but disintegrating agents ( e.g., cellulose derivatives including different mass numbers. By way of general example and microcrystalline cellulose, starches including potato starch, without limitation, isotopes of hydrogen include tritium and croscarmellose sodium, alginates, or alginic acid); binding deuterium, and isotopes of carbon include 13C and 14C. agents (e.g., sucrose, glucose, sorbitol, acacia, alginic acid, Viridicatumtoxin and its derivatives of the present disclo­ 20 sodium alginate, gelatin, starch, pregelatinized starch, sure may also exist in prodrug form. Since prodrugs are microcrystalline cellulose, magnesium aluminum silicate, known to enhance numerous desirable qualities of pharma­ carboxymethylcellulose sodium, methylcellulose, hydroxy­ ceuticals ( e.g., solubility, bioavailability, manufacturing, propyl methylcellulose, ethylcellulose, polyvinylpyrroli­ etc.), the compounds employed in some methods of the done, or polyethylene glycol); and lubricating agents, gli­ disclosure may, if desired, be delivered in prodrug form. 25 dants, and antiadhesives (e.g., magnesium stearate, zinc Thus, the invention contemplates prodrugs ofcompounds of stearate, stearic acid, silicas, hydrogenated vegetable oils, or the present invention as well as methods of delivering talc). Other pharmaceutically acceptable excipients can be prodrugs. Prodrugs of viridicatumtoxin and its derivatives colorants, flavoring agents, plasticizers, humectants, buffer­ employed in the disclosure may be prepared by modifying ing agents, and the like. functional groups present in the compound in such a way 30 The tablets may be uncoated or they may be coated by that the modifications are cleaved, either in routine manipu­ known techniques, optionally to delay disintegration and lation or in vivo, to the parent compound. Accordingly, absorption in the gastrointestinal tract and thereby providing prodrugs include, for example, compounds described herein a sustained action over a longer period. The coating may be in which a hydroxy, amino, or carboxy group is bonded to adapted to release the active drug in a predetermined pattern any group that, when the prodrug is administered to a 35 (e.g., in order to achieve a controlled release formulation) or subject, cleaves to form a hydroxy, amino, or carboxylic it may be adapted not to release the active drug until after acid, respectively. passage of the stomach (enteric coating). The coating may It should be recognized that the particular anion or cation be a sugar coating, a film coating (e.g., based on hydroxy­ forming a part of any salt form of a compound provided propyl methylcellulose, methylcellulose, methyl hydroxy­ herein is not critical, so long as the salt, as a whole, is 40 ethylcellulose, hydroxypropylcellulose, carboxymethylcel­ pharmacologically acceptable. Additional examples ofphar­ lulose, acrylate copolymers, polyethylene glycols and/or maceutically acceptable salts and their methods of prepara­ polyvinylpyrrolidone), or an enteric coating (e.g., based on tion and use are presented in Handbook ofPharmaceutical methacrylic acid copolymer, cellulose acetate phthalate, Salts: Properties, and Use (2002), which is incorporated hydroxypropyl methylcellulose phthalate, hydroxypropyl herein by reference. 45 methylcellulose acetate succinate, polyvinyl acetate phtha­ Those skilled in the art of organic chemistry will appre­ late, shellac, and/or ethylcellulose). Furthermore, a time ciate that many organic compounds can form complexes delay material, such as, e.g., glyceryl monostearate or glyc­ with solvents in which they are reacted or from which they eryl distearate may be employed. are precipitated or crystallized. These complexes are known as "solvates." For example, a complex with water is known 50 II. Microbial Infections as a "hydrate." Solvates of viridicatumtoxin and its deriva­ tives provided herein are within the scope of the invention. A. Bacterial Infections It will also be appreciated by those skilled in organic In some aspects of the present disclosure, the compounds chemistry that many organic compounds can exist in more disclosed herein may be used to treat a bacterial infection. than one crystalline form. For example, crystalline form may 55 While humans contain numerous different bacteria on and vary from solvate to solvate. Thus, all crystalline forms of inside their bodies, an imbalance in bacterial levels or the viridicatumtoxin and its derivatives or the pharmaceutically introduction ofpathogenic bacteria can cause a symptomatic acceptable solvates thereof are within the scope of the bacterial infection. Pathogenic bacteria cause a variety of present invention. different diseases including but not limited to numerous B. Formulations 60 foodborne illness, typhoid fever, tuberculosis, pneumonia, In some embodiments of the present disclosure, the com­ syphilis, and leprosy. pounds are included a pharmaceutical formulation. Materi­ Additionally, different bacteria have a wide range of als for use in the preparation of microspheres and/or micro­ interactions with body and those interactions can modulate capsules are, e.g., biodegradable/bioerodible polymers such ability of the bacteria to cause an infection. For example, as polygalactin, poly-(isobutyl cyanoacrylate ), poly(2-hy­ 65 bacteria can be conditionally pathogenic such that they only droxyethyl-L-glutam-nine) and, poly(lactic acid). Biocom­ cause an infection under specific conditions. For example, patible carriers that may be used when formulating a con­ Staphylococcus and Streptococcus bacteria exist in the nor­ US 10,065,924 B2 23 24 ma! human bacterial biome, but these bacteria when they are vents the bacteria from releasing the stain when dyed with allowed to colonize other parts of the body causing a skin crystal violet. Without being bound by theory, the gram infection, pneumonia, or sepsis. Other bacteria are known as positive bacteria are often more susceptible to antibiotics. opportunistic pathogens and only cause diseases in a patient Generally, gram positive bacteria, in addition to the thick with a weakened immune system or another disease or 5 peptidoglycan layer, also comprise a lipid monolayer and disorder. contain teichoic acids which react with lipids to form Bacteria can also be intracellular pathogens which can lipoteichoic acids that can act as a chelating agent. Addi­ grow and reproduce within the cells of the host organism. tionally, in gram positive bacteria, the peptidoglycan layer is Such bacteria can be divided into two major categories as outer surface of the bacteria. Many gram positive bacteria either obligate intracellular parasites or facultative intracel­ 10 have been known to cause disease including, but are not lular parasites. Obligate intracellular parasites require the limited to, Streptococcus, Straphylococcus, Corynebacte­ host cell in order to reproduce and include such bacteria as rium, Enterococcus, Listeria, Bacillus, Clostridium, Rathy­ but are not limited to Chlamydophila, Rickettsia, and Ehrli­ bacter, Leifsonia, and Clavibacter. chia which are known to cause pneumonia, urinary tract ii. Gram Negative Bacteria infections, typhus, and Rocky Mountain spotted fever. Fae­ 15 In some aspects of the present disclosure, the compounds ultative intracellular parasites can reproduce either intracel­ disclosed herein may be used to treat a bacterial infection by lular or extracellular. Some non-limiting examples of fac­ a gram negative bacteria. Gram negative bacteria do not ultative intracellular parasites include Salmonella, Listeria, retain the crystal violet stain after washing with alcohol. Legionella, Mycobacterium, and Brucella which are known Gram negative bacteria, on the other hand, have a thin to cause food poisoning, typhoid fever, sepsis, meningitis, 20 peptidoglycan layer with an outer membrane of lipopoly­ Legionnaire's disease, tuberculosis, leprosy, and brucellosis. saccharides and phospholipids as well as a space between Finally, bacterial infections could be targeted to a specific the peptidoglycan and the outer cell membrane called the location in or on the body. For example, bacteria could be periplasmic space. Gram negative bacterial generally do not harmless if only exposed to the specific organs, but when it have teichoic acids or lipoteichoic acids in their outer comes in contact with a specific organ or tissue, the bacteria 25 coating. Generally, gram negative bacteria also release some can begin replicating and cause a bacterial infection. endotoxin and contain prions which act as molecular trans­ In particular, the inventors contemplate treatment of bac­ port units for specific compounds. Most bacteria are gram terial infections, including those caused by Staphyloccoccus negative. Some non-limiting examples of gram negative aureus. S. aureus is a major human pathogen, causing a wide bacteria include Bordetella, Borrelia, Burcelia, Campy­ variety of illnesses ranging from mild skin and soft tissue 30 lobacteria, Escherichia, Francisella, Haemophilus, Helico­ infections and food poisoning to life-threatening illnesses bacter, Legionella, Leptospira, Neisseria, Pseudomonas, such as deep post-surgical infections, septicaemia, endo­ Rickettsia, Salmonella, Shigella, Treponema, Vibrio, and carditis, necrotizing pneumonia, and toxic shock syndrome. Yersinia. These organisms have a remarkable ability to accumulate Iii. Gram Indeterminate Bacteria additional antibiotic resistance determinants, resulting in the 35 In some aspects of the present disclosure, the compounds formation of multiply-drug-resistant strains. disclosed herein may be used to treat a bacterial infection by Methicillin, being the first semi-synthetic penicillin to be a gram indeterminate bacteria. Gram indeterminate bacteria developed, was introduced in 1959 to overcome the problem do not full stain or partially stain when exposed to crystal of penicillin-resistant S. aureus due to ~-lactamase (penicil­ violet. Without being bound by theory, a gram indeterimi­ linase) production (Livermore, 2000). However, methicillin­ 40 nate bacteria may exhibit some ofthe properties of the gram resistant S. aureus (MRSA) strains were identified soon after positive and gram negative bacteria. Anon-limiting example the introduction ofmethicillin (Barber, 1961; Jevons, 1961 ). of a gram indeterminate bacteria include mycobacterium MRSA have acquired and integrated into their genome a 21­ tuberculosis or mycobacterium leprae. to 67-kb mobile genetic element, termed the staphylococcal B. Viral Infections cassette chromosome mec (SCCmec) that harbors the methi­ 45 In some aspects of the present disclosure, the compounds cillin resistance (mecA) gene and other antibiotic resistance disclosed herein may be used to treat a viral infection. determinants (Ito et al., 2001; Ito et al., 2004; Ma et al., Similarly, virus can also exist in pathogenic form which can 2002). The mecA gene encodes an altered additional low lead to human diseases. Viral infections are typically not affinity penicillin-binding protein (PBP2a) that confers treated directly but rather symptomatically since virus often broad resistance to all penicillin-related compounds includ­ 50 have a self-limiting life cycle. Viral infections can also be ing cephalosporins and carbapenems that are currently some more difficult to diagnosis than a bacterial infection since of the most potent broad-spectrum drugs available (Hack­ viral infections often do result in the concombinent increase barth & Chambers, 1989). Since their first identification, in white blood cell counts. Some non-limiting examples of strains of MRSA have spread and become established as pathogenic virus include influenza virus, smallpox, BK major nosocomial (hospital-acquired (HA)-MRSA) patho­ 55 virus, JC virus, human papillomavirus, adenovirus, herpes gens worldwide (Ayliffe, 1997; Crossley et al., 1979; Pan­ simplex type 1, herpes simplex type 2, varicella-zoster virus, lilio et al., 1992; Voss et al., 1994). These organisms have Epstein barr virus, human cytomegalovirus, human herpes­ evolved and emerged as a major cause of community­ virus type 8, Norwalk virus, human bocavirus, rubella virus, acquired infections (CA-MRSA) in healthy individuals lack­ hepatitis E virus, hepatitis B virus, human immunodefi­ ing traditional risk factors for infection, and are causing 60 ciency virus (HIV), Ebola virus, rabies virus, rotavirus, and community-outbreaks, which pose a significant threat to hepatitis D virus. public health. i. Gram Positive Bacteria III. Hyperproliferative Diseases In some aspects of the present disclosure, the compounds disclosed herein may be used to treat a bacterial infection by 65 A. Cancer and Other Hyperproliferative Disease a gram positive bacteria. Gram positive bacteria contain a While hyperproliferative diseases can be associated with thick peptidoglycan layer within the cell wall which pre­ any disease which causes a cell to begin to reproduce US 10,065,924 B2 25 26 uncontrollably, the prototypical example is cancer. One of enchymal chondrosarcoma; giant cell tumor of bone; the key elements ofcancer is that the cell's normal apoptotic ewing's sarcoma; odontogenic tumor, malignant; ameloblas­ cycle is interrupted and thus agents that interrupt the growth tic odontosarcoma; ameloblastoma, malignant; ameloblastic of the cells are important as therapeutic agents for treating fibrosarcoma; pinealoma, malignant; chordoma; glioma, these diseases. In this disclosure, the viridicatumtoxin 5 malignant; ependymoma; astrocytoma; protoplasmic astro­ derivatives may be used to lead to decreased cell counts and cytoma; fibrillary astrocytoma; astroblastoma; glioblastoma; as such can potentially be used to treat a variety of types of oligodendroglioma; oligodendroblastoma; primitive neu­ cancer lines. In various aspects, it is anticipated that the roectodermal; cerebellar sarcoma; ganglioneuroblastoma; viridicatumtoxin derivatives of the present disclosure may neuroblastoma; retinoblastoma; olfactory neurogenic tumor; be used to treat virtually any malignancy. 10 meningioma, malignant; neurofibrosarcoma; neurilem­ Cancer cells that may be treated with the compounds of moma, malignant; granular cell tumor, malignant; malignant the present disclosure include but are not limited to cells lymphoma; hodgkin's disease; hodgkin's; paragranuloma; from the bladder, blood, bone, bone marrow, brain, breast, malignant lymphoma, small lymphocytic; malignant lym­ colon, esophagus, gastrointestine, gum, head, kidney, liver, phoma, large cell, diffuse; malignant lymphoma, follicular; lung, nasopharynx, neck, ovary, prostate, skin, stomach, 15 mycosis fungoides; other specified non-hodgkin's lympho­ pancreas, testis, tongue, cervix, or uterus. In addition, the mas; malignant histiocytosis; multiple myeloma; mast cell cancer may specifically be of the following histological sarcoma; immunoproliferative small intestinal disease; leu­ type, though it is not limited to these: neoplasm, malignant; kemia; lymphoid leukemia; plasma cell leukemia; erythro­ carcinoma; carcinoma, undifferentiated; giant and spindle leukemia; lymphosarcoma cell leukemia; myeloid leukemia; cell carcinoma; small cell carcinoma; papillary carcinoma; 20 basophilic leukemia; eosinophilic leukemia; monocytic leu­ squamous cell carcinoma; lymphoepithelial carcinoma; kemia; mast cell leukemia; megakaryoblastic leukemia; basal cell carcinoma; pilomatrix carcinoma; transitional cell myeloid sarcoma; and hairy cell leukemia. In certain carcinoma; papillary transitional cell carcinoma; adenocar­ aspects, the tumor may comprise an osteosarcoma, angiosar­ cinoma; gastrinoma, malignant; cholangiocarcinoma; coma, rhabdosarcoma, leiomyosarcoma, Ewing sarcoma, hepatocellular carcinoma; combined hepatocellular carci­ 25 glioblastoma, neuroblastoma, or leukemia. noma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous III. Therapies polyp; adenocarcinoma, familial polyposis coli; solid carci­ noma; carcinoid tumor, malignant; branchiolo-alveolar A. Pharmaceutical Formulations and Routes of Adminis­ adenocarcinoma; papillary adenocarcinoma; chromophobe 30 tration carcinoma; acidophil carcinoma; oxyphilic adenocarci­ Where clinical applications are contemplated, it will be noma; basophil carcinoma; clear cell adenocarcinoma; necessary to prepare pharmaceutical compositions in a form granular cell carcinoma; follicular adenocarcinoma; papil­ appropriate for the intended application. In some embodi­ lary and follicular adenocarcinoma; nonencapsulating scle­ ments, such formulation with the compounds of the present rosing carcinoma; adrenal cortical carcinoma; endometroid 35 disclosure is contemplated. Generally, this will entail pre­ carcinoma; skin appendage carcinoma; apocrine adenocar­ paring compositions that are essentially free of pyrogens, as cinoma; sebaceous adenocarcinoma; ceruminous adenocar­ well as other impurities that could be harmful to humans or cinoma; mucoepidermoid carcinoma; cystadenocarcinoma; animals. papillary cystadenocarcinoma; papillary serous cystadeno­ One will generally desire to employ appropriate salts and carcinoma; mucinous cystadenocarcinoma; mucinous 40 buffers to render delivery vectors stable and allow for uptake adenocarcinoma; signet ring cell carcinoma; infiltrating duct by target cells. Buffers also will be employed when recom­ carcinoma; medullary carcinoma; lobular carcinoma; binant cells are introduced into a patient. Aqueous compo­ inflammatory carcinoma; paget's disease, mannnary; acinar sitions ofthe present invention comprise an effective amount cell carcinoma; adenosquamous carcinoma; adenocarci­ of the vector to cells, dissolved or dispersed in a pharma­ noma w/squamous metaplasia; thymoma, malignant; ovar­ 45 ceutically acceptable carrier or aqueous medium. Such com­ ian stromal tumor, malignant; thecoma, malignant; granu­ positions also are referred to as inocula. The phrase "phar­ losa cell tumor, malignant; androblastoma, malignant; maceutically or pharmacologically acceptable" refers to sertoli cell carcinoma; leydig cell tumor, malignant; lipid molecular entities and compositions that do not produce cell tumor, malignant; paraganglioma, malignant; extra­ adverse, allergic, or other untoward reactions when admin­ mammary paraganglioma, malignant; pheochromocytoma; 50 istered to an animal or a human. As used herein, "pharma­ glomangiosarcoma; malignant melanoma; amelanotic mela­ ceutically acceptable carrier" includes any and all solvents, noma; superficial spreading melanoma; malig melanoma in dispersion media, coatings, antibacterial and antifungal giant pigmented nevus; epithelioid cell melanoma; blue agents, isotonic and absorption delaying agents and the like. nevus, malignant; sarcoma; fibrosarcoma; fibrous histiocy­ The use of such media and agents for pharmaceutically toma, malignant; myxosarcoma; liposarcoma; leiomyosar­ 55 active substances is well known in the art. Except insofar as coma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; any conventional media or agent is incompatible with the alveolar rhabdomyosarcoma; stromal sarcoma; mixed vectors or cells of the present invention, its use in therapeu­ tumor, malignant; mullerian mixed tumor; nephroblastoma; tic compositions is contemplated. Supplementary active hepatoblastoma; carcinosarcoma; mesenchymoma, malig­ ingredients also can be incorporated into the compositions. nant; brenner tumor, malignant; phyllodes tumor, malignant; 60 The active compositions of the present invention may synovial sarcoma; mesothelioma, malignant; dysgermi­ include classic pharmaceutical preparations. Administration noma; embryonal carcinoma; teratoma, malignant; struma ofthese compositions according to the present invention will ovarii, malignant; choriocarcinoma; mesonephroma, malig­ be via any common route so long as the target tissue is nant; hemangiosarcoma; hemangioendothelioma, malig­ available via that route. Such routes include oral, nasal, nant; kaposi's sarcoma; hemangiopericytoma, malignant; 65 buccal, rectal, vaginal or topical route. Alternatively, admin­ lymphangiosarcoma; osteosarcoma; juxtacortical osteosar­ istration may be by orthotopic, intradermal, subcutaneous, coma; chondrosarcoma; chondroblastoma, malignant; mes­ intramuscular, intratumoral, intraperitoneal, or intravenous US 10,065,924 B2 27 28 injection. Such compositions would normally be adminis­ potassium bicarbonate. The active ingredient may also be tered as pharmaceutically acceptable compositions, dispersed in dentifrices, including: gels, pastes, powders and described supra. slurries. The active ingredient may be added in a therapeu­ The active compounds may also be administered paren­ tically effective amount to a paste dentifrice that may include terally or intraperitoneally. Solutions of the active com­ 5 water, binders, abrasives, flavoring agents, foaming agents, pounds as free base or pharmacologically acceptable salts and humectants. can be prepared in water suitably mixed with a surfactant, The compositions of the present disclosure may be for­ such as hydroxypropylcellulose. Dispersions can also be mulated in a neutral or salt form. Pharmaceutically-accept­ prepared in glycerol, liquid polyethylene glycols, and mix­ able salts include the acid addition salts (formed with the tures thereof and in oils. Under ordinary conditions of 10 free amino groups ofthe protein) and which are formed with storage and use, these preparations contain a preservative to inorganic acids such as, for example, hydrochloric or phos­ prevent the growth of microorganisms. phoric acids, or such organic acids as acetic, oxalic, tartaric, The pharmaceutical forms suitable for injectable use mandelic, and the like. Salts formed with the free carboxyl include sterile aqueous solutions or dispersions and sterile groups can also be derived from inorganic bases such as, for powders for the extemporaneous preparation of sterile 15 example, sodium, potassium, animonium, calcium, or ferric injectable solutions or dispersions. In all cases the form must hydroxides, and such organic bases as isopropylamine, be sterile and must be fluid to the extent that easy syring­ trimethylamine, histidine, procaine and the like. ability exists. It must be stable under the conditions of Upon formulation, solutions will be administered in a manufacture and storage and must be preserved against the manner compatible with the dosage formulation and in such contaminating action of microorganisms, such as bacteria 20 amount as is therapeutically effective. The formulations are and fungi. The carrier can be a solvent or dispersion medium easily administered in a variety of dosage forms such as containing, for example, water, ethanol, polyol (for example, injectable solutions, drug release capsules and the like. For glycerol, propylene glycol, and liquid polyethylene glycol, parenteral administration in an aqueous solution, for and the like), suitable mixtures thereof, and vegetable oils. example, the solution should be suitably buffered if neces­ The proper fluidity can be maintained, for example, by the 25 sary and the liquid diluent first rendered isotonic with use of a coating, such as lecithin, by the maintenance of the sufficient saline or glucose. These particular aqueous solu­ required particle size in the case ofdispersion and by the use tions are especially suitable for intravenous, intramuscular, of surfactants. The prevention of the action of microorgan­ subcutaneous and intraperitoneal administration. In this con­ isms can be brought about by various antibacterial and nection, sterile aqueous media which can be employed will antifungal agents, for example, parabens, chlorobutanol, 30 be known to those of skill in the art in light of the present phenol, sorbic acid, thimerosal, and the like. In many cases, disclosure. For example, one dosage could be dissolved in 1 it will be preferable to include isotonic agents, for example, ml of isotonic NaCl solution and either added to 1000 ml of sugars or sodium chloride. Prolonged absorption of the hypodermoclysis fluid or injected at the proposed site of injectable compositions can be brought about by the use in infusion, (see for example, "Remington's Pharmaceutical the compositions of agents delaying absorption, for 35 Sciences," 15th Edition, pages 1035-1038 and 1570-1580). example, aluminum monostearate and gelatin. Some variation in dosage will necessarily occur depending Sterile injectable solutions are prepared by incorporating on the condition of the subject being treated. The person the active compounds in the required amount in the appro­ responsible for administration will, in any event, determine priate solvent with various of the other ingredients enumer­ the appropriate dose for the individual subject. Moreover, ated above, as required, followed by filtered sterilization. 40 for human administration, preparations should meet sterility, Generally, dispersions are prepared by incorporating the pyrogenicity, general safety and purity standards as required various sterilized active ingredients into a sterile vehicle by FDA Office of Biologics standards. which contains the basic dispersion medium and the B. Methods of Treatment required other ingredients from those enumerated above. In In particular, the compositions that may be used in treat­ the case of sterile powders for the preparation of sterile 45 ing microbial infections and cancer in a subject (e.g., a injectable solutions, the preferred methods of preparation human subject) are disclosed herein. The compositions are vacuum-drying and freeze-drying techniques which described above are preferably administered to a mammal yield a powder of the active ingredient plus any additional (e.g., rodent, human, non-human primates, canine, bovine, desired ingredient from a previously sterile-filtered solution ovine, equine, feline, etc.) in an effective amount, that is, an thereof. 50 amount capable of producing a desirable result in a treated As used herein, "pharmaceutically acceptable carrier" subject ( e.g., causing apoptosis of cancerous cells or killing includes any and all solvents, dispersion media, coatings, bacterial cells). Toxicity and therapeutic efficacy of the antibacterial and antifungal agents, isotonic and absorption compositions utilized in methods of the invention can be delaying agents and the like. The use of such media and determined by standard pharmaceutical procedures. As is agents for pharmaceutical active substances is well known in 55 well known in the medical and veterinary arts, dosage for the art. Except insofar as any conventional media or agent is any one animal depends on many factors, including the incompatible with the active ingredient, its use in the thera­ subject's size, body surface area, body weight, age, the peutic compositions is contemplated. Supplementary active particular composition to be administered, time and route of ingredients can also be incorporated into the compositions. administration, general health, the clinical symptoms of the For oral administration viridicatumtoxin and its deriva­ 60 infection or cancer and other drugs being administered tives may be incorporated with excipients and used in the concurrently. A composition as described herein is typically form of non-ingestible mouthwashes and dentifrices. A administered at a dosage that inhibits the growth or prolif­ mouthwash may be prepared incorporating the active ingre­ eration ofa bacterial cell, inhibits the growth ofa biofilm, or dient in the required amount in an appropriate solvent, such induces death of cancerous cells (e.g., induces apoptosis of as a sodium borate solution (Dobell's Solution). Alterna­ 65 a cancer cell), as assayed by identifying a reduction in tively, the active ingredient may be incorporated into an hematological parameters (Complete blood count (CBC)), antiseptic wash containing sodium borate, glycerin and or cancer cell growth or proliferation. In some embodiments, US 10,065,924 B2 29 30 in the experiments described herein and based upon dosing amount effective to achieve a reduction in one or more of other tetracycline compounds, the amount of the viridi­ disease parameter. This process may involve contacting the catumtoxin derivatives used to inhibit bacterial growth, treat cells/subjects with the both agents/therapies at the same a viral infection, or induce apoptosis of the cancer cells is time, e.g., using a single composition or pharmacological calculated to be from about 0.01 mg to about 10,000 mg/day. 5 formulation that includes both agents, or by contacting the In some embodiments, the amount is from about 1 mg to cell/subject with two distinct compositions or formulations, about 1,000 mg/day. In some embodiments, these aminoglycosides, toms thereof associated with cancer (e.g., leukemia) in quinolones (including fluoroquinolones), sulfonamides and which the subject has been administered a therapeutic tetracylcines. In particular, one may use combination thera­ amount of a composition as described herein. The level of 40 pies for treating MRSA. Both CA-MRSA and HA-MRSA marker determined in the method can be compared to known are resistant to traditional anti-staphylococcal beta-lactam levels ofmarker in either healthy normal controls or in other antibiotics, such as cephalexin. CA-MRSA has a greater afflicted patients to establish the subject's disease status. In spectrum of antimicrobial susceptibility, including to sulfa preferred embodiments, a second level of marker in the drugs (like co-trimoxazole/trimethoprim-sulfamethox­ subject is determined at a time point later than the determi­ 45 azole), tetracyclines (like doxycycline and minocycline) and nation of the first level, and the two levels are compared to clindamycin (for osteomyelitis), but the drug of choice for monitor the course of disease or the efficacy of the therapy. treating CA-MRSA is now believed to be vancomycin, In certain preferred embodiments, a pre-treatment level of according to a Henry Ford Hospital Study. HA-MRSA is marker in the subject is determined prior to beginning resistant even to these antibiotics and often is susceptible treatment according to the methods described herein; this 50 only to vancomycin. Newer drugs, such as linezolid (belong­ pre-treatment level of marker can then be compared to the ing to the newer oxazolidinones class) and daptomycin, are level of marker in the subject after the treatment com­ effective against both CA-MRSA and HA-MRSA. The mences, to determine the efficacy of the treatment. Infectious Disease Society of America recommends vanco­ C. Combination Therapies mycin, linezolid, or clindamycin (if susceptible) for treating It is envisioned that the viridicatumtoxin derivatives 55 patients with MRSA pneumonia. Ceftaroline, a fifth genera­ described herein may be used in combination therapies with tion cephalosporin, is the first beta-lactam antibiotic an additional antimicrobial agent such as anti-viral, antibi­ approved in the U.S. to treat MRSA infections (skin and soft otic, or a compound which mitigates one or more ofthe side tissue or community acquired pneumonia only). effects experienced by the patient. Quinolone-resistant S. aureus is another emerging patho­ Furthermore, it is very common in the field of cancer 60 gen that may be treated with compounds according to the therapy to combine therapeutic modalities. The following is present invention, optionally in combination with vancomy­ a general discussion of therapies that may be used in cin, teicoplanin and linezolid. conjunction with the therapies of the present disclosure. Vancomycin and teicoplanin are glycopeptide antibiotics To treat cancers using the methods and compositions of used to treat MRSA infections. Teicoplanin is a structural the present disclosure, one would generally contact a tumor 65 congener ofvancomycin that has a similar activity spectrum cell or subject with a compound and at least one other but a longer half-life. Because the oral absorption of van­ therapy. These therapies would be provided in a combined comycin and teicoplanin is very low, these agents must be US 10,065,924 B2 31 32 administered intravenously to control systemic infections. oxytetracycline, or tetracycline. In some embodiments, the Treatment of MRSA infection with vancomycin can be compounds could be combined with a drug which acts complicated, due to its inconvenient route of administration. against mycobacteria such as cycloserine, capreomycin, Moreover, many clinicians believe that the efficacy of van­ ethionamide, rifampicin, rifabutin, rifapentine, and strepto­ comycin against MRSA is inferior to that of anti-staphylo­ 5 mycin. Other antibiotics that are contemplated for combi­ coccal beta-lactam antibiotics against methicillin-suscep­ nation therapies may include arsphenamine, chlorampheni­ tible Staphylococcus aureus (MSSA). col, fosfomycin, fusidic acid, metronidazole, mupirocin, Several newly discovered strains of MRSA show antibi­ platensimycin, quinupristin, dalfopristin, thiamphenicol, otic resistance even to vancomycin and teicoplanin. These tigecycline, tinidazole, or trimethoprim. new evolutions of the MRSA bacterium have been dubbed 10 2. Antivirals Vancomycin intermediate-resistant Staphylococcus aureus The term "antiviral" or "antiviral agents" are drugs which (VISA). Linezolid, quinupristin/dalfopristin, daptomycin, may be used to treat a viral infection. In general, antiviral ceftaroline, and tigecycline are used to treat more severe agents act via two major mechanisms: preventing viral entry infections that do not respond to glycopeptides such as into the cell and inhibiting viral synthesis. Without being vancomycin. Current guidelines recommend daptomycin for 15 bound by theory, viral replication can be inhibited by using VISA bloodstream infections and endocarditis. agents that mimic either the virus-associated proteins and Studies suggest that allicin, a compound found in garlic, thus block the cellular receptors or using agents that mimic may prove to be effective in the treatment of MRSA. the cellular receptors and thus block the virus-associated Other combinations are contemplated. The following is a proteins. Furthermore, agents which cause an uncoating of general discussion of antibiotic, antiviral, and cancer thera­ 20 the virus can also be used as antiviral agents. pies that may be used in combination with the compounds of The second mechanism ofviral inhibition is preventing or the present disclosure. interrupting viral synthesis. Such drugs can target different 1. Antibiotics proteins associated with the replication ofviral DNA includ­ The term "antibiotics" are drugs which may be used to ing reverse transcriptase, integrase, transcription factors, or treat a bacterial infection through either inhibiting the 25 ribozymes. Additionally, the therapeutic agent interrupts growth of bacteria or killing bacteria. Without being bound translation by acting as an antisense DNA strain, inhibiting by theory, it is believed that antibiotics can be classified into the formation ofprotein processing or assembly, or acting as two major classes: bactericidal agents that kill bacteria or virus protease inhibitors. Finally, an anti-viral agent could bacteriostatic agents that slow down or prevent the growth additionally inhibit the release of the virus after viral pro­ of bacteria. 30 duction in the cell. The first commercially available antibiotic was released in Additionally, anti-viral agents could modulate the bodies the 1930's. Since then, many different antibiotics have been own immune system to fight a viral infection. Without being developed and widely prescribed. In 2010, on average, 4 in bound by theory, the anti-viral agent which stimulates the 5 Americans are prescribed antibiotics annually. Given the immune system may be used with a wide variety of viral prevalence of anitbiotics, bacteria have started to develop 35 infections. resistance to specific antibiotics and antibiotic mechanisms. In some embodiments, the present disclosure provides Without being bound by theory, the use of antibiotics in methods ofusing the disclosed compounds in a combination combination with another antibiotic may modulate resis­ therapy with an anti-viral agent as described above. In some tance and enhance the efficacy of one or both agents. non-limiting examples, the anti-viral agent is abacavir, aci­ In some embodiments, antibiotics can fall into a wide 40 clovir, acyclovir, adefovir, amantadine, amprenavir, ampli­ range of classes. In some embodiments, the compounds of gen, arbidol, atazanavir, atripla, balavir, boceprevirertet, the present disclosure may be used in conjunction with cidofovir, combivir, dolutegravir, daruavir, delavirdine, another antibiotic. In some embodiments, the compounds didanosine, docosanol, edoxudine, efavirenz, emtricitabine, may be used in conjunction with a narrow spectrum antibi­ enfuvirtide, entecavir, ecoliever, famciclovir, fomivirsen, otic which targets a specific bacteria type. In some non- 45 fosamprenavir, foscarnet, fosfonet, ganciclovir, ibacitabine, limiting examples of bactericidal antibiotics include peni­ imunovir, idoxuridine, imiquimod, indinavir, inosine, inter­ cillin, cephalosporin, polymyxin, rifamycin, lipiarmycin, feron type I, type II, and type III, lamivudine, lopinavir, quinolones, and sulfonamides. In some non-limiting loviride, maraviroc, moroxydine, methisazone, nelfinavir, examples of bacteriostatic antibiotics include macrolides, nevirapine, nexavir, oseltamivir, penciclovir, peramivir, ple­ lincosamides, or tetracyclines. In some embodiments, the 50 conaril, podophyllotoxin, raltegravir, ribavirin, rimantadine, antibiotic is an aminoglycoside such as kanamycin and ritonavir, pyramidine, saquinavir, sofosbuvir, stavudine, tel­ streptomycin, an ansamycin such as rifaximin and geldan­ aprevir, tenofovir, tenofovir disoproxil, tipranavir, trifluri­ amycin, a carbacephem such as loracarbef, a carbapenem dine, trizivir, tromantadine, truvada, traporved, valaciclovir, such as ertapenem, imipenem, a cephalosporin such as valganciclovir, vicriviroc, vidarabine, viramidine, zalcit­ cephalexin, cefixime, cefepime, and ceftobiprole, a glyco­ 55 abine, zanamivir, or zidovudine. In some embodiments, the peptide such as vancomycin or teicoplanin, a lincosamide anti-viral agents is an anti-retroviral, a fusion inhibitor, an such as lincomycin and clindamycin, a lipopeptide such as integrase inhibitor, an interferon, a nucleoside analogues, a daptomycin, a macrolide such as clarithromycin, spiramy­ protease inhibitor, a reverse transcriptase inhibitor, a syner­ cin, azithromycin, and telithromycin, a monobactam such as gistic enhancer, or a natural product such as tea tree oil. aztreonam, a nitrofuran such as furazolidone and nitrofuran­ 60 3. Chemotherapy toin, an oxazolidonones such as linezolid, a penicillin such The term "chemotherapy" refers to the use of drugs to as amoxicillin, azlocillin, flucloxacillin, and penicillin G, an treat cancer. A "chemotherapeutic agent" is used to connote antibiotic polypeptide such as bacitracin, polymyxin B, and a compound or composition that is administered in the colistin, a quinolone such as ciprofloxacin, levofloxacin, and treatment of cancer. These agents or drugs are categorized gatifloxacin, a sulfonamide such as silver sulfadiazine, 65 by their mode of activity within a cell, for example, whether mefenide, sulfadimethoxine, or sulfasalazine, or a tetracy­ and at what stage they affect the cell cycle. Alternatively, an cline such as demeclocycline, doxycycline, minocycline, agent may be characterized based on its ability to directly US 10,065,924 B2 33 34 cross-link DNA, to intercalate into DNA, or to induce doxetaxel; chlorambucil; gemcitabine; 6-thioguanine; mer­ chromosomal and mitotic aberrations by affecting nucleic captopurine; methotrexate; platinum coordination com­ acid synthesis. Most chemotherapeutic agents fall into the plexes such as cisplatin, oxaliplatin and carboplatin; vin­ following categories: alkylating agents, antimetabolites, blastine; platinum; etoposide (VP-16); ifosfamide; antitumor antibiotics, mitotic inhibitors, and nitrosoureas. 5 mitoxantrone; vincristine; vinorelbine; novantrone; tenipo­ Examples of chemotherapeutic agents include alkylating side; edatrexate; daunomycin; aminopterin; xeloda; ibandro­ agents such as thiotepa and cyclosphosphamide; alkyl sul­ nate; irinotecan (e.g., CPT-11 ); topoisomerase inhibitor RFS fonates such as busulfan, improsulfan and piposulfan; aziri­ 2000; difluorometlhylomithine (DMFO); retinoids such as dines such as benzodopa, carboquone, meturedopa, and retinoic acid; capecitabine; cisplatin (CDDP), carboplatin, uredopa; ethylenimines and methylamelamines including 10 procarbazine, mechlorethamine, cyclophosphamide, camp­ altretamine, triethy lenemelamine, triety lenephosphoramide, tothecin, ifosfamide, melphalan, chlorambucil, busulfan, triethiylenethiophosphoramide and trimethylolomelamine; nitrosurea, dactinomycin, daunorubicin, doxorubicin, bleo­ acetogenins (especially bullatacin and bullatacinone); a mycin, plicomycin, mitomycin, etoposide (VP16), tamox­ camptothecin (including the synthetic analogue topotecan); ifen, raloxifene, estrogen receptor binding agents, taxol, bryostatin; callystatin; CC-1065 (including its adozelesin, 15 paclitaxel, docetaxel, gemcitabien, navelbine, famesyl-pro­ carzelesin and bizelesin synthetic analogues); cryptophycins tein tansferase inhibitors, transplatinum, 5-fluorouracil, vin­ (particularly cryptophycin 1 and cryptophycin 8); dolastatin; cristin, vinblastin and methotrexate and pharmaceutically duocarmycin (including the synthetic analogues, KW-2189 acceptable salts, acids or derivatives of any of the above. and CBl-TMl); eleutherobin; pancratistatin; a sarcodictyin; 4. Radiotherapy spongistatin; nitrogen mustards such as chlorambucil, chlo­ 20 Radiotherapy, also called radiation therapy, is the treat­ maphazine, cholophosphamide, estramustine, ifosfamide, ment of cancer and other diseases with ionizing radiation. mechlorethamine, mechlorethamine oxide hydrochloride, Ionizing radiation deposits energy that injures or destroys melphalan, novembichin, phenesterine, prednimustine, tro­ cells in the area being treated by damaging their genetic fosfamide, uracil mustard; nitrosureas such as carmustine, material, making it impossible for these cells to continue to chlorozotocin, fotemustine, lomustine, nimustine, and ran­ 25 grow. Although radiation damages both cancer cells and imnustine; antibiotics such as the enediyne antibiotics (e.g., normal cells, the latter are able to repair themselves and calicheamicin, especially calicheamicin gamma!I and cali­ function properly. cheamicin omegall; dynemicin, including dynemicin A Radiation therapy used according to the present invention uncialamycin and derivatives thereof; bisphosphonates, may include, but is not limited to, the use ofy-rays, X-rays, such as clodronate; an esperamicin; as well as neocarzinos­ 30 and/or the directed delivery of radioisotopes to tumor cells. tatin chromophore and related chromoprotein enediyne Other forms of DNA damaging factors are also contem­ antiobiotic chromophores, aclacinomysins, actinomycin, plated such as microwaves and UV-irradiation. It is most authramycin, azaserine, bleomycins, cactinomycin, carabi­ likely that all of these factors induce a broad range of cin, carminomycin, carzinophilin, chromomycinis, dactino­ damage on DNA, on the precursors of DNA, on the repli­ mycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleu­ 35 cation and repair of DNA, and on the assembly and main­ cine, doxorubicin (including morpholino-doxorubicin, tenance of chromosomes. Dosage ranges for X-rays range cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin from daily doses of 50 to 200 roentgens for prolonged and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, periods of time (3 to 4 wk), to single doses of 2000 to 6000 marcellomycin, mitomycins such as mitomycin C, myco­ roentgens. Dosage ranges for radioisotopes vary widely, and phenolic acid, nogalamycin, olivomycins, peplomycin, pot­ 40 depend on the half-life of the isotope, the strength and type firomycin, puromycin, quelamycin, rodorubicin, streptoni­ of radiation emitted, and the uptake by the neoplastic cells. grin, streptozocin, tubercidin, ubenimex, zinostatin, Radiotherapy may comprise the use of radiolabeled anti­ zorubicin; anti-metabolites such as methotrexate and 5-fluo­ bodies to deliver doses ofradiation directly to the cancer site rouracil (5-FU); folic acid analogues such as denopterin, (radioimmunotherapy). Antibodies are highly specific pro­ methotrexate, pteropterin, trimetrexate; purine analogs such 45 teins that are made by the body in response to the presence as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; ofantigens (substances recognized as foreign by the immune pyrimidine analogs such as ancitabine, azacitidine, 6-azau­ system). Some tumor cells contain specific antigens that ridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, trigger the production of tumor-specific antibodies. Large enocitabine, floxuridine; androgens such as calusterone, quantities of these antibodies can be made in the laboratory dromostanolone propionate, epitiostanol, mepitiostane, tes­ 50 and attached to radioactive substances (a process known as tolactone; anti-adrenals such as aminoglutethimide, mito­ radiolabeling). Once injected into the body, the antibodies tane, trilostane; folic acid replenisher such as frolinic acid; actively seek out the cancer cells, which are destroyed by the aceglatone; aldophosphamide glycoside; aminolevulinic cell-killing (cytotoxic) action ofthe radiation. This approach acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatrax­ can minimize the risk of radiation damage to healthy cells. ate; defofamine; demecolcine; diaziquone; elformithine; 55 Conformal radiotherapy uses the same radiotherapy elliptinium acetate; an epothilone; etoglucid; gallium nitrate; machine, a linear accelerator, as the normal radiotherapy hydroxyurea; lentinan; lonidainine; maytansinoids such as treatment but metal blocks are placed in the path ofthe x-ray maytansine and ansamitocins; mitoguazone; mitoxantrone; beam to alter its shape to match that of the cancer. This mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; ensures that a higher radiation dose is given to the tumor. losoxantrone; podophyllinic acid; 2-ethylhydrazide; procar­ 60 Healthy surrounding cells and nearby structures receive a bazine; PSK polysaccharide complex); razoxane; rhizoxin; lower dose of radiation, so the possibility of side effects is sizofiran; spirogermanium; tenuazonic acid; triaziquone; reduced. A device called a multi-leaf collimator has been 2,2' ,2"-trichlorotriethylamine; trichothecenes ( especially developed and may be used as an alternative to the metal T-2 toxin, verracurin A, roridin A and anguidine); urethan; blocks. The multi-leaf collimator consists of a number of vindesine; dacarbazine; mannomustine; mitobronitol; mito­ 65 metal sheets which are fixed to the linear accelerator. Each lactol; pipobroman; gacytosine; arabinoside ("Ara-C"); layer can be adjusted so that the radiotherapy beams can be cyclophosphamide; thiotepa; taxoids, e.g., paclitaxel and shaped to the treatment area without the need for metal US 10,065,924 B2 35 36 blocks. Precise positioning of the radiotherapy machine is bovis, Plasmodium falciparum, dinitrochlorobenzene and very important for conformal radiotherapy treatment and a aromatic compounds (U.S. Pat. Nos. 5,801,005 and 5,739, special scanning machine may be used to check the position 169; Hui and Hashimoto, 1998; Christodoulides et al., of internal organs at the beginning of each treatment. 1998), cytokine therapy, e.g., interferons a, ~' and y; IL-1, High-resolution intensity modulated radiotherapy also 5 GM-CSF and TNF (Bukowski et al., 1998; Davidson et al., uses a multi-leaf collimator. During this treatment the layers 1998; Hellstrand et al., 1998) gene therapy, e.g., TNF, IL-1, ofthe multi-leaf collimator are moved while the treatment is IL-2, p53 (Qin et al., 1998; Austin-Ward and Villaseca, being given. This method is likely to achieve even more 1998; U.S. Pat. Nos. 5,830,880 and 5,846,945) and mono­ precise shaping of the treatment beams and allows the dose clonal antibodies, e.g., anti-ganglioside GM2, anti-HER-2, ofradiotherapy to be constant over the whole treatment area. 10 anti-p185 (Pietras et al., 1998; Hanibuchi et al., 1998; U.S. Although research studies have shown that conformal Pat. No. 5,824,311). It is contemplated that one or more radiotherapy and intensity modulated radiotherapy may anti-cancer therapies may be employed with the gene silenc­ reduce the side effects of radiotherapy treatment, it is ing therapies described herein. possible that shaping the treatment area so precisely could In active immunotherapy, an antigenic peptide, polypep­ stop microscopic cancer cells just outside the treatment area 15 tide or protein, or an autologous or allogenic tumor cell being destroyed. This means that the risk of the cancer composition or "vaccine" is administered, generally with a coming back in the future may be higher with these spe­ distinct bacterial adjuvant (Ravindranath and Morton, 1991; cialized radiotherapy techniques. Morton et al., 1992; Mitchell et al., 1990; Mitchell et al., Scientists also are looking for ways to increase the 1993). effectiveness of radiation therapy. Two types of investiga­ 20 In adoptive immunotherapy, the patient's circulating lym­ tional drugs are being studied for their effect on cells phocytes, or tumor infiltrated lymphocytes, are isolated in undergoing radiation. Radiosensitizers make the tumor cells vitro, activated by lymphokines such as IL-2 or transduced more likely to be damaged, and radioprotectors protect with genes for tumor necrosis, and readministered (Rosen­ normal tissues from the effects of radiation. Hyperthermia, berg et al., 1988; 1989). the use of heat, is also being studied for its effectiveness in 25 6. Surgery sensitizing tissue to radiation. Approximately 60% of persons with cancer will undergo 5. Immunotherapy surgery of some type, which includes preventative, diagnos­ In the context of cancer treatment, immunotherapeutics, tic or staging, curative, and palliative surgery. Curative generally, rely on the use of immune effector cells and surgery is a cancer treatment that may be used in conjunction molecules to target and destroy cancer cells. Trastuzumab 30 with other therapies, such as the treatment of the present (HERCEPTIN™) is such an example. The immune effector invention, chemotherapy, radiotherapy, hormonal therapy, may be, for example, an antibody specific for some marker gene therapy, immunotherapy and/or alternative therapies. on the surface ofa tumor cell. The antibody alone may serve Curative surgery includes resection in which all or part of as an effector of therapy or it may recruit other cells to cancerous tissue is physically removed, excised, and/or actually affect cell killing. The antibody also may be con­ 35 destroyed. Tumor resection refers to physical removal of at jugated to a drug or toxin ( chemotherapeutic, radionuclide, least part of a tumor. In addition to tumor resection, treat­ ricin A chain, cholera toxin, pertussis toxin, etc.) and serve ment by surgery includes laser surgery, cryosurgery, elec­ merely as a targeting agent. Alternatively, the effector may trosurgery, and microscopically controlled surgery (Mohs' be a lymphocyte carrying a surface molecule that interacts, surgery). It is further contemplated that the present invention either directly or indirectly, with a tumor cell target. Various 40 may be used in conjunction with removal of superficial effector cells include cytotoxic T cells and NK cells. The cancers, precancers, or incidental amounts of normal tissue. combination of therapeutic modalities, i.e., direct cytotoxic Upon excision of part or all of cancerous cells, tissue, or activity and inhibition or reduction of ErbB2 would provide tumor, a cavity may be formed in the body. Treatment may therapeutic benefit in the treatment ofErbB2 overexpressing be accomplished by perfusion, direct injection or local cancers. 45 application of the area with an additional anti-cancer In one aspect ofimmunotherapy, the tumor cell must bear therapy. Such treatment may be repeated, for example, every some marker that is amenable to targeting, i.e., is not present 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5 weeks on the majority ofother cells. Many tumor markers exist and or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. These any of these may be suitable for targeting in the context of treatments may be of varying dosages as well. the present invention. Common tumor markers include 50 In some particular embodiments, after removal of the carcinoembryonic antigen, prostate specific antigen, urinary tumor, an adjuvant treatment with a compound ofthe present tumor associated antigen, fetal antigen, tyrosinase (p97), disclosure is believe to be particularly efficacious in reduc­ gp68, TAG-72, HMFG, Sialyl Lewis Antigen, MucA, ing the reoccurance of the tumor. Additionally, the com­ MucB, PLAP, estrogen receptor, laminin receptor, erb B and pounds of the present disclosure can also be used in a p 155. An alternative aspect ofimmunotherapy is to combine 55 neoadjuvant setting. anticancer effects with immune stimulatory effects. Immune 7. Other Agents stimulating molecules also exist including: cytokines such It is contemplated that other agents may be used with the as IL-2, IL-4, IL-12, GM-CSF, y-IFN, chemokines such as present invention. These additional agents include immuno­ MIP-1, MCP-1, IL-8 and growth factors such as FLT3 modulatory agents, agents that affect the upregulation ofcell ligand. Combining immune stimulating molecules, either as 60 surface receptors and GAP junctions, cytostatic and differ­ proteins or using gene delivery in combination with a tumor entiation agents, inhibitors of cell adhesion, agents that suppressor has been shown to enhance anti-tumor effects (Ju increase the sensitivity of the hyperproliferative cells to et al., 2000). Moreover, antibodies against any of these apoptotic inducers, or other biological agents. Immuno­ compounds may be used to target the anti-cancer agents modulatory agents include tumor necrosis factor; interferon discussed herein. 65 alpha, beta, and gamma; IL-2 and other cytokines; F42K and Examples of immunotherapies currently under investiga­ other cytokine analogs; or MIP-1, MIP-1~, MCP-1, tion or in use are immune adjuvants e.g., Mycobacterium RANTES, and other chemokines. It is further contemplated US 10,065,924 B2 37 38 that the upregulation ofcell surface receptors or their ligands oforganic chemistry as applied by a person skilled in the art. such as Fas/Fas ligand, DR4 or DRS/TRAIL (Apo-2 ligand) Such principles and techniques are taught, for example, in would potentiate the apoptotic inducing abilities of the March's Advanced Organic Chemistry: Reactions, Mecha­ present invention by establishment of an autocrine or para­ nisms, and Structure (2007), which is incorporated by ref­ crine effect on hyperproliferative cells. Increases intercellu­ 5 erence herein lar signaling by elevating the number of GAP junctions A. Process Scale-Up would increase the anti-hyperproliferative effects on the The synthetic methods described herein can be further neighboring hyperproliferative cell population. In other modified and optimized for preparative, pilot- or large-scale embodiments, cytostatic or differentiation agents may be production, either batch of continuous, using the principles used in combination with the present invention to improve 10 and techniques of process chemistry as applied by a person the anti-hyerproliferative efficacy of the treatments. Inhibi­ skilled in the art. Such principles and techniques are taught, tors of cell adhesion are contemplated to improve the for example, in Practical Process Research & Development efficacy of the present invention. Examples of cell adhesion (2000), which is incorporated by reference herein. The inhibitors are focal adhesion kinase (FAKs) inhibitors and synthetic method described herein may be used to produce Lovastatin. It is further contemplated that other agents that 15 increase the sensitivity of a hyperproliferative cell to apop­ preparative scale amounts of viridicatumtoxin and deriva­ tosis, such as the antibody c225, could be used in combi­ tives thereof. nation with the present invention to improve the treatment B. Chemical Definitions efficacy. When used in the context of a chemical group: "hydro­ gen" means -H; "hydroxy" means --OH; "oxo" means There have been many advances in the therapy of cancer 20 following the introduction of cytotoxic chemotherapeutic =0; "carbonyl" means -C(=O)-; "carboxy" means ----C(=O)OH (also written as -COOR or----CO H); "halo" drugs. However, one of the consequences of chemotherapy 2 means independently -F, -Cl, -Br or -I; "amino" is the development/acquisition of drug-resistant phenotypes means -NH ; "hydroxyamino" means -NHOH; "nitro" and the development of multiple drug resistance. The devel­ 2 means -NO ; imino means NH; "cyano" means -CN; opment of drug resistance remains a major obstacle in the 25 2 "isocyanate" means -N=C=O; "azido" means -N ; in a treatment of such tumors and therefore, there is an obvious 3 monovalent context "phosphate" means -OP(O)(OH) or a need for alternative approaches such as gene therapy. 2 Another form of therapy for use in conjunction with deprotonated form thereof in a divalent context "phosphate" chemotherapy, radiation therapy or biological therapy means -OP(O)(OH)O- or a deprotonated form thereof "mercapto" means -SH; and "thio" means =S; "sulfa" includes hyperthermia, which is a procedure in which a 30 means -SO H, "sulfonyl" means -S(O) -; and "sulfi­ patient's tissue is exposed to high temperatures (up to 106° 3 2 nyl" means -S(O)-. F.). External or internal heating devices may be involved in the application of local, regional, or whole-body hyperther­ In the context of chemical formulas, the symbol "-" mia. Local hyperthermia involves the application of heat to means a single bond, "=" means a double bond, and "=" means triple bond. The symbol " - - - - " represents an a small area, such as a tumor. Heat may be generated 35 externally with high-frequency waves targeting a tumor optional bond, which if present is either single or double. from a device outside the body. Internal heat may involve a The symbol " =" represents a single bond or a double sterile probe, including thin, heated wires or hollow tubes bond. Thus, for example, the formula filled with warm water, implanted microwave antennae, or radiofrequency electrodes. 40 A patient's organ or a limb is heated for regional therapy, which is accomplished using devices that produce high ) energy, such as magnets. Alternatively, some ofthe patient's 0 blood may be removed and heated before being perfused into an area that will be internally heated. Whole-body 45 heating may also be implemented in cases where cancer has includes spread throughout the body. Warm-water blankets, hot wax, inductive coils, and thermal chambers may be used for this purpose. The skilled artisan is directed to "Remington's Pharma­ ceutical Sciences" 15th Edition, chapter 33, in particular pages 624-652. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in And it is understood that no one such ring atom forms part of more than one double bond. Furthermore, it is noted that any event, determine the appropriate dose for the individual 55 the covalent bond symbol"-", when connecting one or two subject. Moreover, for human administration, preparations should meet sterility, pyrogenicity, general safety and purity stereogenic atoms, does not indicate any preferred stereo­ standards as required by FDA Office of Biologics standards. chemistry. Instead, it covers all stereoisomers as well as It also should be pointed out that any of the foregoing mixtures thereof The symbol "= ", when drawn perpen­ dicularly across a bond therapies may prove useful by themselves in treating cancer. 60

IV. Synthetic Methods

In some aspects, the compounds of this invention can be synthesized using the methods of organic chemistry as 65 described in this application. These methods can be further modified and optimized using the principles and techniques US 10,065,924 B2 39 40 for methyl) indicates a point of attachment of the group. It (Cn-n') defines both the minimum (n) and maximum number is noted that the point of attachment is typically only (n') of carbon atoms in the group. Similarly, "alkylcc2 _10i" identified in this manner for larger groups in order to assist designates those alkyl groups having from 2 to 10 carbon the reader in unambiguously identifying a point of attach­ atoms. ment. The symbol "....,.." means a single bond where the 5 The term "saturated" as used herein means the compound group attached to the thick end of the wedge is "out of the or group so modified has no carbon-carbon double and no page." The symbol ".,,1111" means a single bond where the carbon-carbon triple bonds, except as noted below. In the group attached to the thick end of the wedge is "into the case ofsubstituted versions of saturated groups, one or more page". The symbol "= " means a single bond where the carbon oxygen double bond or a carbon nitrogen double bond may be present. And when such a bond is present, then geometry around a double bond (e.g., either E or Z) is 10 carbon-carbon double bonds that may occur as part of undefined. Both options, as well as combinations thereof are keto-enol tautomerism or imine/enamine tautomerism are therefore intended. Any undefined valency on an atom of a not precluded. structure shown in this application implicitly represents a The term "aliphatic" when used without the "substituted" hydrogen atom bonded to that atom. A bold dot on a carbon modifier signifies that the compound/group so modified is an atom indicates that the hydrogen attached to that carbon is 15 acyclic or cyclic, but non-aromatic hydrocarbon compound oriented out of the plane of the paper. or group. In aliphatic compounds/groups, the carbon atoms When a group "R" is depicted as a "floating group" on a can be joined together in straight chains, branched chains, or ring system, for example, in the formula: non-aromatic rings (alicyclic). Aliphatic compounds/groups can be saturated, that is joined by single bonds (alkanes/ 20 alkyl), or unsaturated, with one or more double bonds (alkenes/alkenyl) or with one or more triple bonds (alkynes/ alkynyl). The term "alkyl" when used without the "substituted" modifier refers to a monovalent saturated aliphatic group 25 with a carbon atom as the point of attachment, a linear or branched acyclic structure, and no atoms other than carbon then R may replace any hydrogen atom attached to any of and hydrogen. The groups ----CH3 (Me), ----CH2 CH3 (Et), the ring atoms, including a depicted, implied, or expressly ----CH2 CH2 CH3 (n-Pr or propyl), ----CH(CH3 ) 2 (i-Pr, 'Pr or defined hydrogen, so long as a stable structure is formed. isopropyl), -CH2 CH2 CH2 CH3 (n-Bu), -CH(CH3 ) When a group "R" is depicted as a "floating group" on a 30 CH2 CH3 (sec-butyl), -CH2 CH(CH3 ) 2 (isobutyl), fused ring system, as for example in the formula: ----C(CH3 ) 3 (tert-butyl, t-butyl, t-Bu or 'Bu), and -CH2 C (CH3 ) 3 (neo-pentyl) are non-limiting examples of alkyl groups. The term "alkanediyl" when used without the "sub­ stituted" modifier refers to a divalent saturated aliphatic 35 group, with one or two saturated carbon atom(s) as the point(s) ofattachment, a linear or branched acyclic structure, no carbon-carbon double or triple bonds, and no atoms other

than carbon and hydrogen. The groups, -CH2- (methyl­ ene), ----CH2 CH2-, ----CH2 C(CH3 ) 2 CH2-, and 40 ----CH2 CH2 CH-, are non-limiting examples of alkanediyl then R may replace any hydrogen attached to any of the ring groups. The term "alkylidene" when used without the "sub­ atoms ofeither ofthe fused rings unless specified otherwise. stituted" modifier refers to the divalent group =CRR' in Replaceable hydrogens include depicted hydrogens ( e.g., the which R and R' are independently hydrogen or alkyl. Non­ hydrogen attached to the nitrogen in the formula above), limiting examples of alkylidene groups include: =CH2 , implied hydrogens (e.g., a hydrogen of the formula above 45 =CH(CH2 CH3 ), and =C(CH3 ) 2 . An "alkane" refers to the that is not shown but understood to be present), expressly compound H-R, wherein R is alkyl as this term is defined defined hydrogens, and optional hydrogens whose presence above. When any of these terms is used with the "substi­ depends on the identity of a ring atom (e.g., a hydrogen tuted" modifier one or more hydrogen atom has been inde­ attached to group X, when X equals -CH-), so long as a pendently replaced by -OH, -F, -Cl, -Br, -I, -NH2 , stable structure is formed. In the example depicted, R may 50 -NO2 , ----CO2 H, -CO2 CH3 , ----CN, -SH, ---OCH3 , reside on either the 5-membered or the 6-membered ring of ---OCH2 CH3 , ----C(O)CH3 , -NHCH3 , -NHCH2 CH3 , the fused ring system. In the formula above, the subscript -N(CH3 ) 2 , ----C(O)NH2 , ---OC(O)CH3 , or -S(O)2NH2 . letter "y" immediately following the group "R" enclosed in The following groups are non-limiting examples of substi­ parentheses, represents a numeric variable. Unless specified tuted alkyl groups: ----CH2 OH, ----CH2 Cl, ----CF 3 , ----CH2 CN, otherwise, this variable can be 0, 1, 2, or any integer greater 55 ----CH2 C(O)OH, ----CH2 C(O)OCH3 , -CH2 C(O)NH2 , than 2, only limited by the maximum number of replaceable ----CH2 C(O)CH3 , ----CH2 OCH3 , -CH2 OC(O)CH3 , hydrogen atoms of the ring or ring system. ----CH2 NH2 , ----CH2 N(CH3 ) 2 , and ----CH2 CH2 Cl. The term For the groups and classes below, the following paren­ "haloalkyl" is a subset of substituted alkyl, in which one or thetical subscripts further define the group/class as follows: more hydrogen atoms has been substituted with a halo group "(Cn)" defines the exact number (n) of carbon atoms in the 60 and no other atoms aside from carbon, hydrogen and halo­ group/class. "(Cn)" defines the maximum number (n) of gen are present. The group, ----CH2Cl is a non-limiting carbon atoms that can be in the group/class, with the example ofa haloalkyl. The term "fluoroalkyl" is a subset of minimum number as small as possible for the group in substituted alkyl, in which one or more hydrogen has been question, e.g., it is understood that the minimum number of substituted with a fluoro group and no other atoms aside carbon atoms in the group "alkenylcc,si" or the class 65 from carbon, hydrogen and fluorine are present. The groups,

"alkenecc,si" is two. For example, "alkoxyccsrni" desig­ ----CH2 F, -CF3 , and ----CH2 CF3 are non-limiting examples nates those alkoxy groups having from 1 to 10 carbon atoms. of fluoroalkyl groups. US 10,065,924 B2 41 42

The term "cycloalkyl" when used without the "substi­ ----CH=CHCH2-, are non-limiting examples ofalkenediyl tuted" modifier refers to a monovalent saturated aliphatic groups. It is noted that while the alkenediyl group is ali­ group with a carbon atom as the point of attachment, said phatic, once connected at both ends, this group is not carbon atom forms part of one or more non-aromatic ring precluded from forming part of an aromatic structure. The structures, a cyclo or cyclic structure, no carbon-carbon 5 terms "alkene" and refer to a compound having the formula double or triple bonds, and no atoms other than carbon and H-R, wherein R is alkenyl as this term is defined above. A hydrogen. Non-limiting examples of cycloalkyl groups "terminal alkene" refers to an alkene having just one carbon­ include: -CH(CH2 ) 2 ( cyclopropyl), cyclobutyl, cyclopen­ carbon double bond, wherein that bond forms a vinyl group tyl, or cyclohexyl. The term "cycloalkanediyl" when used at one end ofthe molecule. When any ofthese terms are used without the "substituted" modifier refers to a divalent satu­ 10 with the "substituted" modifier one or more hydrogen atom rated aliphatic group with one or two carbon atom as the has been independently replaced by -OH, -F, -Cl, -Br, point(s) ofattachment, said carbon atom(s) forms part ofone -I, -NH2 , -NO2 , -CO2 H, -CO2 CH3 , ----CN, -SH, or more non-aromatic ring structures, a cyclo or cyclic ---OCH3 , ---OCH2 CH3 , ----C(O)CH3 , -NHCH3 , structure, no carbon-carbon double or triple bonds, and no -NHCH CH , -N(CH ) , ----C(O)NH , ---OC(O)CH , or 15 2 3 3 2 2 3 atoms other than carbon and hydrogen. -S(O)2 NH2 . The groups, ----CH=CHF, -CH=CHCI and ----CH=CHBr, are non-limiting examples of substituted alkenyl groups. The term "cycloalkenyl" when used without the "substi­ 20 tuted" modifier refers to a monovalent unsaturated aliphatic group with a carbon atom as the point of attachment, said carbon atom forms part of one or more non-aromatic ring structures, a cyclo or cyclic structure, at least one non­ aromatic carbon-carbon double bond, no carbon-carbon triple bonds, and no atoms other than carbon and hydrogen. 25 In some non-limiting examples of cycloalkenyl groups include

30 and are non-limiting examples of cycloalkanediyl groups. A "cycloalkane" refers to the compound H-R, wherein R is cycloalkyl as this term is defined above. When any of these terms is used with the "substituted" modifier one or more 35 The term "cycloalkenediyl" when used without the "substi­ hydrogen atom has been independently replaced by -OH, tuted" modifier refers to a divalent unsaturated aliphatic -F, --Cl, -Br, -I, -NH2 , -NO2 , ----CO2 H, -CO2 CH3 , group with one or two carbon atom(s) as the point(s) of -CN, -SH, -OCH3 , -OCH2 CH3 , ----C(O)CH3 , attachment, said carbon atom(s) forms part of one or more -NHCH3 , -NHCH2 CH3 , -N(CH3 ) 2 , ----C(O)NH2 , -DC non-aromatic ring structures, a cyclo or cyclic structure, at 40 (O)CH3 , or -S(O)2NH2 . The following groups are non- least one non-aromatic carbon-carbon double bond, no car­ limiting examples of substituted cycloalkyl groups: bon-carbon triple bonds, and no atoms other than carbon and -C(OH)(CH2 ) 2 , hydrogen.

45

50

The term "alkenyl" when used without the "substituted" modifier refers to a monovalent unsaturated aliphatic group are non-limiting examples of cycloalkenediyl. It is noted with a carbon atom as the point of attachment, a linear or that while the cycloalkenediyl group is aliphatic, once branched, acyclic structure, at least one nonaromatic carbon- 55 connected at both ends, this group is not precluded from carbon double bond, no carbon-carbon triple bonds, and no forming part of an aromatic structure. The terms "cycloalk­ atoms other than carbon and hydrogen. Non-limiting ene" and refer to a compound having the formula H-R, examples of alkenyl groups include: ----CH=CH2 (vinyl), wherein R is cycloalkenyl as this term is defined above. The -CH=CHCH3 , ----CH=CHCH2 CH3 , -CH2 CH=CH2 term "olefin" is synonymous with the terms "alkene" or a (ally!), ----CH2 CH=CHCH3 , and -CH=CHCH=CH2 . 60 "cycloalkane" as those terms are defined above. When any The term "alkenediyl" when used without the "substituted" of these terms are used with the "substituted" modifier one modifier refers to a divalent unsaturated aliphatic group, or more hydrogen atom has been independently replaced by with two carbon atoms as points of attachment, a linear or -OH, -F, --Cl, -Br, -I, -NH2 , -NO2 , ----CO2 H, branched, cyclo, cyclic or acyclic structure, at least one ----CO2 CH3 , -CN, -SH, -OCH3 , -OCH2 CH3 , ----C(O) nonaromatic carbon-carbon double bond, no carbon-carbon 65 CH3 , -NHCH3 , -NHCH2 CH3 , -N(CH3 ) 2 , ----C(O)NH2 , triple bonds, and no atoms other than carbon and hydrogen. ---OC(O)CH3 , or -S(O)2 NH2 . In some non-limiting The groups, ----CH=CH-, -CH=C(CH3 )CH2-, and examples of substituted cycloalkenyl include US 10,065,924 B2 43 44 -continued

and

The term "alkynyl" when used without the "substituted" 10 \ j \ j modifier refers to a monovalent unsaturated aliphatic group with a carbon atom as the point of attachment, a linear or and branched, acyclic structure, at least one carbon-carbon triple bond, and no atoms other than carbon and hydrogen. As used -to-~'-ot herein, the term alkynyl does not preclude the presence of 15 one or more non-aromatic carbon-carbon double bonds. The groups, ----C=CH, -C=CCH3 , and -CH2 C .. CCH3 , are non-limiting examples of alkynyl groups. An "alkyne" refers to the compound H-R, wherein R is alkynyl. When any of 20 these terms are used with the "substituted" modifier one or more hydrogen atom has been independently replaced by An "arene" refers to the compound H-R, wherein R is aryl

-OH, -F, --Cl, -Br, -I, -NH2 , -NO2 , ----CO2 H, as that term is defined above. Benzene and toluene are

-CO2 CH3 , ----CN, -SH, -OCH3 , -OCH2 CH3 , ----C(O) non-limiting examples of arenes. When any of these terms

CH3 , -NHCH3 , -NHCH2 CH3 , -N(CH3 ) 2 , ----C(O)NH2 , 25 are used with the "substituted" modifier one or more hydro­

-OC(O)CH3 , or -S(O)2NH2 . gen atom has been independently replaced by -OH, -F, The term "aryl" when used without the "substituted" --Cl, -Br, -I, -NH2 , -NO2 , -CO2 H, -CO2 CH3 , ----CN, -SH, ---OCH , -OCH CH , ----C(O)CH , modifier refers to a monovalent unsaturated aromatic group 3 2 3 3 with an aromatic carbon atom as the point of attachment, -NHCH3 , -NHCH2 CH3 , -N(CH3 ) 2 , ----C(O)NH2 , -DC 30 (O)CH , or -S(O) NH . said carbon atom forming part of a one or more six­ 3 2 2 The term "aralkyl" when used without the "substituted" membered aromatic ring structure, wherein the ring atoms modifier refers to the monovalent group -alkanediyl-aryl, in are all carbon, and wherein the group consists of no atoms which the terms alkanediyl and aryl are each used in a other than carbon and hydrogen. If more than one ring is manner consistent with the definitions provided above. present, the rings may be fused or unfused. As used herein, 35 Non-limiting examples of aralkyls are: phenylmethyl (ben­ the term does not preclude the presence of one or more alkyl zyl, Bn) and 2-phenyl-ethyl. When the term aralkyl is used or aralkyl groups (carbon number limitation permitting) with the "substituted" modifier one or more hydrogen atom attached to the first aromatic ring or any additional aromatic from the alkanediyl and/or the aryl group has been indepen­ ring present. Non-limiting examples of aryl groups include dently replaced by -OH, -F, --Cl, -Br, -I, -NH2 , phenyl (Ph), methylphenyl, (dimethyl)phenyl, 40 -NO2 , ----CO2 H, -CO2 CH3 , ----CN, -SH, ---OCH3 , -C6 H4 CH2 CH3 (ethylphenyl), naphthyl, and a monovalent ---OCH2 CH3 , ----C(O)CH3 , -NHCH3 , -NHCH2 CH3 , group derived from biphenyl. The term "arenediyl" when -N(CH3 ) 2 , ----C(O)NH2 , ---OC(O)CH3 , or -S(O)2NH2 . used without the "substituted" modifier refers to a divalent Non-limiting examples of substituted aralkyls are: (3-chlo­ aromatic group with two aromatic carbon atoms as points of ropheny !)-methyl, and 2-chloro-2-pheny l-eth-1-y I. attachment, said carbon atoms forming part of one or more 45 The term "heteroaryl" when used without the "substi­ six-membered aromatic ring structure(s) wherein the ring tuted" modifier refers to a monovalent aromatic group with atoms are all carbon, and wherein the monovalent group an aromatic carbon atom or nitrogen atom as the point of consists of no atoms other than carbon and hydrogen. As attachment, said carbon atom or nitrogen atom forming part used herein, the term does not preclude the presence of one of one or more aromatic ring structures wherein at least one or more alkyl, aryl or aralkyl groups (carbon number limi­ 50 of the ring atoms is nitrogen, oxygen or sulfur, and wherein tation permitting) attached to the first aromatic ring or any the heteroaryl group consists of no atoms other than carbon, hydrogen, aromatic nitrogen, aromatic oxygen and aromatic additional aromatic ring present. If more than one ring is sulfur. If more than one ring is present, the rings may be present, the rings may be fused or unfused. U nfused rings fused or unfused. As used herein, the term does not preclude may be connected via one or more of the following: a 55 the presence ofone or more alkyl, aryl, and/or aralkyl groups covalent bond, alkanediyl, or alkenediyl groups (carbon (carbon number limitation permitting) attached to the aro­ number limitation permitting). Non-limiting examples of matic ring or aromatic ring system. Non-limiting examples arenediyl groups include: of heteroaryl groups include furanyl, imidazolyl, indolyl, indazolyl (Im), isoxazolyl, methylpyridinyl, oxazolyl, phe­ 60 nylpyridinyl, pyridinyl, pyrrolyl, pyrimidinyl, pyrazinyl, quinolyl, quinazolyl, quinoxalinyl, triazinyl, tetrazolyl, thi­ azolyl, thienyl, and triazolyl. The term "N-heteroaryl" refers to a heteroaryl group with a nitrogen atom as the point of attachment. The term "heteroarenediyl" when used without 65 the "substituted" modifier refers to an divalent aromatic group, with two aromatic carbon atoms, two aromatic nitro­ gen atoms, or one aromatic carbon atom and one aromatic US 10,065,924 B2 45 46 nitrogen atom as the two points of attachment, said atoms Also, the term does not preclude the presence of one or more forming part of one or more aromatic ring structure(s) double bonds in the ring or ring system, provided that the wherein at least one ofthe ring atoms is nitrogen, oxygen or resulting group remains non-aromatic. Non-limiting sulfur, and wherein the divalent group consists of no atoms examples of heterocycloalkyl groups include aziridinyl, other than carbon, hydrogen, aromatic nitrogen, aromatic 5 azetidinyl, pyrrolidinyl, piperidinyl, piperazinyl, morpholi­ oxygen and aromatic sulfur. Ifmore than one ring is present, nyl, thiomorpholinyl, tetrahydrofuranyl, tetrahydrothiofura­ the rings may be fused or unfused. Unfused rings may be nyl, tetrahydropyranyl, pyranyl, oxiranyl, and oxetanyl. The connected via one or more ofthe following: a covalent bond, term "N-heterocycloalkyl" refers to a heterocycloalkyl alkanediyl, or alkenediyl groups (carbon number limitation group with a nitrogen atom as the point of attachment. The permitting). As used herein, the term does not preclude the 10 term "heterocycloalkanediyl" when used without the "sub­ presence of one or more alkyl, aryl, and/or aralkyl groups stituted" modifier refers to an divalent cyclic group, with ( carbon number limitation permitting) attached to the aro­ two carbon atoms, two nitrogen atoms, or one carbon atom matic ring or aromatic ring system. Non-limiting examples and one nitrogen atom as the two points of attachment, said of heteroarenediyl groups include: atoms forming part of one or more ring structure(s) wherein 15 at least one of the ring atoms is nitrogen, oxygen or sulfur, and wherein the divalent group consists of no atoms other than carbon, hydrogen, nitrogen, oxygen and sulfur. Ifmore than one ring is present, the rings may be fused or unfused. Unfused rings may be connected via one or more of the +o+ 20 following: a covalent bond, alkanediyl, or alkenediyl groups (carbon number limitation permitting). As used herein, the term does not preclude the presence of one or more alkyl groups (carbon number limitation permitting) attached to the ring or ring system. Also, the term does not preclude the 25 presence of one or more double bonds in the ring or ring system, provided that the resulting group remains non­ aromatic. Non-limiting examples of heterocycloalkanediyl groups include: and 30

A "heteroarene" refers to the compound H-R, wherein R is heteroaryl. Pyridine and quinoline are non-limiting and examples of heteroarenes. When these terms are used with the "substituted" modifier one or more hydrogen atom has 35 +o+p/ been independently replaced by -OH, -F, --Cl, -Br, -I,

-NH2 , -NO2 , -CO2 H, -CO2 CH3 , ----CN, -SH, -OCH3 , ---OCH2 CH3 , -C(O)CH3 , -NHCH3 , -NHCH2 CH3 , -N(CH3 ) 2 , ----C(O)NH2 , ---OC(O)CH3 , or -S(O)2 NH2 . 40 ~1' The term "heteroaralkyl" when used without the "substi­ tuted" modifier refers to the monovalent group -alkanediyl­ When these terms are used with the "substituted" modifier heteroaryl, in which the terms alkanediyl and heteroaryl are one or more hydrogen atom has been independently replaced each used in a manner consistent with the definitions pro­ by -OH, -F, -Cl, -Br, -I, -NH2 , -NO2 , ----CO2 H, vided above. Non-limiting examples of heteroaralkyls are: 45 ----CO2 CH3 , -CN, -SH, -OCH3 , -OCH2 CH3 , ----C(O) 2-pyridylmethyl and 2-indazolyl-ethyl. When the term het­ CH3 , -NHCH3 , -NHCH2 CH3 , -N(CH3 ) 2 , ----C(O)NH2 , eroaralkyl is used with the "substituted" modifier one or ---OC(O)CH3 , -S(O)2NH2 , or ----C(O)OC(CH3 ) 3 (tert-bu­ more hydrogen atom from the alkanediyl and/or the het­ tyloxycarbonyl, BOC). eroaryl group has been independently replaced by -OH, The term "acyl" when used without the "substituted"

-F, --Cl, -Br, -I, -NH2 , -NO2 , ----CO2 H, -CO2 CH3 , 50 modifier refers to the group -C(O)R, in which R is a -CN, -SH, -OCH3 , -OCH2 CH3 , ----C(O)CH3 , hydrogen, alkyl, cycloalkyl, aryl, aralkyl or heteroaryl, as -NHCH3 , -NHCH2 CH3 , -N(CH3 ) 2 , ----C(O)NH2 , -DC those terms are defined above. The groups, --CHO, ----C(O) (O)CH3 , or -S(O)2 NH2 . Non-limiting examples of substi­ CH3 (acetyl, Ac), ----C(O)CH2 CH3 , ----C(O)CH2 CH2 CH3 , tuted heteroaralkyls are: (3-chloroquinolyl)-methyl, and ----C(O)CH(CH3 ) 2 , ----C(O)CH(CH2 ) 2 , -C(O)C6H5 , 2-chloro-2-thienyl-eth-1-yl. 55 ----C(O)C 6 H4 CH3 , -C(O)CH2 C6 H5 , ----C(O)(imidazolyl) The term "heterocycloalkyl" when used without the "sub­ are non-limiting examples of acyl groups. A "thioacyl" is stituted" modifier refers to a monovalent non-aromatic defined in an analogous manner, except that the oxygen atom group with a carbon atom or nitrogen atom as the point of ofthe group ----C(O)R has been replaced with a sulfur atom, attachment, said carbon atom or nitrogen atom forming part ----C(S)R. The term "aldehyde" corresponds to an alkane, as of one or more non-aromatic ring structures wherein at least 60 defined above, wherein at least one of the hydrogen atoms one of the ring atoms is nitrogen, oxygen or sulfur, and has been replaced with a -CHO group. When any of these wherein the heterocycloalkyl group consists of no atoms terms are used with the "substituted" modifier one or more other than carbon, hydrogen, nitrogen, oxygen and sulfur. If hydrogen atom (including a hydrogen atom directly attached more than one ring is present, the rings may be fused or the carbonyl or thiocarbonyl group, if any) has been inde­ unfused. As used herein, the term does not preclude the 65 pendently replaced by -OH, -F, --Cl, -Br, -I, -NH2 , presence of one or more alkyl groups (carbon number -NO2 , ----CO2 H, -CO2 CH3 , ----CN, -SH, ---OCH3 , limitation permitting) attached to the ring or ring system. ---OCH2 CH3 , ----C(O)CH3 , -NHCH3 , -NHCH2 CH3 , US 10,065,924 B2 47 48

-N(CH3 ) 2 , ----C(O)NH2 , ---OC(O)CH3 , or -S(O)2NH2 . CH3 , -NHCH3 , -NHCH2 CH3 , -N(CH3 ) 2 , ----C(O)NH2 , The groups, ----C(O)CH2 CF3 , ----CO2 H (carboxyl), ---OC(O)CH3 , or -S(O)2 NH2 . -CO2 CH3 (methylcarboxyl), -CO2 CH2 CH3 , -C(O)NH2 A "reducing agent" in the context of this application is a ( carbamoyl), and ----CON(CH3 ) 2 , are non-limiting examples compound which causes the reduction of a compound of substituted acyl groups. 5 through the donation of an electron. A soft reducing agent is The term "alkylamino" when used without the "substi­ a reducing agent which contains electron delocalizing tuted" modifier refers to the group -NHR, in which R is an ligands which weaken the nucleophilic strength of the alkyl, as that term is defined above. Non-limiting examples hydride. Some non-limiting examples ofreducing agents are of alkylamino groups include: -NHCH3 and sodium borohydride, sodium triacetoxyborohydride, sodium -NHCH2 CH3 . The term "dialkylamino" when used with­ 10 cyanoborohydride, lithium aluminum hydride, diisobutyl­ out the "substituted" modifier refers to the group -NRR', in aluminum hydride, hydrogen gas, or metal hydride. which R and R' can each independently be the same or A "base" in the context of this application is a compound different alkyl groups, or R and R' can be taken together to which has a lone pair of electron that can accept a proton. represent an alkanediyl. Non-limiting examples of dialky­ Non-limiting examples of a base can include triethylamine, lamino groups include: -N(CH3 ) 2 , -N(CH3 )(CH2 CH3 ), 15 a metal hydroxide, a metal alkoxide, a metal hydride, or a and N-pyrrolidinyl. The terms "alkoxyamino", "cycloalky­ metal alkane. An alkyllithium or organolithium is a com­ lamino", "alkenylamino", "cycloalkenylamino", "alky­ pound of the formula alkylccsi 2 )-Li. A nitrogenous base is nylamino", "arylamino", "aralkylamino", "heteroary­ an alkylamine, dialkylamino, trialkylamine, nitrogen con­ lamino", "heterocycloalkylamino" and taining heterocycloalkane or heteroarene wherein the base "alkylsulfonylamino" when used without the "substituted" 20 can accept a proton to form a positively charged species. For modifier, refers to groups, defined as -NHR, in which R is example, but not limited to, a nitrogenous base could be alkoxy, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, 4,4-dimethylpyridine, pyridine, 1,8-diazabicyclo[5.4.0]un­ aralkyl, heteroaryl, heterocycloalkyl, and alkylsulfonyl, dec-7-ene, diisopropylethylamine, or triethylamine. A metal respectively. A non-limiting example of an arylamino group alkoxide is an alkoxy group wherein rather than the oxygen is -NHC6H5 . The term "amido" (acylamino), when used 25 atom which was the point of connectivity has an extra without the "substituted" modifier, refers to the group electron and thus a negative charge which is charged bal­ -NHR, in which R is acyl, as that term is defined above. A anced by the metal ion. For example, a metal alkoxide could non-limiting example of an amido group is -NHC(O)CHy be a sodium tert-butoxide or potassium methoxide. The term "alkylimino" when used without the "substituted" An "oxidizing agent" in the context of this application is modifier refers to the divalent group NR, in which R is an 30 a compound which causes the oxidation of a compound by alkyl, as that term is defined above. The term "alkylamin­ accepting an electron. Some non-limiting examples of oxi­ odiyl" refers to the divalent group -NH-alkanediyl-, dizing agent are oxygen gas, peroxides, chlorite, hypochlo­ -NH-alkanediyl-NH-, or -alkanediyl-NH-alkanediyl-. rite, or a chromium compound such as pyridinium chloro­ When any of these terms is used with the "substituted" chromate or hydrochromic acid. modifier one or more hydrogen atom has been independently 35 A "metal" in the context of this application is a transition replaced by -OH, -F, -Cl, -Br, -I, -NH2 , -NO2 , metal or a metal of groups I or II. In some embodiments, a -CO2 H, ----CO2 CH3 , -CN, -SH, ---OCH3 , -OCH2 CH3 , metal is lithium, sodium, or potassium. In other embodi­ -C(O)CH3 , -NHCH3 , -NHCH2 CH3 , -N(CH3 ) 2 , ments, a metal is calcium or magnesium. -C(O)NH2 , -OC(O)CH3 , or -S(O)2 NH2 . The groups An "alkylaluminium" in the context of this application is -NHC(O)OCH3 and -NHC(O)NHCH3 are non-limiting 40 a reagent which contains one, two, three, or four alkyl examples of substituted amido groups. groups as that group is defined above to a central aluminum The term "alkoxy" when used without the "substituted" atom. Some non-limiting examples of alkylaluminiums are modifier refers to the group -OR, in which R is an alkyl, trimethylaluminum or tetramethylaluminium. as that term is defined above. Non-limiting examples A "linker" in the context of this application is divalent include: ---OCH3 (methoxy), ---OCH2 CH3 (ethoxy), 45 chemical group which may be used to join one or more -OCH2 CH2 CH3 , ---OCH(CH3 ) 2 (isopropoxy), and -DC molecules to the compound of the instant disclosure. In (CH3 ) 3 (tert-butoxy). The terms "cycloalkoxy", "alkeny­ some embodiments, the linker contains a reactive functional loxy", "alkynyloxy", "aryloxy", "aralkoxy", "heteroary­ group, such as a carboxyl, an amide, a amine, a hydroxy, a loxy", "heterocycloalkoxy", and "acyloxy", when used mercapto, an aldehyde, or a ketone on each end that be used without the "substituted" modifier, refers to groups, defined 50 to join one or more molecules to the compounds of the as -OR, in which R is cycloalkyl, alkenyl, alkynyl, aryl, instant disclosure. In some non-limiting examples, aralkyl, heteroaryl, heterocycloalkyl, and acyl, respectively. ----CH2 CH2 CH2 CH2-, -C(O)CH2 CH2 CH2-, The term "alkoxydiyl" refers to the divalent group -O­ ---OCH2 CH2NH-, -NHCH2 CH2 NH-, and alkanediyl- , -O-alkanediyl---0-, or -alkanediyl---0-al­ -(OCH2 CH2 )n- wherein n is between 1-1000, are linkers. kanediyl-. The term "alkylthio" and "acylthio" when used 55 A "stereoisomer" or "optical isomer" is an isomer of a without the "substituted" modifier refers to the group -SR, given compound in which the same atoms are bonded to the in which R is an alkyl and acyl, respectively. The term same other atoms, but where the configuration of those "alcohol" corresponds to an alkane, as defined above, atoms in three dimensions differs. "Enantiomers" are ste­ wherein at least one of the hydrogen atoms has been reoisomers of a given compound that are mirror images of replaced with a hydroxy group. The term "ether" corre­ 60 each other, like left and right hands. "Diastereomers" are sponds to an alkane or cycloalkane, as defined above, stereoisomers ofa given compound that are not enantiomers. wherein at least one of the hydrogen atoms has been Chiral molecules contain a chiral center, also referred to as replaced with an alkoxy or cycloalkoxy group. When any of a stereocenter or stereogenic center, which is any point, these terms is used with the "substituted" modifier one or though not necessarily an atom, in a molecule bearing more hydrogen atom has been independently replaced by 65 groups such that an interchanging of any two groups leads

-OH, -F, -Cl, -Br, -I, -NH2 , -NO2 , ----CO2 H, to a stereoisomer. In organic compounds, the chiral center is -CO2 CH3 , ----CN, -SH, -OCH3 , -OCH2 CH3 , ----C(O) typically a carbon, phosphorus or sulfur atom, though it is US 10,065,924 B2 49 50 also possible for other atoms to be stereocenters in organic that the composition contains s15%, more preferably s10%, and inorganic compounds. A molecule can have multiple even more preferably s5%, or most preferably sl% of stereocenters, giving it many stereoisomers. In compounds another stereoisomer(s ). whose stereoisomerism is due to tetrahedral stereogenic centers (e.g., tetrahedral carbon), the total number of hypo­ 5 V. Examples thetically possible stereoisomers will not exceed r, where n is the number of tetrahedral stereocenters. Molecules with The following examples are included to demonstrate symmetry frequently have fewer than the maximum possible preferred embodiments of the invention. It should be appre­ number of stereoisomers. A 50:50 mixture of enantiomers is ciated by those of skill in the art that the techniques referred to as a racemic mixture. Alternatively, a mixture of 10 disclosed in the examples which follow represent techniques enantiomers can be enantiomerically enriched so that one discovered by the inventor to function well in the practice of enantiomer is present in an amount greater than 50%. the invention, and thus can be considered to constitute Typically, enantiomers and/or diastereomers can be resolved preferred modes for its practice. However, those of skill in or separated using techniques known in the art. It is con­ the art should, in light of the present disclosure, appreciate templated that that for any stereocenter or axis of chirality 15 that many changes can be made in the specific embodiments for which stereochemistry has not been defined, that stereo­ which are disclosed and still obtain a like or similar result center or axis of chirality can be present in its R form, S without departing from the spirit and scope of the invention. form, or as a mixture of the R and S forms, including racemic and non-racemic mixtures. As used herein, the Example I-Synthesis of Viridicatumtoxin and phrase "substantially free from other stereoisomers" means Analogs Thereof

Scheme 1: Synthesis ofviridicatumtoxin B(l).0

TBSO MeO OMe OMe ~'Me a)NaH b)DBU MeO Me Br M~WO c)CSA 6 I # o d)Na2C03 e) BF3•0Et2 OBn 0 Q 0 OBn 0 OH 3 4 5

OBn 0 OH

7

g)CSAoemAj h) PIDA

Teoc0N PhO A k)DMDO O OBn Ni(OAc)i 9 Me l)NABH3CN i)tBuOK MeO j) TBAF, NI¼F

OBn 0 OH 0 OBn 0 0 11 10 8

m)HCl n) NaHB(OAc)3 jo) TBSOTf US 10,065,924 B2 51 52 -continued

p) KHMDS; Davis

OBn OH 0 OTBS

12

q) HF-pyr r)DMP s) H2, Pd MeO

OBn OH 0 OTBS

13

MeO

0

"Reagents and conditions: a) 4 (3.0 equiv), NaH (3.0 equiv), THF, 0° C., 45 min; then 25° C., 1 h; b) DBU (5.0 equiv). toluene, 65° C., 4.5 h, 54% for 2 steps; c) CSA (0.02 equiv), CH2Cl2, 25° C., 30 min, 99%; d) 6 (1.1 equiv), Na2C03 (10 equiv), DMF, 25° C., 1 h, 77%, d.r. ca. 1: 1; e) BF3•0Et2 (0.15 equiv), CH2Cl2, 0° C., 20 min, 73%; f) FIDA (1.2 equiv), MeOH:CH2Cl2 1: 1, 0-25° C., 1 h; g) CSA (0.07 equiv), CH2CI2, 0° C., 5 min, 85% for 2 steps; h) FIDA (1.2 equiv), MeOH:CH2C12 10:1, 25° C., 1.5 h, 90%; i) 8 (1.1 equiv), tBuOK (1.2 equiv), toluene, 25°C., 15 min, 91 %, d.r. ca. 2: 1; j) TBAF (10 equiv), NJ¼F (20 equiv), degassed THF, 25° C., 5 min, 86%; k) Ni(acac), (0.2 equiv), DMDO (5.1 equiv), CH2Cl2, -78--60° C., 6.5 h, 36%,

60% brsm, 50% after one recycle; 1) NaCNBH3 (10 equiv), THF, -78-----s,.60° C., 90 min, 39% for 11, 19% for 15-epi-11, chromatographically separated; m) 2N aq. HCI:THF 1: 10, 25° C., 5 h, quant.: n) NaBH(OAch (1.2 equiv), EtOAc:acetone 1:1, 40° C., 105 min, 47%; o) TBSOTf(40 equiv), 2,6-lutidine (60 equiv), CH2CI2, 0-25° C., 1 h, 61 %; p) KHMDS (3.4 equiv), THF, -78° C., 1 h; then Davis ox. (3.9 equiv), -78° C., 1.7 h, 20% + 45% recovered 12; q) HF•py (excess), MeCN, 0-50° C., 25 h, 61 %; r) DMP (3.0 equiv), DCE, 0-50° C.,

7.5 h, 66%; s) H2, Pd black:(4.9 equiv), 1,4-dioxane:MeOH 1:1, 25° C., 8 min, 98%. Abbreviations: DBU = 1,8-diazabicyclo[5.4.0]undec-7-ene, CSA= (±)-camphor-10-sulfonic acid, PIDA = iodobenzene diacetate; TBAF = tetra-n-butylammonium fluoride; acac = acetylacetonate, DMDO = dimethyldioxirane; TBSOTf= tert-butyldimethylsilyl trifluoromethanesulfonate; KHMDS = potassium hexamethyldisilazide, Davis ox. = (±)­ trans-2-(phenylsulfonyl)-3-phenyloxaziridine, py = pyridine, DMP = Dess-Martin periodinane, DCE = 1,2-dichloroethane. US 10,065,924 B2 53 54 -continued Scheme 2: Synthesis ofviridicatumtoxin analogs V2, V3 and V4. 0

a) H2, Pd black 0 10 OBn OH 0

11 (±)-V3:R~H (±)-V4:R~Me 15

aReagents and conditions: a) H2 (1 atrn), Pd-black (5.6 equiv.) 1,4-dioxane:methanol; b)

H2 (1 atrn), Pd-black (7.0 equiv.), 1,4-dioxane:methanol.

The synthesis ofviridicatumtoxin analogs V2-V6 is based on the recently disclosed synthetic approach to racemic viridicatumtoxin B [( ±)-1] as shown in scheme 1. (Nicolaou, (±)-V2 et al., 2013, which is incorporated herein by reference) Specifically, intermediate (±)-11 of our synthetic route (see 25 scheme 1) was hydrogenated in the presence of palladium black to provide access to analog (±)-V2 (scheme 2). Inter­ mediate (±)-15-epi-11, which had been obtained as a side­ product in our synthesis ofviridicatumtoxin B [(±)-1] (see b) H2, scheme 1), was transformed into analog (±)-V2 accordingly Pd black 30 (scheme 2). Intermediate (±)-15-epi-14, which was obtained analogously to (±)-15-epi-11, (Nicolaou, et al., 2014) was subjected to hydrogenolysis in the presence of palladium 15-epi-11:R~Bn black to provide access to analog (±)-V4 (scheme 2). For all 15-epi-14: R ~ Me of these analogs, ketal hydrolysis occurred during HPLC purification.

Scheme 3: Synthesis ofviridicatumtoxin analogs V5 and V6. 0

11

a) NaCNBH3, I AcOH •

[chromatographically separated] 15 5-epi-15

ic) H2, Pd black US 10,065,924 B2 55 56 -continued

OMe w ~

0

OH OH

V5 V6

"Reagents and conditions: a) NaCNBH3 (4.0 equiv), AcOH, 25° C., 25 minutes, 31 % for 15,

27% for 5-epi-15; b) H2 balloon, Pd black (4.1 equiv), THF:MeOH 1:1, 25° C., 10 min, 96%; c) H2, Pd black (4.5 equiv), THF:MeOH 1: 1, 25° C., 10 min, quant.

The synthesis of analogs (±)-V5 and (±)-V6 commenced using a Bruker Smart-APEX instrument (CCD detector) or with intermediate (±)-11 (scheme 3). Reduction with 20 a Bruker Kappa APEX-II instrument (CCD detector).

NaCNBH3 (4.0 equiv.) in acetic acid led to a mixture of Preparative HPLC separations were performed using a methyl ethers (±)-15 and (±)-5-epi-15, which was chromato­ Waters 2767 prep LC system equipped with a Waters Atlan­ graphically separated. Intermediate (±)-15 was then trans­ tis prep T3 OBD column (16x150 mm, 5 µm particle size) formed into (±)-V5 through hydrogenolysis in the presence and monitored using a Waters 2996 photodiode array detec­ of palladium black (scheme 3). (±)-V6 was obtained from 25 tor. (±)-5-epi-15 accordingly (scheme 3). Example 3-Compound Characterization Example 2---General Methods and Materials

All reactions were carried out under an argon atmosphere 30 unless otherwise noted. Methylene chloride, tetrahydro­ furan, toluene, methanol, dimethylformamide, acetonitrile, diisopropylamine, and triethylamine were dried prior to use by passage through an activated alumina colunm unless otherwise noted (Pangborn, et al., 1996). Anhydrous 35 acetone, ethyl acetate, and 1,2-dichloro-ethane were pur­ 0 chased from commercial suppliers and stored under argon. Yields refer to chromatographically and spectroscopically

(1H NMR) homogenous material, unless otherwise stated. 40 Reactions were monitored by thin-layer chromatography (TLC) carried out on 0.25 mm E. Merck silica gel plates Viridicatumtoxin analog V2: To a stirred solution of (60E-254) and were visualized using UV light and an (±)-11 (30 mg, 0.048 mmol, 1.0 equiv) in 1,4-dioxane: ethanolic solution of phosphomolybdic acid and cerium MeOH ( 4 mL, 1: 1) was added Pd black (28 mg, 0.27 mmol, sulfate or an aqueous solution of potassium permanganate. 45 5.6 equiv) under argon. The flask headspace was exchanged

Flash colunm chromatography using E. Merck silica gel (60, for H2 , and the reaction mixture was stirred for 10 minutes particle size 0.040-0.063 mm) was performed as described at room temperature. The flask headspace was re-exchanged by Still, et al. (1978). NMR spectra were recorded on a for argon, and the mixture was filtered through Celite® and Bruker DRX-600 equipped with a 5 mm DCH cryoprobe, concentrated (crude product: 30 mg). A portion of this Bruker DRX-500, Bruker AV-400, or Varian INOVA-400 50 material was purified by reverse-phase prep-HPLC [Waters instrument and calibrated using residual undeuterated sol­ Atlantis prep T3 OBD, 16x150 mm, 5 µm particle size, 20 1 vent for H NMR [oH=7.26 (CHCl3 ), 7.16 (C 6 D5 H), 2.05 mL/min, 50% MeCN in H20 (0-15 min), then ramp to 70% (D5 H-acetone), 2.50 (D5 H-DMSO), and 5.32(CDHCl2 ) MeCN (15-25 min), 0.07% TFA buffer, A=288 nm]: ppm] and 13C deuterated solvent for 13C NMR [oc=77.16 tR=17.59 min, to provide pure (±)-V2 (9.5 mg, 17 µmo!).

(CDCl3 ), 128.06 (C6D6 ), 206.68 (dcacetone), and 53.84 55 (±)-V2: Rf=0.1 (silica gel, MeOH:CH2 Cl2 1:19); FT-IR (CD2 Cl2 ) ppm] as an internal reference at 298 K (Fulmer, et (neat) V max=3435, 2921, 1685, 1623, 1585, 1492, 1448, al., 2010). The following abbreviations were used to desig­ 1399, 1281, 1259, 1198, 1162, 1133, 1069, 904, 733 cm-1; 1 nate the multiplicities: s=singlet, d=doublet, t=triplet, H NMR (CDCl3 , 600 MHz) 0=18.21 (s, 1 H), 14.49 (s, 1 q=quartet, m=multiplet, b=broad, ap=apparent. H), 9.02 (s, 1 H), 8.71 (s, 1 H), 6.79 (s, 1 H), 5.73 (s, 1 H), ATR-Infrared (IR) spectra were recorded on a Perkin- 60 5.50 (s, 1 H), 4.82 (s, 1 H), 3.88 (s, 3 H), 3.71 (d, J=20.1 Hz, Elmer 100 series FT-IR spectrometer. High-resolution mass 1 H), 3.56 (t, J=4.4 Hz, 1 H), 3.35 (dd, J=18.0, 3.9 Hz, 1 H), spectra (HRMS) were recorded on an Agilent LC/MSD/TOF 3.20 (d, J=20.1 Hz, 1 H), 3.15 (dd, J=18.0, 5.0 Hz, 1 H), 2.20 mass spectrometer using ESI (electrospray ionization) or a (m, 1 H), 2.04 (m, 1 H), 1.81 (td, J=12.6, 6.1 Hz, 1 H), 1.45 Shimadzu Ion Trap-TOP using ESL Melting points were (s, 3 H), 1.32 (dd, J=12.6, 6.1 Hz, 1 H), 0.90 (s, 3 H), 0.34 13 recorded on a Fisher-Johns 12-144 melting point apparatus 65 (s, 3 H) ppm; C NMR (CDCl3 , 151 MHz) 0=196.6, 196.5, or a Thomson Hoover uni-melt capillary melting point 192.6, 189.8, 173.7, 163.5, 160.9, 158.1, 146.7, 143.6, apparatus. X-Ray crystallographic structures were collected 135.9, 127.0, 121.7, 120.7, 106.9, 106.9, 102.4, 99.8, 77.4, US 10,065,924 B2 57 58 60.4, 55.8, 50.4, 45.6, 38.5, 34.1, 29.3, 25.5, 24.3, 22.9, 21.0 of this material was purified by reverse-phase prep-HPLC ppm; HRMS (ESI) calcd for C34H29NO9 H+ [M+H+] [Waters Atlantis prep T3 OBD, 16x150 mm, 5 µm particle

548.1915, found 548.1902. size, 20 mL/min, 50% MeCN in H2 O (0-15 min), then ramp to 70% MeCN (15-25 min), 0.07% TFA buffer, 5 A=288 nm]: tR=14.25 min, to provide pure (±)-V4 (8.8 mg,

16 µmo!). (±)-V4: Rf=0.1 (silica gel, MeOH: CH2 Cl2 1:19); FT-IR (neat) vmax=3400, 2939, 1683, 1585, 1466, 1400, 1342, 1285, 1216, 1187, 1163, 1132, 1068, 914, 824, 731 1 cm-1; H NMR (CDCl3 , 600 MHz) 0=18.19 (s, 1 H), 14.20 10 (s, 1 H), 9.06 (s, 1 H), 6.72 (s, 1 H), 5.72 (s, 1 H), 5.49 (s, 1 H), 4.93 (s, 1 H), 4.09 (s, 3 H), 3.94 (s, 3 H), 3.86 (d, 1=19.9 Hz, 1 H), 3.44 (t, 1=4.5 Hz, 1 H), 3.36 (dd, 1=18.1, 4.0 Hz, 1 H), 3.13 (dd, 1=18.1, 5.1 Hz, 1 H), 3.08 (d, 1=19.9

15 Hz, 1 H), 2.20 (m, 1 H), 2.05 (m, 1 H), 1.85 (td, 1=12.5, 6.1 Hz, 1 H), 1.42 (s, 3 H), 1.34 (dd, 1=12.5, 6.2 Hz, 1 H), 0.91 13 (s, 3 H), 0.37 (s, 3 H) ppm; C NMR (CDCl , 151 MHz) Viridicatumtoxin analog V3: To a stirred solution of 3 0=196.2, 196.1, 193.1, 190.0, 173.6, 165.0, 160.4, 159.6, (±)-15-epi-11 (44 mg, 0.057 mmol, 1.0 equiv) in 1,4­ 148.1, 142.1, 136.2, 126.9, 121.6, 121.5, 110.0, 107.9, 99.9, dioxane:MeOH (5 mL, 1:1) was added Pd black (42 mg, 20 98.1, 77.2, 59.7, 56.8, 55.6, 50.5, 45.2, 38.5, 34.0, 29.4, 25.5, 0.40 mmol, 7 equiv) under argon. The flask headspace was 24.5, 23.0, 20.8 ppm; HRMS (ESI) calcd for C31H31NO9 H+ exchanged for H , and the reaction mixture was stirred for 2 [M+W] 562.2072, found 562.2082. 10 minutes at room temperature. The flask headspace was re-exchanged for argon, and the mixture was filtered through Celite® and concentrated (crude product: 40 mg). A portion 25 15 of this material was purified by reverse-phase prep-HPLC [Waters Atlantis prep T3 OBD, 16x150 mm, 5 µm particle size, 20 mL/min, 50% MeCN in H2 O (0-15 min), then ramp to 70% MeCN (15-25 min), 0.07% TFA buffer, A=288 nm]: tR=17.51 min, to provide pure (±)-V3 (11 mg, 30

20 µmo!). (±)-V3: Rf=0.1 (silica gel, MeOH:CH2 Cl 2 1:19); FT-IR (neat) vmax=34l4, 2923, 1681, 1622, 1585, 1452, 1401, 1283, 1261, 1203, 1134, 1070, 909, 730 cm -1; 1H

NMR (CDCl3 , 600 MHz) 0=18.22 (s, 1 H), 14.60 (s, 1 H), 9.05 (s, 1 H), 8.74 (s, 1 H), 6.79 (s, 1 H), 5.73 (s, 1 H), 5.49 35 (s, 1 H), 5.47 (bs, 1 H), 3.88 (s, 3 H), 3.85 (m, 1 H), 3.44 (t, 1=4.6 Hz, 1 H), 3.36 (dd, 1=18.1, 2.3 Hz, 1 H), 3.14 (dd, 1=18.1, 5.0 Hz, 1 H), 3.08 (d, 1=20.0 Hz, 1 H), 2.19 (m, 1 Me H), 2.04 (m, 1 H), 1.83 (td, 1=12.6, 6.2 Hz, 1 H), 1.41 (s, 3 MeO H), 1.36 (dd, 1=12.6, 5.9 Hz, 1 H), 0.91 (s, 3 H), 0.39 (s, 3 40 13 H) ppm; C NMR (CDCl3 , 151 MHz) 0=196.5, 196.3, 192.6, 189.8, 173.6, 164.0, 161.0, 158.1, 146.9, 143.6, 135.9, 127.1, 121.8, 120.6, 107.0, 106.9, 102.4, 99.9, 77.3, 60.4, 55.8, 50.5, 45.4, 38.5, 34.1, 29.5, 25.5, 24.6, 23.0, 20.8 OBn OH ppm; HRMS (ESI) calcd for C30H29NO9 H+ [M+H+] 45 5-epi-15 548.1915, found 548.1894.

(±)-15 and (±)-5-epi-15: This reaction was run twice in parallel. To two separate batches of substrate (±)-11 (25 mg 50 each, 0.032 mmol each, 1.0 equiv each) was added solid

NaCNBH3 (8 mg each, 0.13 mmol each, 4.0 equiv each). Then, AcOH (2.5 mL each) was rapidly injected into the reaction vessel, and the mixture was vigorously stirred at room temperature for 25 minutes. The two reaction mixtures 55 were combined for work up by pouring them into water (25 mL). Brine ( 5 mL) was added, and the mixture was extracted with EtOAc (35 mL). The organic phase was washed with additional brine (2x25 mL), and the organic layer was dried 60 Viridicatumtoxin analog V4: To a stirred solution of over Na SO , filtered and concentrated. The crude residue (±)-15-epi-14 (40 mg, 0.058 mmol, 1.0 equiv) in 1,4­ 2 4 was purified by preparative TLC (silica gel, 10% acetone: dioxane:MeOH (5 mL, 1:1) was added Pd black (43 mg, 0.40 mmol, 7 equiv) under argon. The flask headspace was toluene) to provide (±)-15 (26 mg, not pure) and isomer (±)-5-epi-15 (13 mg, 0.017 mmol, 27%, yellow foam). exchanged for H2 , and the reaction mixture was stirred for 10 minutes at room temperature. The flask headspace was 65 Product (±)-15 was further purified by preparative TLC re-exchanged for argon, and the mixture was filtered through (silica gel, 5% EtOAc:CH2 Cl2 ) to give pure compound Celite® and concentrated (crude product: 44 mg). A portion (±)-15 (15 mg, 0.020 mmol, 31 %) as a yellow foam. US 10,065,924 B2 59 60

15

OH Me ~ H MeO

0

10 OH OH 0 0

Viridicatumtoxin analog V5: Following conditions simi­ (±)-15: Rf=0.6 (silica gel, acetone:toluene 3:17); FT-IR lar to those of Stork, et al. (1996), Pd black (12 mg, 0.11 (neat) vmax=3397, 2916, 1702, 1591, 1512, 1481, 1448, 15 mmol, 4.1 equiv) was added to a stirred solution of (±)-15 1405, 1371, 1339, 1319, 1254, 1195, 1143, 1109, 1081, (20 mg, 0.027 mmol, 1.0 equiv) in THF:MeOH (1.2 mL, 1 1 1032, 986, 914, 812, 734, 695 cm- ; H NMR (C D , 500 6 6 1: 1) under argon. The flask headspace was exchanged for MHz) 0=15.00 (s, 1 H), 7.58-7.55 (m, 2 H), 7.28-7.25 (m, 2 H2 , and the reaction mixture was stirred for 10 minutes at H), 7.24-7.20 (m, 2 H), 7.12 (m, 1 H), 7.05-6.98 (m, 3 H), room temperature. The flask headspace was re-exchanged 20 6.26 (s, 1 H), 5.56 (s, 1 H), 5.14 (d, J=12.2 Hz, 1 H, AB for argon, and the mixture was filtered through cotton and system), 5.10 (d, J=12.2 Hz, 1 H, AB system), 4.89 (ap s, 3 concentrated to give (±)-V5 (15 mg, 0.026 mmol, 96%) as H), 4.34 (d, J=8.6 Hz, 1 H), 3.56 (d, J=17.6 Hz, 1 H), 3.26 a yellow powder. (±)-V5: Rf=0.1 (silica gel, MeOH:CH2 Cl2 (s, 3 H), 2.85 (s, 3 H), 2.83 (m, 1 H), 2.77-2.64 (m, 3 H), 2.31 1:19); FT-IR (neat) vmax=3399, 2917, 1625, 1595, 1490, (m, 1 H), 2.04 (m, 1 H), 1.91 (ddd, J=12.3, 12.3, 6.1 Hz, 1 1 25 1471, 1451, 1401, 1309, 1273, 1200, 1084, 909, 732 cm - ; H), 1.65 (s, 3 H), 1.37 (dd, J=13.2, 6.0 Hz, 1 H), 1.15 (s, 3 1 , 13 H NMR (CDC13 600 MHz) 0=18.00 (s, 1 H), 15.12 (s, 1 H), 0.69 (s, 3 H) ppm; C NMR (C 6 D6 , 126 MHz) 0=197.0, H), 9.25 (bs, 1 H), 8.80 (s, 1 H), 6.63 (s, 1 H), 5.91 (bs, 1 185.4, 180.3, 168.6, 166.3, 159.7, 158.8, 149.2, 137.9, H), 5.49 (bs, 1 H), 5.16 (bs, 1 H), 4.39 (d, J=4.5 Hz, 1 H), 137.2, 135.7, 132.5, 128.9, 128.7, 128.6, 128.51, 128.48, 3.85 (s, 3 H), 3.57 (s, 3 H), 3.36 (d, J=17.5 Hz, 1 H), 3.13 127.2, 127.0, 122.5, 121.1, 109.2, 109.0, 106.2, 97.5, 78.6, 30 (m, 1 H), 2.88 (d, J=17.5 Hz, 1 H), 2.85 (dd, J=18.9, 5.4 Hz, 75.3, 72.3, 71.2, 59.3, 54.9, 54.7, 42.8, 42.6, 38.7, 34.4, 26.1, 1 H), 2.58 (dd, J=18.9, 10.0 Hz, 1 H), 2.22 (m, 1 H), 2.04 24.6, 23.4, 22.7, 21.5 ppm; HRMS (ESI) calcd for (m, 1 H), 1.77 (ddd, J=12.7, 12.7, 6.1 Hz, 1 H), 1.48 (s, 3 H),

C45H43NO9 Na+ [M+Na+] 764.2830, found 764.2853. 1.38 (dd, J=12.7, 6.2 Hz, 1 H), 0.93 (s, 3 H), 0.50 (s, 3 H) 13 ppm; C NMR (CDC1 3 , 151 MHz) 0=195.0, 194.0, 191.5, 35 173.2, 166.1, 160.5, 158.2, 147.6, 137.1, 135.7, 123.5, 122.5, 121.2, 106.4, 105.7, 100.6, 99.7, 76.7, 76.5, 60.0, 57.1, 55.6, 41.9, 38.4, 38.0, 34.2, 32.8, 25.8, 24.3, 23.0, 21.1

ppm; HRMS (ESI) calcd for C31H33NO9 Na+ [M+Na+] 586.2048, found 586.2045. 40

45 5-epi-15

0 (±)-5-epi-15: Rf=0.7 (silica gel, acetone:toluene 3:17); FT-IR (neat) vmax=3447, 2917, 1708, 1593, 1514, 1485, 50 1449, 1407, 1373, 1345, 1317, 1259, 1192, 1138, 1115, 1 1086, 994, 913, 813, 735, 696 cm-1; H NMR (C 6 D6 , 500 MHz)* 0=15.33 (s, 1 H), 7.57-7.54 (m, 2 H), 7.35-7.32 (m, Viridicatumtoxin analog V6: Following conditions simi­ 2 H), 7.22-7.18 (m, 2 H), 7.12-7.06 (m, 3 H), 7.04 (m, 1 H), lar to those of Stork, et al., (1996), Pd black (8.6 mg, 0.081 6.25 (s, 1 H), 5.59 (s, 1 H), 5.25 (d, J=12.1 Hz, 1 H, AB 55 mmol, 4.5 equiv) was added to a stirred solution of (±)-5­ system), 5.13 (d, J=12.1 Hz, 1 H,AB system), 4.91 (s, 2 H), epi-15 (13 mg, 0.018 mmol, 1.0 equiv) in THF:MeOH (1.0 4.75 (bs, 1 H), 3.44 (bs, 1 H), 3.36 (m, 1 H), 3.24 (s, 3 H), mL, 1: 1) under argon. The flask headspace was exchanged

2.91 (bs, 3 H), 2.64-2.56 (m, 2 H), 2.38-2.25 (m, 2 H), for H2 , and the reaction mixture was stirred for 10 minutes 2.10-1.97 (m, 2 H), 1.78 (s, 3 H), 1.38 (dd, J=12.5, 6.0 Hz, at room temperature. The flask headspace was re-exchanged 13 1 H), 1.12 (s, 3 H), 0.59 (s, 3 H) ppm; C NMR (C6D6 , 126 60 for argon, and the mixture was filtered through cotton and MHz)* 0=167.5, 159.7, 158.9, 138.0, 137.3, 135.8, 132.5, concentrated to give (±)-V6 (10 mg, 0.018 mmol, quant.) as

128.9, 128.7, 128.63, 128.56, 127.1, 126.4, 122.5, 121.2, a yellow powder. (±)-V6: Rf=0.1 silica gel, MeOH:CH2 Cl2 108.6, 106.7, 97.5, 72.4, 71.2, 59.1, 56.3, 54.8, 38.5, 34.4, 1:19); FT-IR (neat) vmax=34l9, 2921, 1626, 1594, 1490, 26.0, 24.3, 23.5, 21.5 ppm; HRMS (ESI) calcd for 1471, 1450, 1404, 1301, 1201, 1139, 1088, 907, 733 cm-1; 1 C45H43NO9 Na+ [M+Na+] 764.2830, found 764.2832. 65 H NMR (CDC13 , 600 MHz) 0=17.94 (s, 1 H), 15.34 (s, 1 *Due to signal broadening, 1 proton signal and 12 carbon H), 9.11 (s, 1 H), 8.84 (s, 1 H), 6.60 (s, 1 H), 5.85 (s, 1 H), signals could not be identified. 5.49 (s, 1 H), 5.00 (s, 1 H), 4.86 (s, 1 H), 3.85 (s, 3 H), 3.52 US 10,065,924 B2 61 62 (bs, 3 H), 3.22 (d, J=l 7.9 Hz, 1 H), 3.13 (d, J=l 7.9 Hz, 1 H), skilled in the art are deemed to be within the spirit, scope and 3.10 (m, 1 H), 2.92 (m, 1 H), 2.60 (dd, J=19.6, 10.9 Hz, 1 concept of the invention as defined by the appended claims. H), 2.22 (m, 1 H), 2.03 (m, 1 H), 1.82 (td, J=12.6, 6.1 Hz, 1 H), 1.54 (s, 3 H), 1.32 (dd, J=12.6, 6.1 Hz, 1 H), 0.89 (s, V. References 13 5 3 H), 0.42 (s, 3 H) ppm; C NMR (CDC13 , 151 MHz) 0=195.2, 194.8 (b), 192.1, 173.0, 166.6, 160.6, 158.1, 148.2, The following references, to the extent that they provide 137.6, 134.2, 124.2, 122.1, 120.8, 106.9, 105.3, 100.7, 99.2, exemplary procedural or other details supplementary to 76.6, 75.6, 59.4, 57.0, 55.6, 43.9, 39.2 (b), 38.3, 34.1, 30.8 those set forth herein, are specifically incorporated herein by (b), 25.7, 24.1, 23.0, 21.2 ppm; HRMS (ESI) calcd for reference: 10 JP 06-40995 C31H33NO9 Na+ [M+Na+] 586.2048, found 586.2036. WO 2009/008906 Example 4: Biological Activity Anderson, N. G., Practical Process Research & Develop­ ment-A Guide For Organic Chemists, 2nd ed., Academic A. Bacterial Strains and Growth Media: Press, New York, 2012. Four clinical strains were used for these studies, Entero­ 15 Breinholt, et al., Acta Chem. Scand., 51:855-860, 1997. coccus faecalis S613, Enterococcus faecium isolate 105, Brubaker and Myers, Org. Lett., 9:3523-3525, 2007. Methicillin-Resistant Staphylococcus aureus 371 (MRSA Charest, et al., J. Am. Chem. Soc., 127:8292-8293, 2005. 371) and Acinetobacter baumannii AB210. Enterococcus Charest, et al., Science 2005, 308, 395-398. strains were cultured in 80% Lysogeny Broth (LB) & 20% Brain Heart Infusion (BHI). MRSA 371 and AB210 were 20 Chooi, et al., Chem. Biol., 17:483-494, 2010. cultured in 100% LB. Chooi, et al., J. Am. Chem. Soc., 134:9428-9437, 2012. B. Minimal Inhibitory Concentration (MIC) Assays: Chooi, et al., J. Am. Chem. Soc., 135:16805-16808, 2013. Micro-broth MIC assays were performed in triplicate Chopra and Roberts, Microbial. Mal. Biol. R., 65:232-260, using 96-well plates. Wells were filled with 150 µL of broth 2001. media and inoculated with 2 µL of stationary phase culture. 25 De Jesus, et al., J. Chem. Soc., Chem. Comm., 902-904, The concentration of the test antibiotics increased in 2-fold 1982. increments and ranged from 0.25-128 µg/mL. Plates were Duggar, Ann. N. Y. Acad. Sci., 51:177-181, 1948. incubated overnight at 37° C. and the MICs were defined as Fulmer, et al., Organometallics, 29:2176-2179, 2010. the lowest drug concentration that had no growth after 16-24 Horak, et al., J. Chem. Soc., Chem. Comm., 1562-1564, 30 hours. 1988. Biological Evaluation of Analogs Hutchison, et al., Toxicol. Appl. Pharmacol., 24:507-509, The minimum inhibitory concentrations against both 1973. selected Gram-positive and Gram-negative strains were Inokoshi, et al., J. Antibiot., 66:37-41, 2013. determined for analogs V2-V6 and the results are shown in table 1. 35 Kabuto, et al., J. Chem. Soc., Chem. Comm. 728-729, 1976. Kodukula, et al., J. Antibiot., 48:1055-1059, 1995. TABLE 1 Koyama, et al., Molecules, 18:204-224, 2013. Kummer, et al., Chem. Sci., 2:1710-1718, 2011. Minimum Inhibitory Concentration (MIC) data of compounds March's Advanced Organic Chemistry: Reactions, Mecha­ against Gram-positive and Gram-negative bacteria and comparison with selected literature dataa 4o nisms, and Structure, 2007. Nicolaou, et al., Angew. Chem. Int. Ed., 52:8736-8741, Gram-+ Gram-(-) 2013.

E. faecalis E. faecium MRSA A. baumannii Nicolaou, et al., J. Am. Chem. Soc., 136(34):12137-12160, S613 501 371 AB210 2014. 45 Pangborn, et al., Organometallics, 15: 1518-1520, 1996. (±)-1 0.5 4 64 (+)-2 4 64 Greene's Protective Groups in Organic Chemistry, Wuts and (±)-V2 0.5 0.5 2 64 Greene, Ed., 1973 (±)-V3 4 2 8 64 Remington's Pharmaceutical Sciences, 15 th Ed., 1035-1038 (±)-V4 4 4 4 64 (±)-V5 8 64 50 and 1570-1580, 1990. (±)-V6 0.5 0.5 2 64 Remington's Pharmaceutical Sciences, 15 th Ed., 3:624-652, 1990. aMIC assays were run in triplicate; data are given in units of µg/mL. Shu, et al., J. Antibiot., 48:1060-1065, 1995. All of the compositions and/or methods disclosed and Silverton, et al., Acta Crystallogr. B, 38:3032-3037, 1982. claimed herein can be made and executed without undue 55 Still, et al., J. Org. Chem., 43:2923-2925, 1978. experimentation in light of the present disclosure. While the Stork, et al., J. Am. Chem. Soc., 118:5304-5305, 1996. compositions and methods of this invention have been Sun, et al., J. Am. Chem. Soc., 130:17913-17927, 2008. described in terms of preferred embodiments, it will be Sutcliffe, et al., Antimicrob. Agents Chemother., 57:5548­ apparent to those of skill in the art that variations may be 5558, 2013. applied to the compositions and/or methods and in the steps 60 Tally, et al., J. Antimicrob. Chemother., 35:449-452, 1995. or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the NCI 60 cell line assay for Viridicatumtoxin A (NSC invention. More specifically, it will be apparent that certain 159628). agents which are both chemically and physiologically Wong, et al., J. Antibiot. 46:214-221, 1993. related may be substituted for the agents described herein 65 Wright and Myers, Tetrahedron, 67:9853-9869, 2011. while the same or similar results would be achieved. All Wzorek, et al., Org. Lett., 14:5840-5843, 2012. such similar substitutes and modifications apparent to those Zheng, et al., J. Antibiot. 61:633-637, 2008. US 10,065,924 B2 63 64

What is claimed is: R12 is hydrogen, alkylecss), or substituted 1. A compound of the formula: alkylecss);

R13 is alkanediylecss) or substituted alkanediylecss); and (I) 5 R14 and R1s are each independently selected from: hydrogen, alkylecs12), arylecs12), aralkylecs12), acylecs12), or a substituted version of any of these groups; or R14 and R1s are taken together and are 10 alkanediylecss), alkoxydiylecss), alkylaminodi­ ylecss), or a substituted version of any of these groups; or a pharmaceutically acceptable salt or tautomer

15 thereof. 2. The compound of claim 1 further defined as: wherein: X1 is absent such that atoms 16 and 17 are only (II) connected by the shown single bond, a covalent bond 20 such that a double bond is formed between atoms 16 and 17, ---0-, alkanediylecss), or substituted alkanediylecss);

Yi, Y2, and Y3 are each independently alkylecsi2) or substituted alkylecsi2); 25 R1 is hydrogen, hydroxy, alkoxyecss), substituted alkoxyecss), or oxo, provided that when R1 is oxo, the bond between R1 and atom number 5 is a double bond; 30 R2 is hydrogen, amino, carboxy, cyano, halo, hydroxy, nitro, or sulfa; wherein: alkylecsi2), alkoxyecsi2), alkylaminoecsi2), dialkylam­ inoecsis), or a substituted version of any of these X1 is absent such that atoms 16 and 17 are only groups; connected by the shown single bond, a covalent bond 35 such that a double bond is formed between atoms 16 R3 , R6 , R7 , and R10 are each independently selected from: hydrogen, and 17, -0-, alkanediylecss), or substituted alkanediylecss); alkylecss), alkenylecss), arylecsi2), aralkylecsi2), acylecss), or a substituted version of any of these Yi, Y2, and Y3 are each independently alkylecsi2) or substituted alkylecsi ); groups; 40 2 R is hydrogen, hydroxy, alkoxyecss), substituted R4 and Rs are each independently selected from: hydro­ 1 gen, alkoxyecss), or oxo, provided that when R1 is oxo, alkylecss), alkanediylecss)-heterocycloalkylecss), the bond between R1 and atom number 5 is a double alkanediylecss)-heteroarylecss), alkanediylecss)­ bond; alkylaminoecss), alkanediylecss)-dialkylaminoecss), 45 R2 is hydrogen, amino, carboxy, cyano, halo, hydroxy, or a substituted version of any of these groups; nitro, or sulfa;

R8 is hydrogen, alkylecsi2), alkoxyecsi2), alkylaminoecsi2), dialkylam­ alkylecss), alkenylecss), arylecsi2), aralkylecsi2), inoecss), or a substituted version of any of these acylecss), or a substituted version of any of these groups; groups; or -X2-Ru, wherein: 50 R4 and Rs are each independently selected from: hydro­ X2is alkanediylecsi2) or substituted alkanediylecsi2); gen, and alkylecss), alkanediylecss)-heterocycloalkylecss), Ru is hydroxy, amino, azido, carboxy, or cyano, alkanediylecss)-heteroarylecss), alkanediylecss)­ alkenylecs 6), alkynylecs 6 ), heterocycloalkylecsi2), alkylaminoecss), alkanediylecss)-dialkylaminoecss), alkylaminoecss), dialkylaminoecss), alkoxyecss), 55 or a substituted version of any of these groups;

or a substituted version of any of these groups; or R8 is hydrogen, a -linker-biomolecule wherein the biomolecule is a alkylecss), alkenylecsi2), arylecsi2), aralkylecsi2), protein, a polypeptide, an amino acid, a cofactor, acylecss), or a substituted version of any of these an imaging agent, an antibody, a fatty acid, a groups; or -X2-Ru, wherein: nucleic acid, or a small molecule therapeutic 60 X2is alkanediylecsi2) or substituted alkanediylecsi2); agent; and and

R9 is hydrogen, amino, carboxy, cyano, halo, hydroxy, Ru is hydroxy, amino, azido, carboxy, or cyano, nitro, or sulfa; alkenylecs6 ), alkynylecs6 ), heterocycloalkylecs12), alkylecsi2), alkoxyecsi2), alkylaminoecsi2), dialkylam­ alkylaminoecss), dialkylaminoecss), alkoxyecss), inoecsis), amidoecsi2), or a substituted version ofany 65 or a substituted version of any of these groups; or of these groups; or -NR12C(O)R13-NR14R1s; a -linker-biomolecule wherein the biomolecule is a wherein: protein, a polypeptide, an amino acid, a cofactor, US 10,065,924 B2 65 66 an imaging agent, an antibody, a fatty acid, a a -linker-biomolecule wherein the biomolecule is a nucleic acid, or a small molecule therapeutic protein, a polypeptide, an amino acid, a cofactor, agent; and an imaging agent, an antibody, a fatty acid, a nucleic acid, or a small molecule therapeutic R9 is hydrogen, amino, carboxy, cyano, halo, hydroxy, nitro, or sulfa; 5 agent; and R is hydrogen, amino, carboxy, cyano, halo, hydroxy, alkylecsl2), alkoxyecsl2), alkylaminoecsl2), dialkylam­ 9 nitro, or sulfa; inoecsls), amidoecs 12), or a substituted version ofany of these groups; or -NR C(O)R -NR R ; alkylecsl2), alkoxyecsl2), alkylaminoecsl2), dialkylam­ 12 13 14 15 ino ecs ), amidoecs ), or a substituted version ofany wherein: 18 12 10 of these groups; or -NR12C(O)R13-NR14R15 ; R12 is hydrogen, alkylecss), or substituted wherein: alkylecss); R is hydrogen, alkylecss), or substituted R is alkanediylecss) or substituted 12 13 alkylecss); alkanediylecss); and R13 is alkanediylecss) or substituted R14 and R15 are each independently selected from: 15 alkanediylecss); and hydrogen, R14 and R15 are each independently selected from: alkylecs12), arylecs12), aralkylecs12), acylecs12), or hydrogen, a substituted version of any of these groups; or alkylecsl2), arylecsl2), aralkylecsl2), acylecsl2), or R14 and R15 are taken together and are a substituted version of any of these groups; or alkanediylecss), alkoxydiylecss), alkylaminodi­ 20 R14 and R15 are taken together and are ylecss), or a substituted version of any of these alkanediylecss), alkoxydiylecss), alkylaminodi­ groups; ylecss), or a substituted version of any of these or a pharmaceutically acceptable salt or tautomer groups; thereof. or a pharmaceutically acceptable salt or tautomer thereof. 3. The compound of claim 1 further defined as: 25 4. The compound of claim 1 further defined as:

(IV) (V)

30

35

40 wherein: wherein:

R1 is hydrogen, hydroxy, alkoxyecss), substituted R1 is hydroxy, alkoxyecss), or substituted alkoxyecss); alkoxyecss), or oxo, provided that when R1 is oxo, R2 is hydrogen, amino, carboxy, cyano, halo, hydroxy, the bond between R1 and atom number 5 is a double nitro, or sulfa; bond and when the bond between R1 and atom 45 alkylecsl2), alkoxyecsl2), alkylaminoecsl2), dialkylam­ number 5 is a double bond then R1 is oxo; inoecsls), or a substituted version of any of these R2 is hydrogen, amino, carboxy, cyano, halo, hydroxy, groups; nitro, or sulfa; R4 is hydrogen, alkylecsl2), alkoxyecsl2), alkylaminoecsl2), dialkylam­ alkylecss), alkanediylecss)-heterocycloalkylecss), inoecsls), or a substituted version of any of these 50 alkanediylecss)-heteroarylecss), alkanediylecss)­ groups; alkylaminoecss), alkanediylecss)-dialkylaminoecss), R4 and R5are each independently selected from: hydro­ or a substituted version of any of these groups; gen, R8 is hydrogen, alkylecss), alkanediylecss)-heterocycloalkylecss), alkylecss), alkenylecss), arylecsl2), aralkylecsl2), alkanediylecss)-heteroarylecss), alkanediylecss)­ 55 acylecss), or a substituted version of any of these alkylaminoecss), alkanediylecss)-dialkylaminoecss), groups; or -X2-Ru, wherein: or a substituted version of any of these groups; X2is alkanediylecsl2) or substituted alkanediylecsl2); R8 is hydrogen, and alkylecss), alkenylecss), arylecsl2), aralkylecsl2), Ru is hydroxy, amino, azido, carboxy, or cyano, acylecss), or a substituted version of any of these 60 alkenylecs6 ), alkynylecs6 ), heterocycloalkylecs12), groups; or -X2-Ru, wherein: alkylaminoecss), dialkylaminoecss), alkoxyecss), X2is alkanediylecsl2) or substituted alkanediylecsl2); or a substituted version of any of these groups; or and a -linker-biomolecule wherein the biomolecule is a Ru is hydroxy, amino, azido, carboxy, or cyano, protein, a polypeptide, an amino acid, a cofactor,

alkenylecs 6), alkynylecs 6 ), heterocycloalkylecsl2), 65 an imaging agent, an antibody, a fatty acid, a alkylaminoecss), dialkylaminoecss), alkoxyecss), nucleic acid, or a small molecule therapeutic or a substituted version of any of these groups; or agent; and US 10,065,924 B2 67 68

R9 is hydrogen, amino, carboxy, cyano, halo, hydroxy, the biomolecule is a protein, a polypeptide, an nitro, or sulfa; antibody, an imaging agent, or a small molecule alkylccsl2), alkoxy(Csl2), alkylamino(Csl2), dialkylam­ therapeutic agent; ino(Csls), amidoccsl2), or a substituted version ofany R9 is hydrogen, amino, carboxy, cyano, halo, hydroxy, of these groups; or -NR12C(O)R13-NR14R1s; nitro, or sulfa; wherein: alkylccsl2), alkoxy(Csl2), alkylamino(Csl2), dialkylam­ R12 is hydrogen, alkylccss), or substituted ino ccs 18), amidoccs12), or a substituted version ofany alkyl(css); of these groups; or -NR12C(O)R13-NR14R1s; R13 is alkanediylccss) or substituted wherein: 10 alkanediylccss); and R12 is hydrogen, alkylccss), or substituted R14 and R1s are each independently selected from: alkyl(css); hydrogen, R13 is alkanediylccss) or substituted alkylccs12), arylccs12), aralkylccs12), acylccs12), or alkanediylccss); and a substituted version of any of these groups; or 15 R14 and R1s are each independently selected from: R14 and R1s are taken together and are hydrogen, alkanediylccss), alkoxydiylccss), alkylaminodi­ alkylccs12), arylccs12), aralkylccs12), acylccs12), or yl(css), or a substituted version of any of these a substituted version of any of these groups; or groups; R14 and R1s are taken together and are or a pharmaceutically acceptable salt or tautomer 20 alkanediylccss), alkoxydiylccss), alkylaminodi­ thereof. ylccss), or a substituted version of any of these 5. The compound of claim 1 further defined as: groups; or a pharmaceutically acceptable salt or tautomer thereof. (V) 25 6. The compound of claim 1 further defined as:

(V)

30

0

OH 0 0 _,,..o 35 0 Rs

OH 0 0 wherein: _,,..o Rs R1 is hydroxy, alkoxyccss), or substituted alkoxyccss); 40 R2 is hydrogen, amino, carboxy, cyano, halo, hydroxy, nitro, or sulfa; wherein: R is hydroxy, alkoxy(css), or substituted alkoxy(css); alkylccsl2), alkoxy(Csl2), alkylamino(Csl2), dialkylam­ 1 R is hydrogen, amino, carboxy, cyano, halo, hydroxy, ino(Csls), or a substituted version of any of these 2 nitro, or sulfa; groups; 45 alkylccsl ), alkoxy(Csl ), alkylamino(Csl ), dialkylam­ R is hydrogen, alkylccss), or a substituted alkylccss), 2 2 2 4 ino(Csls), or a substituted version of any of these Rs is hydrogen, groups; alkylccss), alkanediylccss)-heterocycloalkylccss), R4 is hydrogen, alkylccss), or a substituted alkylccss), alkanediylccss)-heteroarylccss), alkanediylccss)­ Rs is hydrogen, alkylaminoccss), alkanediylccss)-dialkylaminoccss), 50 alkylccss), alkanediylccss)-heterocycloalkylccss), or a substituted version of any of these groups; alkanediylccss)-heteroarylccss), alkanediylccss)­ R8 is hydrogen, alkylaminoccss), alkanediylccss)-dialkylaminoccss), alkyl(css), alkenyl(css), aryl(Csl2), aralkyl(Csl2), or a substituted version of any of these groups; acylccss), or a substituted version of any of these R8 is hydrogen or -linker-biomolecule; groups; or -X2-Ru, wherein: 55 wherein: X2is alkanediylccsl2) or substituted alkanediylccsl2); the linker is alkanediylccsl2), alkenediylccs12), and arenediylccsl2), heteroarenediylccsl2), hetero­ Ru is hydroxy, amino, azido, carboxy, or cyano, cycloalkanediylccsl2); and alkenylccs 6), alkynylccs 6 ), heterocycloalkylccsl2), the biomolecule is an antibody; and alkylaminoccss), dialkylaminoccss), alkoxy(css), 60 R9 is hydrogen, amino, carboxy, cyano, halo, hydroxy, or a substituted version of any of these groups; or nitro, or sulfa; a -linker-biomolecule alkylccsl2), alkoxy(Csl2), alkylamino(Csl2), dialkylam­ wherein: ino ccs 18), amidoccs12), or a substituted version ofany the linker is alkanediylccsl2), alkenediylccsl2), of these groups; or -NR12C(O)R13-NR14R1s; arenediylccsl2), heteroarenediylccsl2), hetero- 65 wherein: cycloalkanediylccsl2) or a substituted version of R12 is hydrogen, alkylccss), or substituted any of these groups; and alkyl(css); US 10,065,924 B2 69 70

R13 is alkanediylecss) or substituted or a pharmaceutically acceptable salt or tautomer alkanediylecss); and thereof. R14 and R1s are each independently selected from: 8. The compound of claim 1 further defined as: hydrogen, alkylecsi2), arylecsi2), aralkylecsi2), acylecsi2), or 5 a substituted version of any of these groups; or (V) R14 and R1s are taken together and are alkanediylecss), alkoxydiylecss), alkylaminodi­ ylecss), or a substituted version of any of these groups; 10 or a pharmaceutically acceptable salt or tautomer thereof. 7. The compound of claim 1 further defined as:

15

(V)

wherein: 20 R1 is hydroxy, alkoxyecss), or substituted alkoxyecss);

R2 is hydrogen, amino, halo, or hydroxy; alkylaminoecsi2), dialkylaminoecsis), or a substituted version of any of these groups; 25 R4 is hydrogen, alkylecss), or a substituted alkylecss), Rs is hydrogen, alkylecss), alkanediylecss)-heterocycloalkylecss), alkanediylecss)-heteroarylecss), alkanediylecss)­ wherein: 30 alkylaminoecss), alkanediylecss)-dialkylaminoecss), R1 is hydroxy, alkoxyecss), or substituted alkoxyecss); or a substituted version of any of these groups;

R2 is hydrogen, amino, halo, or hydroxy; R8 is hydrogen, alkylaminoecsi2), dialkylaminoecsis), or a substituted alkylecss), alkenylecss), arylecsi2), aralkylecsi2), version of any of these groups; acylecss), or a substituted version of any of these R4 is hydrogen, alkylecss), or a substituted alkylecss), 35 groups; or -X2-Ru, wherein: Rs is hydrogen, X2is alkanediylecsi2) or substituted alkanediylecsi2); alkylecss), alkanediylecss)-heterocycloalkylecss), and alkanediylecss)-heteroarylecss), alkanediylecss)­ Ru is hydroxy, amino, azido, carboxy, or cyano, alkylaminoecss), alkanediylecss)-dialkylaminoecss), or a substituted version of any of these groups; 40 alkenylecs6 ), alkynylecs6 ), heterocycloalkylecs12), R is hydrogen, alkylaminoecss), dialkylaminoecss), alkoxyecss), 8 or a substituted version of any of these groups; or alkylecss), alkenylecss), arylecsi2), aralkylecsi2), acylecss), or a substituted version of any of these a -linker-biomolecule; and -X -Ru, groups; or 2 wherein: R9 is -NR12C(O)R13-NR14R1s; X2is alkanediylecsi2) or substituted alkanediylecsi2); 45 wherein: and R is hydrogen, alkylecss), or substituted Ru is hydroxy, amino, azido, carboxy, or cyano, 12 alkylecss); alkenylecs 6), alkynylecs 6 ), heterocycloalkylecsi2), alkylaminoecss), dialkylaminoecss), alkoxyecss), R13 is alkanediylecss) or substituted or a substituted version of any of these groups; or 50 alkanediylecss); and a -linker-biomolecule; and R14 and R1s are each independently selected from:

R9 is hydrogen, amino, carboxy, cyano, halo, hydroxy; hydrogen, alkoxyecsi2), amidoecsi2), or a substituted version of alkylecs12), arylecs12), aralkylecs12), acylecs12), or any of these groups; or -NR12C(O)R13-NR14R1s; a substituted version of any of these groups; wherein: 55 or a pharmaceutically acceptable salt or tautomer R is hydrogen, alkylecss), or substituted 12 thereof. alkylecss); 9. The compound of claim 1, wherein R is alkoxyecss) or R13 is alkanediylecss) or substituted 1 alkanediylecss); and substituted alkoxyecss)· R14 and R1s are each independently selected from: 60 10. The compound of claim 9, wherein R1 is methoxy. hydrogen, 11. The compound of claim 1, wherein R1 is oxo. alkylecs12), arylecs12), aralkylecs12), acylecs12), or 12. The compound of claim 1, wherein R2 is hydrogen. a substituted version of any of these groups; or 13. The compound of claim 1, wherein R2 is amino. R14 and R1s are taken together and are alkanediylecss), alkoxydiylecss), alkylaminodi­ 65 14. The compound of claim 1, wherein R8 is hydrogen. ylecss), or a substituted version of any of these 15. The compound of claim 1, wherein the compound is groups; further defined as: US 10,065,924 B2 71 72 18. A method of preparing a compound of the formula:

(VI)

0, 10

15 0

MeO wherein: Y4, Y 5, and Y 6 are each independently hydrogen, 0, 20 alkylecss) or substituted alkYecss); R16 and R16' are each independently hydrogen, alkoxyecss), aralkoxyecsl ), substituted alkoxyecss), OH OH 0 0 2 or substituted aralkoxyecsl2); or R16 and R16' are taken together and are alkoxydiylecsl2); 25 R17 is hydrogen, amino, carboxy, cyano, halo, hydroxy, nitro, or sulfa; 0 alkylecsl2), alkoxyecsl2), alkylaminoecsl2), dialkylam­ inoecsls), or a substituted version of any of these MeO groups; or ----C(0)0CH CH Si(CH ) ; 30 2 2 3 3 X 3 is 0, NH, S, or CH2; 0, X 4 is hydroxy, amino, mercapto, 0, NH, or S provided that when X 4 is 0, NH, or S, the bond to which the OMe OH 0 0 atom is attached is a double bond; 35 X 5 is 0, NH, or S; R18 is hydrogen, alkylecss), alkenylecss), arylecsl2), aralkylecsl2), acylecss), or a substituted version of any of these OH groups; or -X2-Ru, wherein: H 40 X2is alkanediylecsl2) or substituted alkanediylecsl2); MeO and Ru is hydroxy, amino, azido, carboxy, or cyano, 0, or alkenylecs6), alkynylecs6), heterocycloalkylecs12), alkylaminoecss), dialkylaminoecss), alkoxyecss), 45 or a substituted version of any of these groups; OH OH 0 0 R19 is hydrogen, amino, carboxy, cyano, halo, hydroxy, nitro, or sulfa; alkylecsl2), alkoxyecsl2), alkylaminoecsl2), dialkylam­ ino ecs 18), amidoecs12), or a substituted version ofany 50 of these groups; or -NR12C(0)R13-NR14R15 ; wherein:

R12 1s hydrogen, alkylecss), or substituted alkylecss); R is alkanediylecss) or substituted 0, 13 55 alkanediylecss); and R14 and R15 are each independently selected from: hydrogen, alkylecs12), arylecs12), aralkylecs12), acylecs12), or a substituted version of any of these groups; or 60 R14 and R15 taken together and are or a pharmaceutically acceptable salt, or tautomer thereof. alkanediylecss), alkoxydiylecss), alkylaminodi­ ylecss), or a substituted version of any of these 16. A pharmaceutical composition comprising a com­ groups; and pound of claim 1 and an excipient. R20 and R21 are each independently hydrogen, 17. A method of treating a bacterial infection comprising 65 alkylecss), alkenylecss), arylecsl2), aralkylecsl2), administering a pharmaceutically effective amount of a acylecss), or a substituted version of any of these compound or composition of claim 1. groups; US 10,065,924 B2 73 74

or a salt, tautomer, or optical isomer thereof; compris­ or substituted aralkoxyecsl2); or R16 and R16' are ing reacting a compound of the formula: taken together and are alkoxydiylecsl2);

R17 is hydrogen, amino, carboxy, cyano, halo, hydroxy, nitro, or sulfa; (VII) 5 alkylecsl2), alkoxyecsl2), alkylaminoecsl2), dialkylam­ inoecsls), or a substituted version of any of these

groups; or ----C(0)0CH2CH2Si(CH3 ) 3 ; X3 is 0, NH, S, or CH2;

X4 is 0, NH, or S; 10 X5 is 0, NH, or S; R18 is hydrogen, alkylecss), alkenylecss), arylecsl2), aralkylecsl2), acylecss), or a substituted version of any of these 15 groups; or -X2-Ru, wherein: X2is alkanediylecsl2) or substituted alkanediylecsl2); and

wherein: R11 is hydroxy, amino, azido, carboxy, or cyano, alkenylecs ), alkynylecs ), heterocycloalkylecs ), Y4, Y 5, Y 6, R16, R16', X 5, R18, R19, and R20 are as 6 6 12 defined above; and 20 alkylaminoecss), dialkylaminoecss), alkoxyecss), or a substituted version of any of these groups; X 4 is 0, NH, or S; with a compound of the formula: R19 is hydrogen, amino, carboxy, cyano, halo, hydroxy, nitro, or sulfa; alkylecsl2), alkoxyecsl2), alkylaminoecsl2), dialkylam­ (VIII) 25 ino ecs 18), amidoecs12), or a substituted version ofany of these groups; or -NR12C(0)R13-NR14R15 ; wherein:

R12 is hydrogen, alkylecss), or substituted alkylecss); 30 R13 is alkanediylecss) or substituted alkanediylecss); and R14 and R15 are each independently selected from: hydrogen, 35 alkylecs12), arylecs12), aralkylecs12), acylecs12), or wherein: a substituted version of any of these groups; or R and R taken together and are X3 and R17 are as defined above; 14 15 alkanediylecss), alkoxydiylecss), alkylaminodi­ R21 is hydrogen, ylecss), or a substituted version of any of these alkylecss), alkenylecss), arylecsl 2), aralkylecsl2), 40 groups; and acylecss), or a substituted version of any of these groups; and R20 and R21 are each independently hydrogen, alkylecss), alkenylecss), arylecsl ), aralkylecsl ), R22 is arylecsl 2), aralkylecsl2), or a substituted ver­ 2 2 sion of either of these groups; acylecss), or a substituted version of any of these groups; in the presence of a base. 45 19. The method of claim 18, wherein the method further or a salt, tautomer, or optical isomer thereof; compris­ comprises forming a compound of the formula: ing reacting a compound of the formula:

(X) (IX) 50

/0 /0 55 R20 R20

0 Xs Xi X3 /0 Xs Xi X3 /0 60 I R1s R1s R21

wherein: wherein the variables are as defined above; with a metal Y4, Y 5, and Y 6 are each independently hydrogen, catalyst and an oxidizing agent. alkylecss) or substituted alkYecss); 65 20. The method of claim 19, wherein the reaction further R16 and R16' are each independently hydrogen, comprises reacting a compound of formula IX with a alkoxyecss), aralkoxyecsl2), substituted alkoxyecss), reducing agent to form a compound of the formula: US 10,065,924 B2 75 76 X2!;lkanediylecs12) or substituted alkanediylecsl2); (XI)

R11 is hydroxy, amino, azido, carboxy, or cyano, alkenylecs6), alkynylecs6), heterocycloalkylecs12), alkylaminoecss), dialkylaminoecss), alkoxyecss), or a substituted version of any of these groups;

R19 is hydrogen, amino, carboxy, cyano, halo, hydroxy, nitro, or sulfa; al~ylecs 12), alk~XYecsl 2), alkylaminoecsl2), dialkylam­ 10 mo ecs 1s), am1doecs12), or a substituted version ofany of these groups; or -NR12C(0)R13-NR14R15 ; wherein:

R12 is hydrogen, alkylecss), or substituted alkylecss); wherein: 15 R13 is alkanediylecss) or substituted Y , Y , and Y are each independently hydrogen, alkanediylecss); and 4 5 6 R and R are each independently selected from: alkylecss) or substituted alkYecss); 14 15 1 hydrogen, R16 and R16 are each independently hydrogen, alkylecs12), arylecs12), aralkylecs12), acylecs12), or alkoxyec:ss), aralkoxyecsl2), substituted alkoxyecss), or substituted aralkoxyecsl ); or R and R ' are 20 a substituted version of any of these groups; or 2 16 16 R and R are taken together and are taken together and are alkoxydiylecsl2); 14 15 alkanediylecss), alkoxydiylecss), alkylaminodi­ R17 is hydrogen, amino, carboxy, cyano, halo, hydroxy, nitro, or sulfa; ylecss), or a substituted version of any of these groups; al~ylecsl2), alkoxyecsl2), alkylaminoecsl2), dialkylam­ 25 R and R are each independently hydrogen, moecslS)' or a substituted version of any of these 20 21 alkylecss), alkenylecss), arylecsl2), aralkylecsl2), groups; or ----C(0)0CH2CH2Si(CH3 ) 3 ; acylecss), or a substituted version of any of these X 3 is 0, NH, S, or CH2; X is 0, NH, or S; groups; and 4 R is hydroxy, alkoxyecsl ), or substituted R18 is hydrogen, 23 2 30 alkylecss), alkenylecss), arylecsl ), aralkylecsl ), alkoxyecsl 2); 2 2 or a salt, tautomer, or optical isomer thereof. acylecss), or a substituted version of any of these groups; or -X2-Ru, wherein: * * * * * UNITED STATES PATENT AND TRADEMARK OFFICE CERTIFICATE OF CORRECTION

PATENT NO. : 10,065,924 B2 Page 1 of 1 APPLICATION NO. : 15/327912 DATED : September 4, 2018 INVENTOR(S) : Kyriacos C Nicolaou et al.

It is certified that error appears in the above-identified patent and that said Letters Patent is hereby corrected as shown below:

In the Claims

In Claim 2, Column 64, Line 48, delete "(c<:s)°' and insert --(c<:1Sr- therefor.

In Claim 4, Column 66, Lines 48-52, delete the entire contents and insert --~ is hydrogen, alkyl(c<:sJ, or substituted alkyl(c<:sJ, Rs is hydrogen, alkyl(c<:sJ, alkanediyl(c<:sJ-heterocycloalkyl(c<:sJ, alkanediyl(c<:sJ-heteroaryl(c<:sJ, alkanediyl(c<:sJ-alkylamino(c<:sJ, alkanediyl(c<:sJ-dialkylamino(c<:sJ, or a substituted version of any ofthese groups;-- therefor.

In Claim 7, Column 69, Line 52, delete "carboxy,".

In Claim 19, Column 74, Line 37, delete "R1s taken" and insert --R1s are taken-- therefor.

Signed and Sealed this Thirteenth Day ofNovember, 2018

~"""'-...... Andrei Iancu Director ofthe United States Patent and Trademark Office