US 2011 0118274A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2011/0118274 A1 Nathan et al. (43) Pub. Date: May 19, 2011

(54) PROTEASOME INHIBITORS AND THEIR USE (52) U.S. Cl...... 514/252.05: 514/360; 514/378: INTREATING PATHOGEN INFECTION AND 514/374; 514/365: 514/372: 514/364; 514/361; CANCER 514/362; 514/342: 514/256; 514/255.05; 548/122; 514/380: 514/376; 514/377: 514/369; (75) Inventors: Carl Nathan, Larchmont, NY (US); 514/370 Gang Lin, Forest Hills, NY (US) (73) Assignee: CORNELL RESEARCH FOUNDATION, INC., Ithaca, NY (US) (57) ABSTRACT (21) Appl. No.: 12/674,820 The present invention relates to proteasome inhibitors and their use in methods of treating a subject for a pathogen (22) PCT Filed: Aug. 25, 2008 infection or cancer. The hihirov in NR E. subject a compound of Formula (I). (I) where: Q is Formula or (86). PCT No.: PCT/USO8/74.146 Formula, where the crossing dashed line illustrates the bond S371 (c)(1), formed joining Q to the rest of the compound of Formula (I). (2), (4) Date: May 14, 2010 The remainder of substituents of the compound of Formula (I) are defined in the present application. Related U.S. Application Data (60) Provisional application No. 60/957,610, filed on Aug. (I) 23, 2007. o-ky-oO Publication Classification N-S (51) Int. Cl. E (II) A6 IK 3/4I (2006.01) D2 A6 IK3I/422 (2006.01) / A6 IK 3/427 (2006.01) AN A6 IK 3/42.45 (2006.01) (III) A6 IK 3/433 (2006.01) E A6 IK 3/4439 (2006.01) | N A6 IK3I/50 I (2006.01) DNA A6 IK3I/506 (2006.01) (IV) A 6LX 3L/2197 (2006.01) 1's CO7D 49/04 (2006.01) J Y

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PROTEASOME INHIBITORS AND THEIR USE “Proteasome Inhibitors: from Research Tools to Drug Candi INTREATING PATHOGEN INFECTION AND dates.” Chem. Biol. 8:739-758 (2001)). Three of the B sub CANCER units (B1-caspase-like, B2—trypsin-like, and B5—chymot rypsin-like) display proteolytic activity (Orlowski et al., “Evidence for the Presence of Five Distinct Proteolytic Com 0001. This application claims the benefit of U.S. Provi ponents in the Pituitary Multicatalytic Proteinase Complex. sional Patent Application Ser. No. 60/957,610, filed Aug. 23, Properties of Two Components Cleaving Bonds on the Car 2007. boxyl Side of Branched Chain and Small Neutral Amino 0002 The subject matter of this application was made Acids.” Biochemistry 32:1563-1572 (1993)). Prokaryotic with support from the United States Government under The proteasomes usually have only one type of C. Subunits and 1 or National Institutes of Health, Grant No. POI A1056293. The 2 types of B subunits. The Y-OH of the N-terminal threonine of government has certain rights in this invention. the subunits forms a key element of each active site (Seemuller et al., “Autocatalytic Processing of the 20S Pro FIELD OF THE INVENTION teasome,” Nature 382:468-471 (1996)). Because there are few N-terminal threonine-dependent hydrolases, it has been 0003. The present invention relates to proteasome inhibi possible to develop proteasome inhibitors that spare other tors and their use in treating pathogen infection and cancer. proteases to various degrees (Kisselev et al., “Proteasome Inhibitors: from Research Tools to Drug Candidates. Chem. BACKGROUND OF THE INVENTION Biol. 8:739-758 (2001); Goldberg et al., “Not Just Research 0004 Mycobacterium tuberculosis (Mtb) is causing a glo Tools—Proteasome Inhibitors Offer Therapeutic Promise.” bal health emergency that is rapidly worsening through the Nat. Med. 8:338-340 (2002)), such as epoxyketones (Kim et intersection of the tuberculosis pandemic with epidemics of al., “Proteasome Inhibition by the Natural Products Epoxo antibiotic resistance, HIV/AIDS, and obesity-associated dia micin and Dihydroeponemycin: Insights Into Specificity and betes (Corbett et al., “The Growing Burden of Tuberculosis: Potency.” Bioorg. Med. Chem. Lett. 9:3335-3340 (1999)) and Global Trends and Interactions with the HIV Epidemic, peptidyl boronic acids (Adams et al., “Potent and Selective Arch. Intern. Med. 163:1009-1021 (2003); and Restrepo, B. Inhibitors of the Proteasome: Dipeptidyl Boronic Acids.” I., "Convergence of the Tuberculosis and Diabetes Epidem Bioorg Med. Chem. Lett. 8:333-338 (1998)). Studies of the ics: Renewal of Old Acquaintances.” Clin. Infect. Dis. cytotoxicity of proteasome inhibitors have indicated a strong 45:436-438 (2007)). Yet little new chemotherapy against Mtb correlation between inhibitory activity against chymotryptic has emerged in decades. In 1931, Dubos and Avery (Avery et like activity and their cytotoxicity (Adams et al., “Proteasome al., “The Protective Action of a Specific Enzyme Against Inhibitors: A Novel Class of Potent and Effective Antitumor Type III Pneumococcus Infection in Mice.” J. Exp. Med. Agents.” Cancer Res. 59:2615-2622 (1999)). The peptidyl 54:73-89 (1931)) introduced the concept of targeting a path boronate bortezomib (Velcade(R) is in clinical use for the way in the pathogen. The pathway is essential for the patho treatment of multiple myeloma and other malignancies gen Survival in the host, even though the pathogen does not (Kropff et al., “Bortezomib in Combination With Intermedi require that pathway for Survival in a bacteriologic culture ate-Dose Dexamethasone and Continuous Low-Dose Oral medium that Supports its rapid replication. Perhaps because Cyclophosphamide for Relapsed Multiple Myeloma. Br. J. the antimicrobial agent used by Dubos and Avery was a Haematol. 138:330-337 (2007)). microbial enzyme whose efficacy was abolished when the 0006 Although Mtb is a bacterium, it has been shown that host developed antibodies against it, this approach was it expresses a proteasome core consisting of the typical four ignored for over 70 years, until the contemporary crisis in heptameric rings stacked in a cylinder. Cryoelectron micros anti-infectives discovery led to its reconsideration (Nathan, copy, X-ray crystallography with a peptidylboronate inhibi C., “Antibiotics at the Crossroads. Nature 431:899-902 tor and mutation analysis Suggested that the C. Subunits have (2004); and Clatworthy et al., “Targeting Virulence: A New a gating function and confirmed that the B subunits provide Paradigm for Antimicrobial Therapy.” Nat. Chem. Biol. the active site N-terminal threonine hydroxyl (Lin et al., 3:541-548 (2007)) and demonstration with small molecules “Mycobacterium tuberculosis preBA Genes Encode a Gated (Liu et al., “Staphylococcus aureus Golden Pigment Impairs Proteasome With Broad Oligopeptide Specificity. Mol. Neutrophil Killing and Promotes Virulence Through its Anti Microbiol. 59:1405-1416 (2006); Hu et al., “Structure of the oxidant Activity.” J. Exp. Med. 202:209-215 (2005)). Mycobacterium tuberculosis Proteasome and Mechanism of 0005. The proteasome is not essential for Mtb survival Inhibition by a Peptidyl Boronate.” Mol. Microbiol. 59:1417 under in vitro growth-Sustaining conditions, but has emerged 1428 (2006)). A peptidyl boronate and an epoxyketone each as an element in a pathway that Mtb requires in order to prevented growth of Mtb and were mycobactericidal during Survive nitrosative stress and other conditions that the patho recovery of Mtb from exposure to reactive nitrogen interme gen faces during the course of infection (Darwin et al., “The diates (Darwin et al., “The Proteasome of Mycobacterium Proteasome of Mycobacterium tuberculosis is Required for tuberculosis is Required for Resistance to Nitric Oxide.” Sci Resistance to Nitric Oxide.” Science 302:1963-1966 (2003); ence 302:1963-1966 (2003)). Both compounds inhibited a and Gandotra et al., “In vivo Gene Silencing Identifies the peptidolytic activity in Mtb lysates, while an enantiomer of Mycobacterium tuberculosis Proteasome as Essential for the the peptidylboronate neither inhibited the peptidolytic activ Bacteria to Persist in Mice, Nat. Med 13:1515-1520 ity, prevented growth nor killed Mtb (Darwin et al., “The (2007)). Proteasomes are ubiquitous in eukaryotic cells, Proteasome of Mycobacterium tuberculosis is Required for widespread in archaea and rare in eubacteria. In eukaryotes Resistance to Nitric Oxide.” Science 302:1963-1966 (2003)). the proteasome core is a stack of 7 types of C. Subunits forming In vivo silencing of preBA, the proteasome component genes two heteroheptameric outer rings and 7 types of B subunits encoding the B and C. subunits, led to a 500-fold decline in forming two heteroheptameric inner rings (Kisselev et al., viable Mtb in the lungs of mice (Gandotra et al., “In vivo Gene US 2011/011 8274 A1 May 19, 2011

Silencing Identifies the Mycobacterium tuberculosis Protea 0020 R to Rare independently H, substituted or unsub some as Essential for the Bacteria to Persist in Mice.” Nat. stituted C-Coalkyl, Substituted or unsubstituted C-Coalk Med. 13:1515-1520 (2007)). enyl, Substituted or unsubstituted C-C alkynyl, cycloalkyl, 0007. These observations raised the possibility that pro haloalkyl, heterocyclyl, or aryl, teasome inhibitors might be useful in the treatment of tuber culosis. However, the extensive conservation of proteasome wherein if Q is structures militates against species selectivity of proteasome inhibitors. Agents which inhibit the human proteasome might be counterproductive in the treatment of tuberculosis insofar as they might interfere with antigen processing (Yang et al., “The Requirement for Proteasome Activity Class I Major Histocompatibility Complex Antigen Presentation is Dic tated by the Length of Preprocessed Antigen.” J. Exp. Med. 183:1545-1552 (1996); and Hughes et al., “The Protease and E and D or E and G are C, wherein R and R, or R and Inhibitor, N-acetyl-Lleucyl-L-leucyl-leucyl-L-norleucinal, R, respectively, may form a fused substituted or unsubsti Decreases the Pool of Major Histocompatibility Complex tuted aromatic or cyclic hydrocarbon ring and if Q is Class I-Binding Peptides and Inhibits Peptide Trimming in the Endoplasmic Reticulum,” J. Exp. Med. 183:1569-1578 (1996)), a key part of the host immune response, and given D2 that they exert dose-dependent, mechanism-based host tox s icity (Jackson et al., “Bortezomib, a Novel Proteasome A N. 2 Inhibitor, in the Treatment of Hematologic Malignancies.” G Cancer Treat. Rev. 31:591-602 (2005)). 0008. The present invention is directed to overcoming and D and E are C, R, and R may form a fused substituted or these and other deficiencies in the art. unsubstituted aromatic ring or cyclic hydrocarbon ring, under SUMMARY OF THE INVENTION conditions effective to treat the subject for a pathogen infec 0009. One aspect of the present invention relates to a tion, wherein the Substituted alkyl, alkenyl, alkynyl, aromatic, method of treating a subject for a pathogen infection. The or cyclic hydrocarbon ring groups have substituents selected method involves administering to the Subject a compound of from the group consisting of hydroxyl, halogen, —CN. Formula (I). —NO, —SR, —OR, —SOR, —NH2, —NHRs, - NHSOR - NRRs –C(O)NHRs –C(O)NH2, (I) —CONRR, —CHO,-CO)Rs, and —C(O)CR. 0021. Another aspect of the present invention relates to a method of treating cancer. The method involves administer -(- ing to the Subject a compound of Formula (I). where: 0010 Q is -(y- (I) / l NG I N X – where: (0022. Q is

D2 E where the crossing dashed line illustrates the bond formed IXNG - ,NA ) — or joining Q to the rest of the compound of Formula (I); 0011 A is S or O: 1's 0012 D is N or C, wherein C is bound to R: 0013 E is N or C, wherein C is bound to R: 0014) G is N or C, wherein C is bound to R: 0015. X is N or C, wherein C is bound to Rs: 0016 Y is N or C, wherein C is bound to R; 0017 J is N or C, wherein C is bound to R: where the crossing dashed line illustrates the bond formed 0018 T is N or C, wherein C is bound to R: joining Q to the rest of the compound of Formula (I); 0019 Z is N or C, wherein C is bound to R: 0023) A is S or O; wherein R to Rs are independently H, a halogen, —SR —NO. —NR7Rs —SO-Ro. —CONRoR —OR2, Sub (0024 D is N or C, wherein C is bound to R: stituted or unsubstituted C-Co alkyl, Substituted or unsub (0025 E is N or C, wherein C is bound to R; stituted C-Coalkenyl, Substituted or unsubstituted C-Co (0026 G is N or C, wherein C is bound to R: alkynyl, cycloalkyl, haloalkyl, heterocyclyl, or aryl; (0027 X is N or C, wherein C is bound to Rs: US 2011/011 8274 A1 May 19, 2011

0028 Y is N or C, wherein C is bound to R; wherein R to R are independently H, a halogen, —SR, 0029 J is N or C, wherein C is bound to R: —NO. —NR7Rs —SO-Ro. —CONRoR —OR2, Sub 0030 T is N or C, wherein C is bound to R: stituted or unsubstituted C-C alkyl, Substituted or unsub 0031 Z is N or C, wherein C is bound to R: stituted C-C alkenyl, Substituted or unsubstituted alkynyl, wherein R to Rs are independently H, a halogen, —SR cycloalkyl, haloalkyl, heterocyclyl, or aryl; NO. —NR.R. —SOR. - CONRR, —OR, substi 0039 R to Rare independently H, substituted or unsub tuted or unsubstituted C-C alkyl, Substituted or unsubsti stituted C-C alkyl, Substituted or unsubstituted C-C alk tuted C-Coalkenyl, Substituted or unsubstituted alkynyl, enyl, Substituted or unsubstituted C-C alkynyl, cycloalkyl, cycloalkyl, haloalkyl, heterocyclyl, or aryl; haloalkyl, heterocyclyl, or aryl, 0032 R to Rare independently H, substituted or unsub wherein if Q is stituted C-C alkyl, Substituted or unsubstituted C-C alk enyl, Substituted or unsubstituted C-C alkynyl, cycloalkyl, haloalkyl, heterocyclyl, or aryl, wherein if Q is DNA E G Y and E and D or E and G are C, R, and R, or R and R. D-N. respectively, may form a fused substituted or unsubstituted aromatic or cyclic hydrocarbon ring and if Q is and E and D or E and G are C, R, and R, or R and R, may form a fused substituted or unsubstituted aromatic or cyclic hydrocarbon ring and if Q is D2 s AN

and E and Dare C, R, and R may form a fused substituted or unsubstituted aromatic or cyclic hydrocarbon ring, wherein the Substituted alkyl, alkenyl, alkynyl, aromatic, or cyclic and D and E are C, R, and R may form a fused substituted or hydrocarbon ring groups have Substituents selected from the unsubstituted aromatic ring or cyclic hydrocarbon ring, under group consisting of hydroxyl, halogen, —CN, —NO, conditions effective to treat the subject for cancer, wherein the —SR —OR2, —SOR. -NH2, —NHR, Substituted alkyl, alkenyl, alkynyl, aromatic, or cyclic hydro NHSOR - NRRs –C(O)NHRs –C(O)NH, carbon ring groups have substituents selected from the group —CONRR7, CHO, —C(O)Rs, and —C(O)OR: consisting of hydroxyl, halogen, —CN, —NO. —SR, with the proviso that Q is not an isoxazolyl group, and with —OR —SOR, NH, -NHR, NHSOR, the further proviso that at least one of D, E, or G is N if R and —NR,R, —C(O)NHRs, —C(O)NH2. —CONRR, R, or R and R. do not form a fused substituted or unsub —CHO. —C(O)Rs, and —C(O)OR. stituted aromatic hydrocarbon ring. 0033. Another aspect of the present invention relates to 0040. The present invention discloses the identification novel compounds of Formula (I): and mechanistic characterization of a novel class of com pounds that inhibit Mtb proteasome potently and irreversibly carbonylating the active site Thr1 of the Mtb proteasome. (I) BRIEF DESCRIPTION OF THE DRAWINGS -(- 0041 FIGS. 1A-C illustrate structures and modality of inhibition of Mtb PrcAB-OG by oxathiazol-2-ones. FIG. 1A where: shows various structures of GL1, GL3, GL5, and GL6. FIG. 0034. Q is 1B shows GL5, inhibiting the degradation of B-casein by Mtb PrcAB-OG. Mtb PrcAB-OG (12 nM) was incubated with GL5 or GL6 (each 10 uM) at 37° C. for 1 hour prior to addition of B-casein (200 ug/mL). Aliquots were removed Da V immediately or 30, 60, 120 later and degradation of B-casein or || - assessed by SDS-PAGE. PrcAA denotes the C. chain from AN DNA which the N-terminal octapeptide has been deleted. FIG. 1C illustrates the effect of substrate concentration on inactivation where the crossing dashed line illustrates the bond formed of Mtb PrcAB-OG by GL3 and GL5. Curves are fitted to the joining Q to the rest of the compound of Formula (I); equation k-kitextobs missing or illegible when filed/ (1+(K/III)x(1+S/K)) for substrate protection of the 0035 A is S or O: enzyme from inactivation (Copeland, R.A., “Enzymes: A 0036) D is N or C, wherein C is bound to R: Practical Introduction to Structure, Mechanism, and Data 0037 E is N or C, wherein C is bound to R; Analysis. pp. 305-349 (2000), which is hereby incorporated 0038 G is N or C, wherein C is bound to R: by reference in its entirety). US 2011/011 8274 A1 May 19, 2011

0042 FIGS. 2A-D illustrate kinetic analysis of inactiva treated with (open bars) and without (solid bars) DETA-NO tion of Mtb PrcAB-OG and human proteasomes by oxathia (50 uM) and with the indicated oxathiazol-2-one at 10, 25, or 50 uM for four days. Bacteria were then serially diluted in Zol-2-ones. FIG. 2A is the graph of progress curves for inhi saline and plated for enumeration of surviving CFU. DETA bition of Mtb PrcAB-OG with GL5 at indicated NO at this concentration prevented replication but caused no concentrations. FIG. 2B is the plot of pseudo first-order rate killing. FIG. 5B shows the killing of Mtb by GL5 in synergy constants k as function of inhibitor concentration. Values with NO. Mtb was exposed to 100 uMDETANO (open bars) for k, derived from the fit of data in FIG. 2A, to equation or 100 uM DETA (solid bars) overnight prior to addition of (1), were plotted against inhibitor concentration. The plot of GL5 at 25 uM or 50 uM or to GL6 (50 uM) as a control. k Vs GL5 yields a straight line crossing the origin, indi Surviving bacteria were plated 4 days after addition of cating that inactivation is irreversible or very slowly revers oxathiazol-2-one. Initial inocula are indicated. DETA-NO at this concentration prevented replication but caused little or no ible: GL1 and GL3 exhibit a lag at the lower concentrations of killing. The limit of detection was 4 logo CFU/ml inhibitors and approach a maximum at higher concentrations, 0046 FIG. 6 illustrates a mechanism for proteasome inhi indicative of positive cooperativity. FIG. 2C is the inhibition bition by GL5. Irreversible inhibition of the Mtb proteasome of human 20S-B5 by GL5. FIG. 2D is the plot of k of proceeds by the unmarked route. GL5/1/3 against h20S as function of inhibition concentration. Values for k, derived from the fit of data in FIG. 2C, to DETAILED DESCRIPTION OF THE INVENTION equation (1), were plotted against inhibitor concentration. (O) 0047 One aspect of the present invention relates to a method of treating a Subject for a pathogen infection. The GL5; (O) GL1; (x) GL3. method involves administering to the Subject a compound of 0043 FIGS. 3A-D illustrate identification of the modified Formula (I). N-terminus of the Mtb proteasome treated with oxathiazol 2-ones. Oxathiazol-2-ones inactivated the Mtb proteasome (I) by carbonylating the Y-OH and C-NH of the active site Thr1. LC-MS/MS was used to identify the modified N-terminus of the Mtb proteasome treated with oxathiazol-2-ones. Mass g-- spectra of tryptic N-terminal heptapeptides from samples that were untreated (FIG. 3A), GL5-treated (FIG. 3B), treated with glutaraldehyde/Na(CN) BH, after trypsin digestion where: (FIG. 3C), or treated with glutaraldehyde/Na(CN)BH, after 0048 Q is GL5 treatment and trypsin digestion (FIG. 3D). Allions were confirmed by MS/MS fragmentation sequencing. The reac D2 E-1 tion equations illustrate the proposed modification of active IX - ) — or site Thr1 by oxathiazol-2-one and the modification of the NG NA primary amino groups at both Thr1 and Lys7 with glutaral 1's dehyde and Na(CN) BH. Results illustrated for GL5 were identical using GL3. 0044 FIGS. 4A-C illustrate in vivo inactivation of the proteasome in M. bovis BCG by oxathizol-2-ones. FIG. 4A shows the comparison of Oxathiazol-2-ones with a peptidyl boronate. BCG (ODItext missing or illegible when where the crossing dashed line illustrates the bond formed filed 0.6-1) was exposed to vehicle DMSO, 50 uM of GL1, joining Q to the rest of the compound of Formula (I); GL3 or GL5, or 20 uMMLN-273. After 4 hours, the bacteria 0049. A is S or O; were washed twice, lysed mechanically and analyzed for 0050 D is N or C, wherein C is bound to R: proteasome activity with Ac-YQW-AMC as substrate. FIG. 0051 E is N or C, wherein C is bound to R; 4B is a graph of time-course for effect of GL5 (50 uM). 0052 G is N or C, wherein C is bound to R: Experiments were performed as in FIG. 4A except that 0053 X is N or C, wherein C is bound to Rs: removal of extracellular compound began at the indicated 0054 Y is N or C, wherein C is bound to R; times. Exposure to GL5 may have continued for up to 15 0055 J is N or C, wherein C is bound to R: additional minutes during the washing process. “Untreated 0056 T is N or C, wherein C is bound to R: cells were handled in the same manner but without inhibitor 0057 Z is N or C, wherein C is bound to R: or vehicle and were lysed at 60 min. “DMSO cells received wherein R to Rs are independently H, a halogen, —SR vehicle alone (DMSO, <1% vol/vol) at time 0 and were lysed NO. —NR.R. —SOR. - CONRR, OR, substi at 60 min. “TO” cells were treated with inhibitor and then tuted or unsubstituted C-Co alkyl, Substituted or unsubsti washed immediately. FIG. 4C is the concentration-response tuted C-Co alkenyl, Substituted or unsubstituted C-Co for GL5 after 1 hour of exposure. Experiments were also alkynyl, cycloalkyl, haloalkyl, heterocyclyl, or aryl; performed as in FIG. 4A except that the concentration of GL5 0.058 R to Rare independently H, substituted or unsub was varied and lysate was also prepared from an untreated, stituted C-C alkyl, Substituted or unsubstituted C-C alk mutant strain of BCG in which the preBA genes were selec enyl, Substituted or unsubstituted C-C alkynyl, cycloalkyl, tively disrupted. In the control, cells received vehicle alone. haloalkyl, heterocyclyl, or aryl, Data are meansitSD of triplicates in single experiments, rep wherein if Q is resentative of 2 independent experiments. 004.5 FIGS.5A-B illustrate the killing of mycobacteria by oxathiazol-2-ones in synergy with NO. FIG. 5A is a graph of the killing of BCG by GL5, GL1, and GL3 was concentration dependent and augmented following exposure to a Sub-lethal concentration of NO donated by DETA-NO. BCG at an initial number of 2.5x10 CFU/mL (arrow) in Sauton's medium was US 2011/011 8274 A1 May 19, 2011

and E and D or E and G are C, R, and R, or R and R. and D and E are C, R, and R may form a fused substituted or respectively, may form a fused substituted or unsubstituted unsubstituted aromatic ring or cyclic hydrocarbon ring, under aromatic or cyclic hydrocarbon ring and if Q is conditions effective to treat the subject for a pathogen infec tion, wherein the Substituted alkyl, alkenyl, alkynyl, aromatic, or cyclic hydrocarbon ring groups have substituents selected from the group consisting of hydroxyl, halogen, —CN. —NO. —SR —OR —SOR. -NH2, —NHR, - NHSOR - NRRs –C(O)NHRs –C(O)NH2, —CONRR7, CHO, —C(O)Rs, and —C(O)OR. 0059 Specific compounds in accordance with the present invention are set forth in Table 1.

TABLE 1. Kinetic parameters of Oxathiazol-2-ones and Their Mycobacterial Killing Effect C-CT: C -chymotrypsin

kob III.M.'s BCG Mtb H2OS killing killing IC50 M Mtb-OG PA28 Ratio (25 M) (50 iM) (C-CT) 376.4 0.4 1033 >2 log 1.4 log 0.064

HT1016 ON 731.9 12.3 59.3 >3 log 2.2 log 5.87

HT1041 597.5 12.9 46.4 0.8 log 1.7 log 2.08

HT1042 729.0 10.3 71.1 2 log 1.7 log 3.14

HT1043 460.4 O.2 2041 1 log 1.8 log 16.6

HT1044 1074.6 1484 7.2 1.7 log 2 log 8.O2

HT1054 636.O 14.8 43.1 >3 log 2 log 2.19 US 2011/011 8274 A1 May 19, 2011

TABLE 1-continued

Kinetic parameters of Oxathiazol-2-ones and Their Mycobacterial Killing Effect C-CT: C. -chymotrypsin

S kob II). M's BCG Mtb N/ H2OS killing killing IC50 M ID R Mtb-OG PA28 Ratio (25 M) (50 iM) (C-CT) HT1071 CX 1059.3 31.3 33.8 >3 log 2.2 log 2.67 HT1086 154.7 0.4 356.6 ND ND 48.1 FC

HT1113 W \ 434.9 10.1 43.1 1 log 2 log 22.7

O

HT1117 432.7 53.4 8.1 ND ND 3.67

HT1118 132.6 11.3 11.8 0.8 log 1.7 log 15.8

HT1146 408.9 31.3 13.1 ND ND 1O.O

HT1147 45.8 O.O NA ND ND 83.9 1.O cx 1. O

HT1171 ON 3489 10.1 345 ND ND 18.9 N S US 2011/011 8274 A1 May 19, 2011

0060. The infectious pathogens which can be treated in E and G are C, and R and R form a fused substituted or accordance with this aspect of the present invention include unsubstituted aromatic ring or cyclic hydrocarbon ring Mycobacterium tuberculosis, Mycobacterium leprae, and whereby the compound has the following structure of For other disease-causing Mycobacterium. The diseased subject mula (III): can be a mammal especially a human. 0061. In one embodiment of the present invention, Q is (III) O X- S. O R20 2N R21 e A D and E are C, and R and R form a fused substituted or S. unsubstituted aromatic ring or cyclic hydrocarbon ring, R22 whereby the compound has the following structure of For R23 R2 mula (II): 0066. In another embodiment of the present invention, Q is (II) R20 R3 R21 N O ty ( )—o D NA R22 A. N-S and the compound of Formula (I) has the structure of Formula R23 (IV)

(IV) where 0062 seese- is independently a single or double bond and 0063 Ro to R are independently H, a halogen, NO, —NR7Rs – CONRR, substituted or unsubstituted C-C alkyl, Substituted or unsubstituted C-Co alkenyl, Substituted or unsubstituted C-C alkynyl, cycloalkyl, het NN's erocyclyl, or aryl. 0064. Examples of compounds of Formula (II) are 0067 Examples of compounds of Formula (IV) are

0065. In another embodiment of the present invention, Q is US 2011/011 8274 A1 May 19, 2011

-continued -continued / O O

NN SX=0. O-N S 0068. In one embodiment of the present invention, Q is R3 O, O, Da R p-( p-( A N. R2 2 f N- R2 y N S-N O-N and the compound of Formula (I) has the structure of Formula O, O, (V) p-( p-( (V) R2 2N-S-S N R s \, f S-N A. le O R3 X-O. O, O, R3 S R p-( R -(

0069. Examples, of the compounds of Formula (V) are N 2-kN -S a N-SN \S-N f \S | R3 s O O S e O p-( p-( 's'/ no NN'sX=o and % N-, and % N \O-N f \S | e se R3 'Y \- 0071. In another embodiment of the present invention, Q is

0070. In one embodiment of the present invention, Q is

and the compound of Formula (I) is selected from the group consisting of and the compound of Formula (I) is selected from the group consisting of US 2011/011 8274 A1 May 19, 2011

-continued -continued O, O, R O R p-( *NS N- , and - N R N NN1 S R sN S \N-S \N | N 2 Rs R3 2 N R4 O, O, 0072 In yet another embodiment of the present invention, R p- R p Q is N N- S R N N / S l 2 N N R N Rs N. R4 and the compound of Formula (I) has the structure of Formula (VI) N N / S

(VI) N R R3 2

R3 21 R R4 O N R X O R p-( NN's X=0. R2 N N -S , and N 0073. Examples of the compounds of Formula (VI) are NN Rs 5-phenyl-1,3,4-oxathiazol-2-one, 5-(4-nitrophenyl)-1,3,4- O. Oxathiazol-2-one, 5-(3-nitrophenyl)-1,3,4-oxathiazol-2-one, 5-(2-pyridinyl)-1,3,4-oxathiazol-2-one, 5-(3-methoxyphe p-( nyl)-1,3,4-oxathiazol-2-one, 5-(3-fluorophenyl)-1,3,4-ox R2 N N -S athiazol-2-one, 5-(3-(trifluoromethyl)-phenyl)-1,3,4-ox athiazol-2-one, 5-(4-tolyl)-1,3,4-oxathiazol-2-one, 5-(3- N tolyl)-1,3,4-oxathiazol-2-one, and 5-(3,5-dimethoxyphenyl)- R 2 1,3,4-oxathiazol-2-one. R4 0074. In another embodiment of the present invention, Q is 0075 Another aspect of the present invention relates to a method of treating cancer. The method involves administer ing to the Subject a compound of Formula (I).

(I) and the compound of Formula (I) is selected from the group consisting of o-y-

where: 0.076 Q is

NGe/ X - US 2011/011 8274 A1 May 19, 2011 10

dermally, intramuscularly, intraperitoneally, intravenously, -continued Subcutaneously, or intranasally. The compositions of the present invention may be administered alone or with suitable pharmaceutical carriers, and can be in Solid or liquid form, Such as tablets, capsules, powders, solutions, Suspensions, or emulsions. 0090 The agents of the present invention may be orally administered, for example, with an inert diluent, or with an where the crossing dashed line illustrates the bond formed assimilable edible carrier, or it may be enclosed inhard or soft joining Q to the rest of the compound of Formula (I); shell capsules, or it may be compressed into tablets, or they 0077. A is S or O: may be incorporated directly with the food of the diet. Agents 0078 D is N or C, wherein C is bound to R: of the present invention may also be administered in a time 0079 E is N or C, wherein C is bound to R: release manner incorporated within Such devices as time 0080 G is N or C, wherein C is bound to R: release capsules or nanotubes. Such devices afford flexibility 0081 X is N or C, wherein C is bound to Rs: relative to time and dosage. For oral therapeutic administra I0082 Y is N or C, wherein C is bound to R; tion, the agents of the present invention may be incorporated I0083) J is N or C, wherein C is bound to R: with excipients and used in the form of tablets, capsules, I0084. T is N or C, wherein C is bound to R: elixirs, Suspensions, syrups, and the like. Such compositions I0085 Z is N or C, wherein C is bound to R: and preparations should contain at least 0.1% of the agent, wherein R to Rs are independently H, a halogen, —SRs— although lower concentrations may be effective and indeed NO. —NR7Rs - SOR. -CONRoR -OR2, Substi optimal. The percentage of the agent in these compositions tuted or unsubstituted C-C alkyl, substituted or unsubsti may, of course, be varied and may conveniently be between tuted C-Co alkenyl, Substituted or unsubstituted C-Co about 2% to about 60% of the weight of the unit. The amount alkynyl, cycloalkyl, haloalkyl, heterocyclyl, or aryl; of an agent of the present invention in Such therapeutically I0086 R to Rare independently H, substituted or unsub useful compositions is such that a Suitable dosage will be stituted C-Coalkyl, Substituted or unsubstituted C-Coalk obtained. enyl, Substituted or unsubstituted C-C alkynyl, cycloalkyl, 0091 Also specifically contemplated are oral dosage haloalkyl, heterocyclyl, or aryl, forms of the agents of the present invention. The agents may be chemically modified so that oral delivery of the derivative wherein if Q is is efficacious. Generally, the chemical modification contem plated is the attachment of at least one moiety to the compo nent molecule itself, where said moiety permits (a) inhibition N of proteolysis; and (b) uptake into the blood stream from the stomach or intestine. Also desired is the increase in overall DNA stability of the component or components and increase in circulation time in the body. Examples of such moieties and E and D or E and G are C, R, and R, or R and R. include: polyethylene glycol, copolymers of ethylene glycol respectively, may form a fused substituted or unsubstituted and propylene glycol, carboxymethyl cellulose, dextran, aromatic or cyclic hydrocarbon ring and if Q is polyvinyl alcohol, polyvinyl pyrrolidone and polyproline. (Abuchowski and Davis, “Soluble Polymer-Enzyme Adducts. In: Enzymes as Drugs, Hocenberg and Roberts, eds., Wiley-Interscience, New York, N.Y., pp. 367-383 (1981), which are hereby incorporated by reference in their entirety). Other polymers that could be used are poly-1,3- dioxolane and poly-1,3,6-tioxocane. Preferred for pharma ceutical usage, as indicated above, are polyethylene glycol and D and E are C, R, and R form a fused substituted or moieties. unsubstituted aromatic ring or cyclic hydrocarbon ring, under 0092. The tablets, capsules, and the like may also contain conditions effective to treat the subject for a pathogen infec a binder Such as gum tragacanth, acacia, corn starch, or gela tion, wherein the Substituted alkyl, alkenyl, alkynyl, aromatic, tin; excipients such as dicalcium phosphate; a disintegrating or cyclic hydrocarbon ring groups have substituents selected agent Such as corn starch, potato starch, alginic acid; a lubri from the group consisting of hydroxyl, halogen, —CN. cant such as magnesium Stearate; and a Sweetening agent NO. —SR —SOR, NH, -NHR, NHSOR, Such as Sucrose, lactose, Sucrulose, or saccharin. When the —NR7Rs, —C(O)NHRs, —C(O)NH2. —CONRR, dosage unit form is a capsule, it may contain, in addition to —CHO, —C(O)Rs, and —C(O)OR. materials of the above type, a liquid carrier Such as a fatty oil. 0087. In treating cancer, the same compounds are used as 0093 Various other materials may be present as coatings described above for treating pathogen infection. or to modify the physical form of the dosage unit. For 0088. Forms of cancer which can be treated in accordance instance, tablets may be coated with shellac, Sugar, or both. A with the present invention include carcinoma, sarcoma, lym syrup may contain, in addition to active ingredient. Sucrose as phoma, and myeloma. a Sweetening agent, methyl and propylparabens as preserva 0089. In practicing the method of the present invention, tives, a dye, and flavoring Such as cherry or orange flavor. agents suitable for treating a Subject can be administered 0094. The agents of the present invention may also be using any method standard in the art. The agents, in their administered parenterally. Solutions or Suspensions of the appropriate delivery form, can be administered orally, intra agent can be prepared in water Suitably mixed with a Surfac US 2011/011 8274 A1 May 19, 2011

tant Such as hydroxypropylcellulose. Dispersions can also be 0101 Another aspect of the present invention relates to the prepared in glycerol, liquid polyethylene glycols, and mix compound of Formula (I): tures thereof in oils. Illustrative oils are those of petroleum, animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, or mineral oil. In general, water, Saline, (I) aqueous dextrose and related Sugar Solution, and glycols, Such as propylene glycolor polyethylene glycol, are preferred liquid carriers, particularly for injectable solutions. Under ordinary conditions of storage and use, these preparations -(- contain a preservative to prevent the growth of microorgan where: 1SS. 0095. The pharmaceutical forms suitable for injectable 0102 Q is use include sterile aqueous solutions or dispersions and ster ile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must D2 V besterile and must be fluid to the extent that easy syringability M , or | s exists. It must be stable under the conditions of manufacture AN D-N. and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The where the crossing dashed line illustrates the bond formed carrier can be a solvent or dispersion medium containing, for joining Q to the rest of the compound of Formula (I); example, water, ethanol, polyol (e.g., glycerol, propylene (0103) A is S or O; glycol, and liquid polyethylene glycol), Suitable mixtures 0104 D is N or C, wherein C is bound to R: thereof, and vegetable oils. 0105 E is N or C, wherein C is bound to R; 0096. When it is desirable to deliver the agents of the 0106 G is N or C, wherein C is bound to R: present invention systemically, they may be formulated for I0107 wherein R to Rs are independently H, a halogen, parenteral administration by injection, e.g., by bolus injection —SR, NO. —NR,Rs – SOR, CONRR, or continuous infusion. Formulations for injection may be —OR. Substituted or unsubstituted C-C alkyl, substi presented in unit dosage form, e.g., in ampoules or in multi tuted or unsubstituted C-Coalkenyl, Substituted or unsub dose containers, with an added preservative. The composi stituted C-C alkynyl, cycloalkyl, haloalkyl, heterocyclyl, tions may take Such forms as Suspensions, solutions or emul or aryl; sions in oily or aqueous vehicles, and may contain 0.108 R to Rare independently H, substituted or unsub formulatory agents such as Suspending, stabilizing and/or stituted C-C alkyl, Substituted or unsubstituted C-C alk dispersing agents. enyl, Substituted or unsubstituted C-C alkynyl, cycloalkyl, 0097 Intraperitoneal or intrathecal administration of the haloalkyl, heterocyclyl, or aryl, agents of the present invention can also be achieved using wherein if Q is infusion pump devices such as those described by Medtronic, Northridge, Calif. Such devices allow continuous infusion of desired compounds avoiding multiple injections and multiple manipulations. 0098. In addition to the formulations described previously, the agents may also be formulated as a depot preparation. Such long acting formulations may be formulated with Suit able polymeric or hydrophobic materials (for example as an and E and D or E and G are C, R, and R, or R and R. emulsion in an acceptable oil) or ion exchange resins, or as respectively, may form a fused substituted or unsubstituted sparingly soluble derivatives, for example, as a sparingly aromatic or cyclic hydrocarbon ring and if Q is soluble salt 0099. The agents of the present invention may also be administered directly to the airways in the form of an aerosol. 2 For use as aerosols, the agent of the present invention in D Solution or Suspension may be packaged in a pressurized aerosol container together with Suitable propellants, for example, hydrocarbon propellants like propane, butane, or isobutane with conventional adjuvants. The agent of the and D and E are C, R, and R may form a fused substituted or present invention also may be administered in a non-pressur unsubstituted aromatic or cyclic hydrocarbon ring, wherein ized form Such as in a nebulizer or atomizer. the Substituted alkyl, alkenyl, alkynyl, aromatic, or cyclic 0100 Effective doses of the compositions of the present hydrocarbon ring groups have Substituents selected from the invention, for the treatment of cancer or pathogen infection group consisting of hydroxyl, halogen, —CN, —NO, vary depending upon many different factors, including type —SR —OR. -SOR. - NH, -NHR, and stage of cancer or the type of pathogen infection, means - NHSOR - NRRs –C(O)NHRs –C(O)NH2, of administration, target site, physiological state of the —CONRR, —CHO,-CO)Rs, and —C(O)CRs: patient, other medications or therapies administered, and with the proviso that Q is not an isoxazolyl group, and with physical state of the patient relative to other medical compli the further proviso that at least one of D, E, or G is N if R and cations. Treatment dosages need to be titrated to optimize R, or R and R. do not form a fused substituted or unsub safety and efficacy. stituted aromatic hydrocarbon ring. US 2011/011 8274 A1 May 19, 2011

0109 To the extent within the scope of the compound preBA Genes Encode a Gated Proteasome With Broad Oli recited in the preceding paragraph, that compound can take gopeptide Specificity.” Mol. Microbiol. 59:1405-1416 the form of the Sub-classes of compounds and specific com (2006), which is hereby incorporated by reference in its pounds described above entirety). Human red blood cell 20S and PA28 were pur 0110. In the embodiment of the present invention, where Q chased from Boston Biochem (Cambridge, Mass.). GL1, 1S GL2, GL3, GL4 were purchased from TimTec LLC (DE, USA) and GL5, GL6, GL7 from ChemDiv, Inc. (CA, USA). Bortezomib was purchased from LC Laboratories (MA, USA). Substrates Ac-RFW-AMC, Ac-YQW-AMC were syn thesized by AnaSpec (CA, USA) and used for detailed kinetic analyses. Different Substrates were chosen according to the nature of the experiments. suitable compounds of Formula (I) have the following struc tures: Example 2 High Throughput Screen 0113. The screening was conducted with compounds from ChemDiv, Chembridge, Spectrum, Preswick, and Cerep. Example 3 O S Kinetics R3 R3 0114 Kinetic measurements were made on a Hitachi O, O, F-2500 fluorescence spectrophotometer with 0.238 nM PreAB-OG in 20 mM HEPES, 0.5 mM EDTA, pH 7.5, 0.1 p-( p-( mg/mL BSA and 25 uM Ac-RFWAMC for Mtb PrcAB-OG R S R N S or 25 M Suc-LLVY-AMC for human 20S at 37° C. After 2 2 N- ne NY steady state conditions were achieved, inhibitors were added S-N O-N and Substrate cleavage was monitored ( 360 nm, we 460 O, O, nm) at 5-second intervals for 60 minutes or until no activity remained. The data were fitted to equation (Corbett et al., “The Growing Burden of Tuberculosis: Global Trends and N S R N S Interactions with the HIV Epidemic. Arch. Intern. Med. 163: N22 f NN1 2 2 f N N1 1009-1021 (2003), which is hereby incorporated by reference in its entirety) to determine kitext missing or illegible O S-N when filed using Prism (GraphPad Software, Inc. San R3 Diego, Calif.). O, O, Example 4 R p-( R p-( LC-MS/MS Analysis % N-S 4 NY S 0115 Mtb PrcAB-OG (415ug/mL; 8.2 uMactive sites) in \ f \ | 20 mM HEPES, 0.5 mM EDTA, pH 7.5, was incubated with S-N S 500 LMGL5 or GL3 at room temperature until the activity assay demonstrated that inactivation was complete. A control R3 sample was incubated for the same time with an equivalent O O volume of DMSO. The samples were then run on SDS-page R p-( p-( to separate C. and B subunits. The gel bands corresponding to N untreated and inhibitor-treated Prch were excised from the N1 N- , and N2 N gel, reduced with 10 mM DTT, alkylated with 55 mMiodoac \ f \ / etamide and digested with sequence grade modified trypsin O-N S (Promega) in ammonium bicarbonate buffer at 37 overnight. R3 The digestion products were analyzed by LC-MS/MS and LC-MS with Thermo LTQ Orbitrap and Applied Biosystems QSTAR mass spectrometers, respectively. One tenth of the digestion products for each sample were also analyzed by EXAMPLES MALDI-TOF with a PerSeptive MALDI-TOF DE-STR mass 0111. The Examples set forth below are for illustrative spectrometer. For LC-MS/MS analysis, each digestion prod purposes only and are not intended to limit, in any way, the uct was separated by gradient elution with a Dionex capillary/ Scope of the present invention. nano-HPLC system that is directly interfaced with the mass spectrometer. MS/MS data were searched using the MAS Example 1 COT search engine for identifying proteins and modifica tions. For in-gel modification of primary amine groups, gel Materials slices of untreated- and treated-proteins were incubated with 0112 The “open gate' mutant of recombinant Mtb pro 500 mM sodium cyanoborohydride and 2.5% glutaraldehyde teasome (Pre AB-OG) was over-expressed in E. coli and puri at 37° C. for the desired time. Reactions were stopped by fied as reported (Lin et al., “Mycobacterium tuberculosis addition of 1M Tris-HC1. US 2011/011 8274 A1 May 19, 2011

Example 5 “Comparison of Biochemical and Biological Effects of ML858 (Salinosporamide A) and Bortezomib. Mol. Cancer Inhibition of Proteasome Activity in Mycobacteria Ther. 5:3052-3061 (2006), which is hereby incorporated by 0116 20-ml cultures of BCG in Sauton's medium at opti reference in its entirety) or NPI-0052 (an irreversible inhibi cal density 0.8-1.0 at 580 nm were treated with GL1, GL3, or tor) (Groll et al., “Crystal Structures of Salinosporamide A GL5 at 50 uM or with MLN-273 (Millennium Pharmaceuti (NPI-0052) and B (NPI-0047) in Complex With the 20S cals Inc.) at 20 uM. After 4 hours, BCG cells were harvested Proteasome Reveal Important Consequences of Beta-Lac by centrifugation at 3,000 g for 10 minutes. Pellets were tone Ring Opening and a Mechanism for Irreversible Bind washed with PBS with 0.02% Tween 80 and again with PBS ing.” J. Am. Chem. Soc. 128:5136-5141 (2006), which is lacking Tween. Pellets were resuspended in assay buffer (20 hereby incorporated by reference in its entirety), for 18 hours mM HEPES, 0.5 mM EDTA, pH 7.4, 100 uM phenylmeth ylsulfonyl fluoride (PMSF)), and lysed by mechanical beat against inhibitor-free buffer, did not restore the activity of ing with Zirconium beads. Lysates were spun at 16,000 g for proteasomes treated with GL3, GL5 or NPI-0052, while 70% 10 minutes and protein concentrations of the Supernatants of activity was recovered in the bortezomib-treated sample. determined by the Bradford assay. Concentration studies These results indicate that oxathiazol-2-ones are either irre used GL5 for 1 hour. Activity assays were performed with versible or practically irreversible inhibitors of the Mtb pro ~10 ug of lysate protein using Ac-YQW-AMC (50 uM) as teasome. However, within the limit of solubility of the substrate in 20 mM HEPES, 0.5 mM EDTA, pH 7.5, 100 uM Oxathiazol-2-ones in aqueous buffer, inhibition of the human PMSF, 0.02% SDS, 0.1 mg/mL BSA at 37° C. proteasome was too limited to make reliable measurements in similar experiments. Thus, kinetic analyses were investigated Example 6 to compare the effects of Oxathiazol-2-ones on proteasomes of the two species. Mycobactericidal Activity I0120 First, irreversible inhibitors can be competitive, 0117 M. bovis BCG (ATCC 35734) and Mtb H37Rv noncompetitive, or uncompetitive with respect to Substrate (ATCC 25618) were cultivated in Sauton's medium pH 7.4 (Copeland, R.A., “Evaluation of Enzyme Inhibitors in Drug with 0.4% L-asparagine, 0.2% glycerol and 0.02% Tween 80. Discovery: A Guide for Medicinal Chemists and Pharma Mid-log phase cultures (Ascotext missing or illegible cologists. pp. 214-248 (2005), which is hereby incorporated when filed 0.8-1.0) were diluted to 0.05-0.1 (Asso) and by reference in its entirety). Accordingly, the effect of the quantified by CFU. Mycobacteria were incubated under indi concentration of substrate (Z-VLR-AMC) on the rate of inac cated conditions in 96 well plates in 200 ul, then serially tivation of the Mtb proteasome by a fixed concentration of diluted in PBS with 0.02% Tween 80, pH 7.2 and plated for inhibitor (5 uM for GL5 and 1 uM for GL3) was tested. CFU on Middlebrook 7H11 agar plates with 10% Middle Increasing Substrate concentration reduced the rate of protea brook OADC enrichment. some inactivation (FIG. 1C). Thus, the oxathiazol-2-ones appear to compete with substrate at the active site of the Mtb Example 7 proteasome. Identification of Oxathiazol-2-Ones as Proteasome Inhibitors Example 9 0118 Mtb PrcAB-OG was screened with Suc-LLVY-7- Kinetics of Proteasome Inhibition by Oxathiazol-2- amido-4-methylcoumarin (AMC) as Substrate, recording the Ones fluorescence of AMC released upon cleavage. From 20,000 I0121 Kinetic analysis provided further evidence of irre commercially available compounds, a potent inhibitor 5-(5- versible inhibition of the Mtb proteasome and showed that methyl-2-(methylthio)thiophen-3-yl)-1,3,4-oxathiazol-2- inhibition of the human proteasome, by contrast, was revers one was identified, here termed GL5 (FIG. 1A). From com ible. Assessment of reaction progress curves in the presence mercial sources, was assembled a total of seven 1.3,4- of various concentrations of GL5 revealed a time-dependent oxathiazol-2-ones for further analysis (FIG. 1A). In conversion from an initial state to a steady-state Velocity of preliminary studies with oligopeptide Substrates, it was deter Zero, characteristic of slow-binding inhibition kinetics due to mined that ICso's of each oxathiazol-2-one except GL6 irreversible enzyme inactivation (FIG. 2A). The progress against the Mtb proteasome were far lower than against the curves were fitted by a nonlinear least-squares method to human proteasome. GL6, the sulfone analog of GL5, inhib equation (1), where P is the product, Vo is the initial Velocity, ited neither. At 10 uM, GL5, but not GL6, prevented the Mtb vtext missing or illegible when filed is the steady-state proteasome from degrading an intact protein, B-casein (FIG. velocity (in the case of irreversible inhibition, V, equals to 1B). Thus, the inhibitory activity of oxathiazol-2-ones is not Zero), ktext missing or illegible when filed is the limited to the action of the proteasome against Small peptides, apparent first order rate constant for conversion from the and is strongly influenced by minor changes in Oxathiazol-2- initial Velocity phase to the steady-state Velocity phase, and t One Structure. is time (Copeland, R.A., “Enzymes: A Practical Introduction to Structure, Mechanism, and Data Analysis. pp. 305-349 Example 8 (2000), which is hereby incorporated by reference in its entirety). Mode of Inhibition P7–1th(o-text missing or illegible 0119 Inhibition of the Mtb proteasome by oxathiazol-2- when filed) kitext missing or ones was irreversible by several criteria. First, no activity was illegible when filed)x1-exp(-kitext recovered by diluting pre-incubated Mtb proteasome with missing or illegible when filed) Equation (1) GL1 633-fold into substrate-containing reaction mixture. I0122. As shown in FIG. 2B, a linear dependence of k, Second, dialysis of the Mtb proteasome treated with GL3, text missing or illegible when filedon GL5 concen GL5, bortezomib (a reversible inhibitor) (Williamson et al., tration I was observed that intersected at the origin, indica US 2011/011 8274 A1 May 19, 2011 tive of one-step irreversible or very slowly reversible This analysis yielded the inhibitory kinetic values shown for inhibition (Copeland, R.A., “Evaluation of Enzyme Inhibi GL1 and GL3 in Table 2. The Hill coefficients (h) of 2.3 and tors in Drug Discovery: A Guide for Medicinal Chemists and Pharmacologists. pp. 214-248 (2005), which is hereby incor 1.9 might imply that binding of GL1 or GL3 to half of the porated by reference in its entirety). The slope gave an appar available active sites of the Mtb proteasome accelerates the ent value of kitext missing or illegible when filed/ inactivation of the whole enzyme complex. III 476+9.3 M'S', also referred to askitext missing or 0.124. As noted, the cytotoxicity of proteasome inhibitors illegible when filed/Ktext missing or illegible is correlated with their inhibition of the proteasome's chy when filed for an irreversible inhibitor, which was then motryptic-like activity (Adams et al., “Proteasome Inhibitors: corrected by correcting Ktext missing or illegible when filed by equation Ktext missing or illegible when A Novel Class of Potent and Effective Antitumor Agents.” filed KPP/(1+S/K) to remove the effect of substrate Cancer Res. 59:2615-2622 (1999), which is hereby incorpo competition (Copeland, R.A., “Enzymes: A Practical Intro rated by reference in its entirety). Accordingly, the next focus duction to Structure, Mechanism, and Data Analysis. pp. was placed on the chymotryptic-like site of the B5 subunit of 305-349 (2000), which is hereby incorporated by reference in proteasomes isolated from human erythrocytes in the pres its entirety). Hence, the kitext missing or illegible when ence of the human proteasome activator PA28 and obtained filed/Ktext missing or illegible when filed for GL5 the kitextobs missing or illegible when filed/L1 values vs Mtb proteasome was 624+12.2 M's (Table 2). for GL5 (tested over the range 50-100 uM, FIG. 2C), GL1

TABLE 2 Kinetic parameters of Oxathiazol-2-ones Hill coefficient Mtb PrcAB-OG H2OS-B5 (Mtb k(s), K(2) (M) k/KG2) (M's) k/III (M's) Selectivity PrcAB-OG) GL1 0.018 + 0.001 14.4 + 1.0 1250 + 111 1945.5 6.4 2.3 GL3 0.020 + 0.002 6.9 + 0.7 2898 + 413 128 12.3 22.6 1.9 GLS NA NA 625 + 12.2 9.8 1.3 63.8 1 : the maximum rate of inactivation achieved at infinite concentration of inactivator. K2: the concentration of inactivator that yields"sing a rate of inactivation. . . . equal to /2k. Error shown is the propagated error from kic and K2). For Mtb proteasome, kis? I equals ki/K). All inactivation constants were converted from apparent values by using equation: The ratio of kina/KM20s OG)versus kois Il H2OSB5), (2) indicates text missing or illegible when filed

0123 GL1 and GL3 also exhibited time-dependent inhi (2.5-20 uM), and GL3 (2.5-10 uM). The plots of kitext bition of the Mtb proteasome, but their kinetics appeared to be missing or illegible when filed vs II for all three com more complex, in that the plots of kitext missing or pounds yielded straight lines, indicating a simple one-step illegible when filed vs I showed a lag at lower concen inactivation mechanism (Copeland, R.A., “Enzymes: A Prac trations of inhibitor, reaching saturation at higher concentra tical Introduction to Structure, Mechanism, and Data Analy tions (FIG. 2B). For a time-dependent irreversible inhibitor, sis. pp. 305-349 (2000), which is hereby incorporated by the dependence of kitext missing or illegible when reference in its entirety). Surprisingly, none of the plots filed inhibitor concentration can be described by a rectangu passed through the origin (FIG. 2D), indicating that inactiva lar hyperbola (equation (3)) (Kitz et al., “Esters of Methane tion of the human proteasome f35 subunit by these three sulfonic Acid as Irreversible Inhibitors of Acetylcholinest oxathiazol-2-ones is reversible. erase.” J. Biol. Chem. 237:3245-3249 (1962), which is hereby 0.125 Species selectivity of the inhibitors was character incorporated by reference in its entirety), from which can be ized kinetically by comparing the second-order rate constants extracted the values of kitext missing or illegible when of kitext missing or illegible when filed/Ktext miss filed, the maximal rate constant for inactivation of the ing or illegible when filed for the Mtb proteasome and enzyme by the inhibitor, and K, the concentration that yields kitext missing or illegible when filed/I for the B5 a rate of inactivation half of kitext missing or illegible Subunit of the human proteasome. The Oxathiazol-2-ones When filed. The lag at lower concentrations may be indica tested ranged from 6- to 64-fold more potent against the Mtb tive of cooperativity, in which the initial binding of inhibitor proteasome than the human B5 subunit (Table 2). accelerates Subsequent inactivation. The mechanistic and kinetic origins and significance of this behavior on the part of Example 10 GL1 and GL3 remain to be determined. Equation (2) was modified to take the “apparent cooperativity into account, Identification of the Covalent Adduct of Mtb Protea yielding equation (3), where h is the Hill coefficient. some f Subunit with Oxathiazol-2-Ones k-kitext missing or illegible when 0.126 The apparent irreversible and competitive manner filed/(1+(Ktext missing or illegible of inhibition suggested that the Oxathiazol-2-ones probably when filed/III) Equation (2) inactivate the Mtb proteasome by covalently bonding with the kitext missing or illegible when active site Thr1. To test this hypothesis, Mtb proteasomes that filed=ktext missing or illegible had been treated or not with oxathiazol-2-ones GL3 or GL5 when filed/(1+Ktext missing or were trypsinized and LC-MS/MS was used to identify pep illegible when filed/H7) Equation (3) tides from the B subunits. Peptides identified by LC-MS/MS US 2011/011 8274 A1 May 19, 2011 collectively covered over 98% of protein sequence. One pep Zol-2-ones each blocked >90% of proteasome activity, tide ion, which had m/z. 771.46, was unique to the GL5 approaching the level of inhibition achieved by disrupting the treated Mtb proteasome B subunit (FIG. 3B); the same ion preBA genes (FIGS. 4A and C). Time course and concentra was also unique to the GL3-treated Mtb proteasome f sub tion-response studies (FIGS. 4B and C) showed that intrabac unit. The mass of this peptide was 26 Da higher than that of terial proteasomes were inhibited 250% after 30 minutes of the N-terminal heptapeptide (TTIVALK (SEQ ID NO:1)) exposure to 50 uMGL5, 250% after 60 minutes of exposure with MW 745.48 (M+1)" that was identified only in the to 10 uMGL5, and by 90% after 60 minutes exposure to 50 untreated samples (FIG. 3A), indicative of addition of a car uMGL5. Thus, the oxathiazol-2-ones enter BCG rapidly and bonyl at the expense of two hydrogen ions. The ion at m/z. inhibit native proteasomes extensively. 771.46 was subjected to LC-MS/MS analysis. Its fragmenta 0.130. The inhibitors were investigated to ascertain that tion pattern confirmed that the altered species corresponded they were mycobactericidal alone and during recovery of to the N-terminal heptapeptide and indicated that the modi mycobacteria from exposure to NO. Provision of NO in vitro fication must occur at the N-terminal Thr1 and/or Thr2. was intended to mimic the nitroxidative stress that limits 0127. To further investigate the modification site, the abil Mtb's replication in wild type mice, as judged by the pro ity to modify primary amino groups by reaction with glut found acceleration of Mtb replication in mice whose alleles araldehyde and sodium cyanoborohydride was exploited, for inducible nitric oxide synthase were disrupted (MacMick resulting in a mass increase of 68.06 Da for each such modi ing et al., “Identification of Nitric Oxide Synthase as a Pro fication. Modifying the Mtb proteasome with glutaraldehyde tective Locus Against Tuberculosis.” Proc. Natl. Acad. Sci. and sodium cyanoborohydride without prior exposure to an USA 94:5243-5248 (1997), which is hereby incorporated by oxathiazol-2-one led to identification of the N-terminal hep reference in its entirety). NO was provided from the decom tapeptide (TTIVALK (SEQ ID NO:1)) with MW 881.48 position of 2.2-(hydroxynitroSohydrazino)-bis-ethanamine (M+H)", a mass increase of 136.13 Da, consistent with (DETA-NO) at 50 uM. By itself, DETA-NO at this concen modification of the primary amino groups of Thr1 and Lys7 tration did not affect bacterial viability. Although DETA-NO (FIG. 3C), as confirmed by the fragmentation pattern in decomposes with a ttext missing or illegible when MS/MS. In contrast, only one glutaraldehyde-dependent filed of 20 hours at 37° C. and pH 7.0, it was added only at the modification of the N-terminal heptapeptide was detected in outset of the 4-day experiment. The oxathiazol-2-ones proved proteasomes that had first been treated with GLS. The corre to be bacteriocidal to BCG both alone and in synergy with DETA-NO. GL1, GL3, and GL5 were eachable to kill BCG. sponding ion had m/z 839.52 (M+H)'", representing a mass GL5 was the most effective, reducing the number of colony increase of 68.06 Da (FIG. 3D). The pattern of the fragmen forming units (CFU) by 400-fold at 25 uMandby more than tation in MS/MS confirmed that only the Lys7 side chain had 2000-fold at 50 uM when the cells had been exposed to been modified. Again, results with GL3 in place of GL5 were DETA-NO (FIG.5A). identical. I0131) An additional investigation on GL5 ability to kill 0128. It was deduced that the 26-Da increase in MW Mtb was performed. Mtb were exposed overnight to 100 uM resulted from carbonylation of NH and OH groups of the DETA-NO or 100 uM DETA, the product that remained after active site or the adjacentamino acid side chains in the N-ter DETA-NO decomposed, followed by incubation with GL5 or minal heptapeptide. The heptapeptide offers three possible its inactive sulfone, GL6. Within 4 days, GL5 (50 uM) carbonylation sites: 1) the C-NH and Y-OH groups of Thr1; reduced the number of CFU by 47-fold if the Mtb had been 2) the C-NH group of the Thr1 and the Y-OH group of Thr2: exposed to DETA-NO but not if they had been exposed to 3) the amide NH group of Thr2 and the Y-OH group of Thr1. DETA. GL6 did not lead to a reduction of CFU in either Because the Y-OH group is the nucleophile of the active site condition (FIG. 5B). Thus, only the oxathiazol-2-one that Thrl, which initiates a nucleophilic attack on carbonyl groups inhibited Mtb's PrcAB could kill Mtb, and only in conjunc of Substrates or oxathiazol-2-ones, the second possibility can tion with exposure to sublethal nitric oxide. be excluded. Given that only the NH group of Lys7 was modified by glutaraldehyde/Na(CN)BH in oxathiazol-2- Example 12 onetreated proteasomes, the third possibility can be ruled out; otherwise the glutaraldehyde/Na(CN)BH, modification Impact of Oxathiazol-2-Ones on Mammalian Cells would have yielded a 162 Da mass shift for the peptide. All I0132) Given that proteasome-inhibiting oxathiazol-2ones these lines of evidence suggest that the N-terminal C-NH, and killed mycobacteria, their impact on the Survival of mamma Y-OH groups of Thrl of the B chain of the Mtb proteasome lian cells was tested. GL5 and GL6 showed no evident cyto participate in a covalent reaction with both GL3 and GL5 toxicity when incubated with human skin fibroblasts, human Oxathiazol-2-ones to form the oxazolidin-2-one species. lung fibroblasts, Vero monkey kidney epithelial cells, or mouse macrophage-like RAW264.7 cells at concentrations Example 11 up to 50 LM for 4 days. In contrast, less than 10% of Vero cells Impact of Oxathiazol-2-Ones on Mycobacteria remained viable after 4-day exposure to 25 nM bortezomib. Finally, because most Mtb in nature resides in human mac 0129. The next step was to examine whether oxathiazol rophages, we exposed human monocyte-derived macroph 2-ones could penetrate the mycobacterial cell wall and inhibit ages (mostly multinucleated giant cells) to 100LMGL3, GL5 native proteasomes in the cytosolic milieu. Mycobacterium or GL6 for 7 days. There was no morphologic sign of cyto bovis var. BCG was then incubated with GL1, GL3, or GL5 toxicity. Again, in contrast, Some of the human macrophages (each at 50 uM) for 4 hours, the cells were washed to remove exposed to as little as 0.5 Mbortezomib for 7 days rounded extracellular compound, lysed, and the residual proteasome up or appeared to disintegrate. activity was measured. The peptidyl boronate MLN-273 0.133 Proteasomes play critical roles in eukaryotes (N-morpholino-(L)-naphthylAla-(L)-Leu-boronic acid) (25 (Ciechanover, A., “Proteolysis: From the Lysosome to Ubiq uM) served as a positive control. Like MLN-273, the oxathia uitin and the Proteasome Nat. Rev. Mol. Cell. Biol. 6:79-87 US 2011/011 8274 A1 May 19, 2011

(2005); and Goldberg, A. L., “Functions of the Proteasome: 0.135 The unique inactivation mechanism of oxathiazol From Protein Degradation and Immune Surveillance to Can 2-ones led to an examination of selectivity of these com cer Therapy,” Biochem. Soc. Trans. 35:12-17 (2007), which pounds among non-proteasomal proteases, such as chymot are hereby incorporated by reference in their entirety). As the rypsin, trypsin, cathepsin B, and matrix metalloproteinase major means of protein turnover, they help cells adapt to (MMP)-2, representatives of the serine-, cysteine-, and changing circumstances, dispose of oxidized (Jung et al., matrix metalloproteinase classes. Most of the Oxathiazol-2- “Oxidized Proteins: Intracellular Distribution and Recogni ones tested were inactive or weak inhibitors of trypsin, cathe psin, B and MMP-2, with ICs values >50 uM. However, all tion by the Proteasome.” Arch. Biochem. Biophys. 462:231 oxathiazol-2-ones reported here except GL2 inhibited C-chy 237 (2007), which is hereby incorporated by reference in its motrypsin, with ICso values ranging from 0.01-2.2 M. GL5 entirety), or nitrosated (Uehara, T., Accumulation of Mis reversibly inhibited C-chymotrypsin in a time-independent folded Protein Through Nitrosative Stress Linked to Neuro manner with K. 64 nM. In contrast to the proteasome's N-ter degenerative Disorders.” Antioxid. Redox. Signal 9:597-601 minal Thractive site residue, chymotrypsin's active site Ser (2007), which is hereby incorporated by reference in its residue is positioned internally. Given the lack of a terminal entirety) proteins, and Survive amino acid limitation and (in NH group, the acyl-enzyme intermediate formed by reaction vertebrates) process antigens (Grune et al., “Peroxynitrite of oxathiazol-2-one with the Y-OH of chymotrypsin's active Increases the Degradation of Aconitase and Other Cellular site Ser can undergo either aqueous hydrolysis to reactivate Proteins by Proteasome,” J. Biol. Chem. 273:10857-10862 the enzyme or regeneration of Oxathiazol-2-one by the (1998), which is hereby incorporated by reference in its released —OH or —SH group attacking —CO-yO-Ser. entirety). In contrast, the functions of proteasomes in bacteria 0.136. The differential kinetic effects of oxathiazol-2-ones are largely unknown. Mtb is unusual among Actinomycetes on Mtb and human proteasomes and on Mtb proteasomes in encoding only two types of chambered, ATP-dependent Versus mammalian O-chymotrypsin will be advantageous in proteases, the proteasome and Clp (Cole et al., “Deciphering the event that Oxathiazol-2 ones find eventual application in the Biology of Mycobacterium tuberculosis From the Com the treatment of tuberculosis. Any inhibitory activity against plete Genome Sequence.” Nature 393:537-544 (1998), which human proteasomes or C-chymotrypsin should be reversed is hereby incorporated by reference in its entirety). Despite a following each dose, while the pathogen's proteasomes lack of information on the specific functions of the Mtb pro would remain inhibited. Mtb may have difficulty replacing teasome, and notwithstanding its dispensability for Mtb to irreversibly blocked proteasomes if bacterial protein synthe Survive under in vitro growth-Sustaining conditions, the pro sis is impaired by other chemotherapeutics or by Mtb's non teasome is indispensable for Mtb to survive in the mouse replicative state (Hu et al., “Protein Synthesis is Shutdown in (Gandotra et al., “Invivo Gene Silencing Identifies the Myco Dormant Mycobacterium Tuberculosis and is Reversed by bacterium tuberculosis Proteasome as Essential for the Bac Oxygen or Heat Shock.” FEMS Microbiol. Lett. 158:139-145 teria to Persist in Mice.” Nat. Med. 13:1515-1520 (2007), (1998), which is hereby incorporated by reference in its which is hereby incorporated by reference in its entirety). entirety). 0134. The oxathiazol-2-ones that emerged in the present 0.137 There is no oxathiazolone chemophore appearing in invention search bear no resemblance to existing proteasome any compound that is known to inhibit a specific enzyme. inhibitors, such as Y-lactam-B-lactones, epoxyketones, vinyl Gezginci et al. found potent antimycobacterial activity in a sulfones, peptidyl aldehydes, or peptidylboronates, which all series of pyrazines (Gezginci et al., “Antimycobacterial form covalent bonds with the Y-OH of the active site Thr. The Activity of Substituted Isosteres of Pyridine- and Pyrazin LC-INAS/MS analyses of oxathiazol-2-one-treated Mtb pro ecarboxylic Acids.” J. Med. Chem. 44:1560-1563 (2001), teasome B subunits are consistent with the mechanism of which is hereby incorporated by reference in its entirety), inactivation illustrated in FIG. 6. The Y-OH of the active site including three compounds bearing an Oxathiazol-2-one moi Thrl attacks the carbonyl group of the Oxathiazol-2-one, ety, but the target was not reported. The compound forming a tetrahedral intermediate 1. Intermediate 1 can 5-(pyrazin-2-yl)-1,3,4-oxathiazol-2-one was synthesized, undergo C-O bond cleavage (route a) or C-S bond cleav and found to inhibit the Mtb proteasome, albeit less potently age (route b) to yield intermediate 2 or 3, respectively. The than GL5. NH group of the Thr1 would then attack the carbonyl group 0.138. The ability of oxathiazol-2-ones to inhibit protea of 2 or 3 to forman oxazolidin-2-one moiety, inactivating the Somes in intact mycobacteria and their selective toxicity for proteasome. In the case of the human proteasome, it was mycobacteria Support the concept that the mycobacterial pro postulated that because of a different disposition of water teasome is both a "druggable' and an accessible target. Inhib molecules, the intermediates 2 or 3 could not undergo the iting macromolecular degradation rather than synthesis nucleophilic attack by the terminal NH group. Instead, they would be unprecedented for an anti-bacterial agent of known would undergo attack by the sulfhydryl or water (route c) to mechanism (Clardy et al., “Lessons From Natural Mol regenerate either the inhibitor or compound 4, with recovery ecules.” Nature 432:829-837 (2004), which is hereby incor of the active proteasome in both cases. Defined water mol porated by reference in its entirety). The similarity in the ecules in the proximity of the active site Thr1 in the yeast concentrations of Oxathiazol-2-ones required to inhibit pro proteasome, especially one located between the Y-OH and teasomes in mycobacteria and to kill mycobacteria is consis NH of the Thr1, are involved in the hydrolysis of peptide tent with proteasome inhibition being a major mechanism for bonds of substrates, and in the slow reactivation of eukaryotic the oxathiazol-2-ones antimycobacterial effects. However, proteasomes following inhibition by omuralide (Mc Cor because oxathiazol-2-ones can also inhibit C-chymotrypsin, a macket al., “Active Site-Directed Inhibitors of Rhodococcus serine protease, they may have (an) additional target(s) in Mtb 20 S. Proteasome. Kinetics and Mechanism,” J. Biol. Chem. besides the proteasome. It would be a boon if, for example, 272:26103-26109 (1997); Borissenko et al., “20S Protea the oxathiazol-2-ones inhibit not only Mtb's proteasome but some and its Inhibitors: Crystallographic Knowledge for also its only other chambered, ATP-dependent protease, Clp Drug Development,” Chem. Rev. 107:687-717 (2007), which (A/X)P. Efforts are underway to diligently test this hypothesis are hereby incorporated by reference in their entirety). genetically and biochemically. US 2011/011 8274 A1 May 19, 2011

0.139. Oxathiazol-2-ones reported herein are the first pro R to R are independently H, substituted or unsubstituted teasome inhibitors found to inhibit proteasomes of Mtb by C-C alkyl, Substituted or unsubstituted C-C alk carbonlyating the Y-OH and C.-NH groups of the active site enyl, Substituted or unsubstituted C-C alkynyl, Thrl, thus forming a stable oxazolidin-2-one species. The cycloalkyl, haloalkyl, heterocyclyl, or aryl, much weaker and reversible inhibition of the human protea Some by the same compounds suggests that inhibitors with an wherein if Q is oxathiazol-2-one warhead can inhibit proteasomes of differ ent species by different modalities. The 64-fold selectivity of GL5 toward the Mtb proteasome over the human B5 protea E-1 G some subunit represents a ~1160-fold improvement in the Y species selectivity ratio relative to bortezomib, a proteasome D-N. inhibitor in clinical use. Irreversible inhibition of the Mtb proteasome together with weak, reversible inhibition of human proteasomes encourages consideration of oxathiazol and E and D or E and G are C, R, and R, or R and R. 2-ones for antimycobacterial drug development. respectively, may form a fused substituted or unsubstituted 0140 Although preferred embodiments have been aromatic or cyclic hydrocarbon ring and if Q is depicted and described in detail herein, it will be apparent to those skilled in the relevant art that various modifications, additions, Substitutions, and the like can be made without departing from the spirit of the invention and these are there fore considered to be within the scope of the invention as defined in the claims which follow. What is claimed: 1. A method of treating a mammalian Subject for a patho and D and E are C, R, and R may form a fused substituted or gen infection, said method comprising: unsubstituted aromatic ring or cyclic hydrocarbon ring, under administering to the Subject a compound of Formula (I): conditions effective to treat the subject for a pathogen infec tion, wherein the Substituted alkyl, alkenyl, alkynyl, aromatic, or cyclic hydrocarbon ring groups have substituents selected (I) from the group consisting of hydroxyl, halogen, —CN, —NO. —SR —OR —SOR. -NH2, —NHR, Q-( )—o - NHSOR - NRRs –C(O)NHRs –C(O)NH2, N-S —CONRR, —CHO,-CO)Rs, and —C(O)CR. 2. The method of claim 1, wherein Q is where:

Q is G E Y D2 1W D NA H, or AN DNA D and E are C, and R and R form a fused substituted or 1's unsubstituted aromatic ring or cyclic hydrocarbon ring, whereby the compound has the following structure of Formula (II):

(II) where the crossing dashed line illustrates the bond formed R20 R3 joining Q to the rest of the compound of Formula (I); R21 O A is S or O: D is N or C, wherein C is bound to R: O X-k=o E is N or C, wherein C is bound to R; R22 A. N-S G is N or C, wherein C is bound to R: X is N or C, wherein C is bound to Rs: R23 Y is N or C, wherein C is bound to R; J is N or C, wherein C is bound to R: where T is N or C, wherein C is bound to R: Z is N or C, wherein C is bound to R: W r is independently a single or double bond and wherein R to Rs are independently H, a halogen, —SR Rao to R2 are independently H, a halogen, —NO, NO. —NR7Rs - SOR. -CONRoR -OR2, Substi —NR7Rs – CONRR7, substituted or unsubstituted tuted or unsubstituted C-C alkyl, Substituted or unsubsti C-C alkyl, Substituted or unsubstituted C-C alk tuted C-Co alkenyl, Substituted or unsubstituted C-Co enyl, Substituted or unsubstituted C-C alkynyl, alkynyl, cycloalkyl, haloalkyl, heterocyclyl, or aryl; cycloalkyl, heterocyclyl, or aryl. US 2011/011 8274 A1 May 19, 2011 18

3. The compound of claim 2, wherein the compound of 6. The compound of claim 5, wherein the compound of Formula (II) is selected from the group consisting of: Formula (IV) is selected for the group consisting of

4. The method of claim 1, wherein Q is

E-1 Gy DNA

E and G are C, and R and R form a fused substituted or unsubstituted aromatic ring or cyclic hydrocarbon ring whereby the compound has the following structure of Formula (III): NN's (III) O X- 7. The method of claim 1, wherein Q is O R20 le N E

R21 e - A A N/ S. R22 R23 R2 and the compound of Formula (I) has the structure of Formula (V) 5. The method of claim 1, wherein Q is R (V)V V s R A D-N. le O R3 X-0. and the compound of Formula (I) has the structure of NS Formula (IV) 8. The compound of claim 7, wherein the compound of (IV) Formula (V) is selected from the group consisting of: R R3

R-62 s A. O S X=o e O US 2011/011 8274 A1 May 19, 2011 19

10. The method of claim 1, wherein Q is -continued ay SirO. / \l S / and the compound of Formula (I) is selected from the group consisting of: 9. The method of claim 1, wherein Q is

D2 O O AN / -( -( N / S, O S. 7. and the compound of Formula (I) is selected from the group riskN N N?V N consisting of N-O N R3 O, O, O O

p- p-( -( S, and -(/ N / S S. % N- 's N R N N M N \ 4 b. 4 N( S N N1 N R3 R3 R3 R p-(O, p-(O,

R 2 N- R e N f f S-N O-N and the compound of Formula (I) has the structure of p-(O, p-(O, Formula (VI) N f NY S R NaN f N/ S (VI) O S-N R3 O, O, R p-( R p-( R -o

N Cy-sN -S 2 N-SN N-N- Y \S-N f \S | R3 12. The compound of claim 11, wherein the compound of O O Formula (VI) is selected from the group consisting of 5-phe nyl-1,3,4-oxathiazol-2-one, 5-(4-nitrophenyl)-1,3,4-oxathia R p-( p-( Zol-2-one, 5-(3-nitrophenyl)-1,3,4-oxathiazol-2-one, 5-(2- pyridinyl)-1,3,4-oxathiazol-2-one, 5-(3-methoxyphenyl)-1, y- , and y N 3,4-oxathiazol-2-one, 5-(3-fluorophenyl)-1,3,4-oxathiazol b- \, 2-one, 5-(3-(trifluoromethyl)-phenyl)-1,3,4-oxathiazol-2- R3 one, 5-(4-tolyl)-1,3,4-oxathiazol-2-one, 5-(3-tolyl)-1,3,4- oxathiazol-2-one, and 5-(3,5-dimethoxyphenyl)-1,3,4- Oxathiazol-2-one. US 2011/011 8274 A1 May 19, 2011 20

13. The method of claim 1, wherein Q is 15. The method of claim 1, wherein the pathogen is selected from the group consisting of Mycobacterium tuber culosis, Mycobacterium leprae, and another disease-causing Mycobacterium. 16. The method of claim 1, wherein the subject is a human. 17. A method of treating a mammalian Subject for cancer, said method comprising: administering to the Subject a compound of Formula (I): (I) and the compound of Formula (I) is selected from the group O consisting of: Q-( )—o O O N-S

R -( R -( where: R N N/ R N N/ Q is D2 V R3 4. Rs R3 2N AN / DSA| : O R4 1. Tsy O O R -( R -( R N N/ R N N/ where the crossing dashed line illustrates the bond formed Na2 R5 R3 2N joining Q to the rest of the compound of Formula (I); A is S or O: R4 D is N or C, wherein C is bound to R: E is N or C, wherein C is bound to R; G is N or C, wherein C is bound to R: X is N or C, wherein C is bound to Rs: Y is N or C, wherein C is bound to R; J is N or C, wherein C is bound to R: T is N or C, wherein C is bound to R: Na2N Z is N or C, wherein C is bound to R: R3 Rs n wherein R to Rs are independently H, a halogen, —SR R4 NO. —NR7Rs - SOR. -CONRoR - OR2, Substi O O tuted or unsubstituted C-C alkyl, Substituted or unsubsti tuted C-Co alkenyl, Substituted or unsubstituted C-Co alkynyl, cycloalkyl, haloalkyl, heterocyclyl, or aryl; R -(S. and R -{S, M M R to R are independently H, substituted or unsubstituted N1 N s R2 s1SN C-C alkyl, Substituted or unsubstituted C-Co alk enyl, substituted or unsubstituted C-C alkynyl, N N 2 cycloalkyl, haloalkyl, heterocyclyl, or aryl, R3 2 NN Rs wherein if Q is R4 -(O S. R N N/ and E and D or E and G are C, R, and R, or R and R. respectively, may form a fused substituted or unsubstituted N R 2 aromatic or cyclic hydrocarbon ring and if Q is

R4 D2 2 s 14. The method of claim 1, wherein said administering is A N. G oral, intradermal, intramuscular, intraperitoneal, intravenous, Subcutaneous, or intranasal. US 2011/011 8274 A1 May 19, 2011

and D and E are C, R, and R may form a fused substituted or unsubstituted aromatic ring or cyclic hydrocarbon ring, under conditions effective to treat the subject for cancer, wherein the (III) Substituted alkyl, alkenyl, alkynyl, aromatic, or cyclic hydro O carbon ring groups have substituents selected from the group X consisting of hydroxyl, halogen, —CN. —NO. —OR, O —SOR, NH, NHR, NHSOR - NR,R, R20 le N —C(O)NHRs –C(O)NH, -CONRR, —CHO, R21 —C(O)Rs, and —C(O)OR. e 18. The method of claim 17, wherein Q is A S. R5 G E Y R3 D NA 21. The method of claim 17, wherein Q is D and E are C, and R and R form a fused substituted or E-1 G unsubstituted aromatic ring or cyclic hydrocarbon ring, Y whereby the compound has the following structure of D-N. Formula (II): and the compound of Formula (I) has the structure of (II) Formula (IV) R20 R3 (IV) R21 O X-ky-oO R5 A N-S R23

where is independently a single or double bond and 22. The compound of claim 21, wherein the compound of Rao to R2 are independently H, a halogen, —NO, Formula (IV) is selected for the group consisting of: —NR7Rs, —CONRR, substituted or unsubsti tuted C-Co alkyl, Substituted or unsubstituted C-C alkenyl, Substituted or unsubstituted C-Co ON alkynyl, cycloalkyl, heterocyclyl, or aryl. 19. The compound of claim 18, wherein the compound of Formula (II) is selected from the group consisting of:

20. The method of claim 17, wherein Q is

G E-1 Y NN's D-N. 23. The method of claim 17, wherein Q is and E and G are C, R, and R form a fused substituted or unsubstituted aromatic ring or cyclic hydrocarbon ring whereby the compound has the following structure of Formula (III): US 2011/011 8274 A1 May 19, 2011 22

and the compound of Formula (I) has the structure of Formula (V) -continued O

(V) R S, s

-(O -{O S / S / z N R N N 24. The compound of claim 23, wherein the compound of ^ / Formula (V) is selected from the group consisting of N N O R3 S O O

25. The method of claim 17, wherein Q is

and the compound of Formula (I) is selected from the group and the compound of Formula (I) is selected from the group consisting of: consisting of -(O -(O S, S, N S. 7. O S. / / N/ R N N/ N NS N N^ N N ) V \ N( ) N- N R3 O R3 O R3 O O -(S. and -( R -(S, -( s N S/ " s N/ N / N N / N s N? NN-S \M R3 US 2011/011 8274 A1 May 19, 2011 23

27. The method of claim 17, wherein Q is -continued O O R -( R -( S, s S- M R S- M N N N N N

and the compound of Formula (I) has the structure of - % Rs N N Formula (VI)

(VI) M s M , and S. R S. N1N 2 N N N N 2 R3 2 NN Rs R4 O NN's -{ S, 28. The compound of claim 27, wherein the compound of R2 N N/ Formula (VI) is selected from the group consisting of 5-phe N nyl-1,3,4-oxathiazol-2-one, 544-nitrophenyl)-1,3,4-oxathia R 2 Zol-2-one, 5-(3-nitrophenyl)-1,3,4-oxathiazol-2-one, 5-(2- pyridinyl)-1,3,4-oxathiazol-2-one, 5-(3-methoxyphenyl)-1, R4 3,4-oxathiazol-2-one, 5-(3-fluorophenyl)-1,3,4-oxathiazol 2-one, 5-(3-(trifluoromethyl)-phenyl)-1,3,4-oxathiazol-2- 30. The method of claim 17, wherein said administering is one, 5-(4-tolyl)-1,3,4-oxathiazol-2-one, 5-(3-tolyl)-1,3,4- oral, intradermal, intramuscular, intraperitoneal, intravenous, oxathiazol-2-one, and 5-(3,5-dimethoxyphenyl)-1,3,4- Subcutaneous, or intranasal. Oxathiazol-2-one. 31. The method of claim 17, wherein the subject is a 29. The method of claim 17, wherein Q is human. 32. The method of claim 17, wherein the cancer is selected from the group consisting of carcinoma, Sarcoma, lymphoma, and myeloma. 33. A compound of Formula (I): (I) and the compound of Formula (I) is selected from the group Q-( )—o consisting of: N-S where: O O Q is R -( R -( S, S, D2P E R S. M R S. M / , or | N s 2 N N 2 N N AN DNA 2 N R3 N Rs R3 2 where the crossing dashed line illustrates the bond formed R4 joining Q to the rest of the compound of Formula (I); O O A is S or O: R -( R -( D is N or C, wherein C is bound to R; S, S, E is N or C, wherein C is bound to R; R N / R N / G is N or C, wherein C is bound to R: 2 N N 2 N N wherein R to R are independently H, a halogen, —SR NO, NRRs – SOR, CONRR, —OR, N 21 Rs R 22N substituted or unsubstituted C-C alkyl, substituted or unsubstituted C-Co alkenyl, Substituted or unsubsti R4 tuted C-Co alkynyl, cycloalkyl, haloalkyl, heterocy clyl, or aryl; US 2011/011 8274 A1 May 19, 2011 24

R to Rio are independently H. Substituted or unsubsti 35. The compound of claim 34, wherein the compound of tuted C-C alkyl, substituted or unsubstituted Formula (II) is selected from the group consisting of: C-C alkenyl, Substituted or unsubstituted C-Co alkynyl, cycloalkyl, haloalkyl, heterocyclyl, or aryl, wherein if Q is S N-S and s' DNA S N

and E and D or E and G are C, R, and R, or R and R. 36. The compound of claim 33, wherein Q is respectively, may form a fused substituted or unsubsti tuted aromatic or cyclic hydrocarbon ring and if Q is E G Y s D2 D-N. A N. and E and G are C, R, and R form a fused substituted or unsubstituted aromatic ring or cyclic hydrocarbon ring and D and E are C, R, and R may form a fused substituted whereby the compound has the following structure of or unsubstituted aromatic or cyclic hydrocarbon ring, Formula (III): wherein the substituted alkyl, alkenyl, alkynyl, aro matic, or cyclic hydrocarbon ring groups have Substitu (III) ents selected from the group consisting of hydroxyl, halogen, —CN, - NO. —SR —OR2, —SOR, OX —NH, -NHR, —NHSOR - NR7Rs —C(O) R20 le N NHRs –C(O)NH, -CONRR, —CHO,-CO) R21 Rs, and —C(O)Ro: e with the proviso that Q is not an isoxazolyl group, and with A the further proviso that at least one of D, E, or G is N if S. RandR, or R and R. do not form a fused substituted R22 or unsubstituted aromatic hydrocarbon ring. R3 34. The compound claim 33, wherein Q is

E G Y - D-N. ) and D and E are C, R, and R form a fused substituted or unsubstituted aromatic ring or cyclic hydrocarbon ring, whereby the compound has the following structure of and the compound of Formula (I) is selected from the group Formula (II): consisting of: (II) R20 O O R21 -( -( N N 7. N s 7. R22 X (S- R23 N N O R3 S R3 O O where W r is independently a single or double bond and S, R S, Rao to R2 are independently H, a halogen, —NO; N S/ N/ —NR7Rs, —CONRR, substituted or unsubsti R2 / tuted C-C alkyl, substituted or unsubstituted N C-Coalkenyl, Substituted or unsubstituted alkynyl, cycloalkyl, heterocyclyl, or aryl. US 2011/011 8274 A1 May 19, 2011 25

38. The compound of claim 33, wherein Q is -continued O O E G -(S, -(S, Y NN-sy. NU-sy DS R2 / / N O R3 and the compound of Formula (I) is selected from the group O O consisting of:

- S, R, -(S, N N/ S/ S, S, s-N \s-N S / S / O "skN N N/S N N-O \ R3 S/ S, N/ S, an K K N S R3 O O O O -( -(S. -( S. 7. and S S/ S. R N N M 2 N/ N( NN K N-S N R3 S R3