FIELD OF THE INVENTION [0002] The present invention generally relates to alkaloid derivatives, pharmaceutical compositions containing these compounds, and methods for their pharmaceutical use.

BACKGROUND OF THE INVENTION [0003] Antibiotic resistance of pathogenic bacteria, including pathogenic actinomycetes, such as M. tuberculosis, is a well-known problem faced by medical practitioners in treatment of bacterial diseases. Mycobacterium tuberculosis, the causative agent of tuberculosis, is a leading pathogenic cause of death worldwide (Zumla, A. et al., Clinical Review : Tuberculosis. Br. Med. J. (1998) 316 : 1962). The rise of mycobacterial resistance to common antituberculars, such as isoniazid and rifampin, along with the high prevalence of tuberculosis and Mycobacterium avium complex in AIDS patients, has led to a renewed interest in the discovery of anti-mycobacterial agents with new modes of action. Therefore, there is a further need in the art for a new class of compounds useful for reducing resistance to existing antibiotics in treatment of bacterial infections in humans and in other mammals, such as domestic and farm animals, and useful as antibiotics in treatment of bacterial diseases, such as tuberculosis.

[0004] Mycothiol (MSH ; AcCys-GlcN-ks ; chemical name l-D-myo-inosityl-2-(N- acetylcysteinyl) amido-2-deoxy-a-D-glucopyranoside) is a low molecular weight thiol found in actinomycetes (G. L. Newton et al. J. Bacteriol. (1996) 178: 1990) : As shown in FIGURE 1, MSH plays a central role in protecting actinomycetes against alkylating agents and other toxins in conjunction with the enzyme, mycothiol S-conjugate amidase, an acyl glucosaminyl inositol amidase (G. L. Newton, et al., Biochemistry (2000) 39: 10739). A second acyl glycosaminyl inositol amidase from Mycobacterium tuberculosis that is highly homologous with mycothiol S-conjugate amidase that is involved in the biosynthesis of MSH has also been described (G. L. Newton et al. J. Bacteriol. (2000) 24: 6958).

[0005] Aerobic organisms are subjected to oxidative stress from many sources, including atmospheric oxygen, basal metabolic activities, and, in the case of pathogenic microorganisms, toxic oxidants from the host phagocytic response intended to destroy the bacterial invader. Glutathione (GSH) is the dominant low molecular weight thiol in most eukaryotes and Gram-negative bacteria, and it plays a key role in protection of the cell against oxygen toxicity and electrophilic toxins (R. C. Fahey and A. R. Sundquist (1991) Adv.

Enzymol., 1991, 64,1-53 ; Dolphin, et al., Glutathione : Chemical, Biochemical, and Medical Aspects, (New York: John Wiley & Sons), 45-84, 1989). Actinomycetes, including Streptomyces and Mycobacteria, do not make GSH but produce millimolar levels of mycothiol (MSH, AcCys-GIcN-Ins), an unusual conjugate of N-acetylcysteine (AcCys) with l-D-myo-inosityl-2-amino-2-deoxy-a-D-glucopyranoside (GlcN-Ins) to carry out detoxification within actinomycetes (G. L. Newton, et al., J. Bacteriol., 1996,178, 1990- 1995; S. Sakuda, et al., Biosci. Biotech. Biochem., 1994 58: 1347-1348; H. S. C. Spies and D. J. Steenkamp, (1994) Eur. J. Biochem. 224: 203-213; G. L. Newton, et al. (1995) Eur. J.

Bioche7 ? Z. 230 : 821-825).

[0006] Because the above-described mycothiol-dependent pathways of actinomycetes are not found in eukaryotes, the enzymes involved represent potentially useful new anti- mycobacterial targets in eukaryotic hosts since inhibition of the enzyme would permit blocking of MSH-dependent detoxification at two distinct levels, namely, biosynthesis and detoxification and would not otherwise interfere with GSH pathways. The present invention is directed to these, as well as other important ends.

DESCRIPTION OF THE FIGURES FIGURE I is a schematic diagram showing the role that mycothiol (MSH) plays in protecting actinomycetes against alkylating agents and other toxins in conjunction with mycothiol S-conjugate amidase.

SUMMARY OF THE INVENTION [0007] The present invention is generally directed to alkaloid derivatives, pharmaceutical compositions containing these compounds, and methods for their pharmaceutical use.

[0008] In certain embodiments, the invention is directed to compounds that in certain useful as inhibitors of acyl glucosaminyl inositol amidases, preferably mycothiol-S-conjugate amidases, wherein the compounds are alkaloid derivatives of the formula: or a prodrug, pharmaceutically acceptable salt, hydrate, solvate, acid salt hydrate, N- oxide or isomorphic crystalline form thereof; wherein Rl is H or halogen selected from F, Cl, Br, and I ; R is H or OH ; R3 is H or halogen selected from F, Cl, Br, and I; R4 is H or CH3 ; R is H, F, NH2; and R6isHorCH3 ; provided that when R'is Cl, Br, or I, then R is not OH; and provided that when Rl is F, then R3 is not F.

[0009] In other embodiments, the invention is directed to a therapeutic drug composition for treatment or prophylaxis of an infection, disease, condition or symptom (s) caused by actinomycetes in a mammalian subject, the composition comprising as an active ingredient a therapeutically effective amount of at least one compound having a formula: or a prodrug, pharmaceutically acceptable salt, hydrate, solvate, acid salt hydrate, N- oxide or isomorphic crystalline form thereof; wherein Rl is H or halogen selected from F, Cl, Br, and I ; R ? is H or OH ; R3 is H or halogen selected from F, Cl, Br, and I ; R4 is H or CH3 ; R5 is H, F, NH2 ; and R6 is H or CH3 ; provided that when Rl is Cl, Br, or I, then W is not OH; and provided that when Rl is F, then R3 is not F.

[0010] In additional embodiments, the invention is directed to a method of inhibiting the activity of an acyl glucosaminyl inositol amidase, preferably a mycothiol S-conjugate amidase, comprising the step of : administering an effective amount of at least one compound having the formula : or a prodrug, pharmaceutically acceptable salt, hydrate, solvate, acid salt hydrate, N- oxide or isomorphic crystalline form thereof; wherein Rl is H or halogen selected from F, Cl, Br, and I ; R2 is H or OH; R3 is H or halogen selected from F, Cl, Br, and I; R4isHorCH3 ; R5 is H, F, NH2 ; and R6 is H or CH3 ; provided that when R'is Cl, Br, or I, then R2 is not OH; and provided that when Rl is F, then R3 is not F.

[0011] In yet additional embodiments, the invention is directed to a method of treating or preventing an infection, disease, condition, or symptom caused by an actinomycete in a mammalian subject, comprising the step of : administering to said subject a therapeutically effective, non-toxic dose of at least one compound having a formula: or a prodrug, pharmaceutically acceptable salt, hydrate, solvate, acid salt hydrate, N- oxide or isomorphic crystalline form thereof; wherein Rl is H or halogen selected from F, Cl, Br, and I; R is H or OH ; R3 is H or halogen selected from F, Cl, Br, and I ; R4 is H or CH3 ; R5 is H, F, NH2; and R6 is H or CH3 ; provided that when Rl is Cl, Br, or I, then W is not OH; and provided that when Rl is F, then R3 is not F.

[0012] In other embodiments, the invention is directed to methods for decreasing the antibiotic-resistance of pathogenic acyl glucosaminyl inositol amidase-producing bacteria, preferably mycothiol S-conjugate amidase-producing bacteria, comprising the step of : exposing said bacteria to an effective amount of at least one compound having the formula : or a prodrug, pharmaceutically acceptable salt, hydrate, solvate, acid salt hydrate, N- oxide or isomorphic crystalline form thereof; wherein Ru ils H or halogen selected from F, Cl, Br, and I; R2 is H or OH; R3 is H or halogen selected from F, Cl, Br, and I ; R4 is H or CH3 ; R5 is H, F, NH2; and R6 is H or CH3 ; provided that when Rl is Cl, Br, or I, then R is not OH; and provided that when Rl is F, then R3 is not F.

[0013] In related aspects, the invention provides methods for reducing the virulence in a mammalian subject of pathogenic acyl glucosaminyl inositol amidase-producing bacteria, preferably mycothiol S-conjugate amidase-producing bacteria, comprising the step of : administering to said subject an effective amount of at least one compound having a formula: or a prodrug, pharmaceutically acceptable salt, hydrate, solvate, acid salt hydrate, N- oxide or isomorphic crystalline form thereof; wherein Rl is H or halogen selected from F, Cl, Br, and I ; R2 is H or OH; R3 is H or halogen selected from F, Cl, Br, and I; R4 is H or CH3 ; R5 is H, F, NH2 ; and R6 is H or CH3 ; provided that when R'is Cl, Br, or I, then R2 is not OH; and provided that when Rl is F, then R3 is not F.

[0014] In another embodiment, the invention is directed to methods of treating or preventing an infection, disease, condition or symptom caused by actinomycetes in a mammalian subject, comprising the steps of : administering to said subject a non-toxic dose of at least one first line antibiotic agent; and administering to said subject a non-toxic dose of at least one compound having the formula: or a prodrug, pharmaceutically acceptable salt, hydrate, solvate, acid salt hydrate, N- oxide or isomorphic crystalline form thereof; wherein Rl is H or halogen selected from F, Cl, Br, and I ; R2 is H or OH ; is H or halogen selected from F, Cl, Br, and I ; R4isHorCH3 ; RS is H, F, NH2 ; and R6isHorCH3 ; provided that when RI is Cl, Br, or I, then R2 is not OH; and provided that when Rl is F, then R3 is not F.

BRIEF DESCRIPTION OF THE FIGURES [0015] Figure 1 is a schematic representation of the MSH-dependent detoxification pathway in mycobacteria.

DETAILED DESCRIPTION OF THE INVENTION [0016] The inhibitors of the invention are chemical compounds that act to inhibit enzymatic activity of an of acyl glucosaminyl inositol amidase. Thus, the inhibitors have anti- mycobacterial activity, especially against actinomycetes. The compounds and formulations of the invention also are effective to inhibit growth of antibiotic-producing bacteria. The inhibitors can be purified from natural organisms using techniques known in the art and as described herein. In addition, the inhibitors can be chemically synthesized using techniques well known in the art.

[0017] As used herein the term"acyl glucosaminyl inositol amidase"refers to polypeptides with enzymatic amidase activity for glucosaminyl inositol (GIcN-Ins)-containing substrates.

Also contemplated within this term are polypeptide analogs, mimetic and chemically modified derivatives (e. g, conjugates) of these polypeptides that retain enzymatic amidase activity for glucosaminyl inositol (GlcN-Ins)-containing substrates. The term typically refers to an amidase that hydrolyzes a substrate having the chemical formula R-CONH-GIcN-Ins wherein R is CH3 (CH2) n- where n is 0-6. Included in the group of substrate compounds is the natural substrate, acetyl-GlcN-Ins, where nisO. Members of this group of substrate compounds are typically derived from an alkanoic acid. In addition, compounds having the chemical structure R-CONH-GIcN-Ins wherein R is aryl- (CH2) n, where n is 0-6 are encompassed by the term. Members of this group of substrate compounds are typically derived from an aryl alkanoic acid such as phenyl-(CH2) nCOOH.

[0018] The acyl glucosaminyl inositols of the above chemical formula can also be derived from the reaction of GlcN-Ins with any commercial acid chloride in the form RCOC1, wherein R is: o-tolyl-, 4-ethylphenyl-, 4-propylphenyl-, 4-biphenyl-, 3, 4-dimethoxyphenyl-, 3,4, 5-trimethoxyphenyl-, 2-furyl-, and the like.

[0019] The acyl glucosaminyl inositols of the above chemical formula can also be derived from an amino acid or an N-acetyl amino acid. Preferred amino acids include N- acetylcysteinyl-S-R'or cysteinyl-S-R', wherein R'is an organic group attached to the cysteine sulfur, such as may be derived from commercial thiol labeling reagents like 2- bromoacetophenone, monobromobimane, N-ethylmaleimide, 7-diethylamino-3- (4'- maleimidyl phenyl)-4-methyl cumarin, 3 (N-maleimidopropionyl) biocytin, and from naturally occurring antibiotics, including cerulenin, granaticin A, naphthomycin A, naphthomycin H, and the like.

[0020] A subset of the acyl glucosaminyl inositol amidases that are inhibited by the inhibitors of the invention are referred to herein as mycothiol S-conjugate amidases, whose substrate is an S-conjugate of a cysteine amide. As used herein, the term"S-conjugate" means that the molecule is a thioether or thioester containing two chemical moieties joined by a sulfur (i. e.,-S-) moiety. In a preferred embodiment, the S-conjugate molecule is derived from mycothiol by the reaction shown in Figure 1, wherein RX is an electrophile and R is an alkyl or alkyloid moiety. However, inhibitors of acyl glucosaminyl inositol amidases of the invention do not require a sulfur-containing amide substrate and instead cleave a GlcN-Ins- containing amide substrate.

[0021] As used herein, the terms"GlcN-Ins-containing amide"and"glucosaminyl inositol- containing amide"are interchangeable when used to describe a substrate molecule for which an acyl glucosaminyl inositol amidase has enzymatic activity, resulting in cleavage of the molecule. Similarly, the term"amide-containing S-conjugate"and"S-conjugate-containing amide"are interchangeable when used to describe a substrate molecule for which an S- conjugate amidase has enzymatic activity, resulting in cleavage of the molecule. If a particular amidase is an amide hydrolase, cleavage of the substrate molecule will form breakdown products wherein one product is a carboxylic acid, (e. g., a carboxylic acid containing at least one sulfur moiety) and the other product is an amine (e. g., GIcN-Ins). If the substrate is a mycothiol-derived S-conjugate amide of the type illustrated in Figure 1, one of the breakdown products will be l-D-myo-inosityl-2-amino-2-deoxy-oc-D-glucopyranoside (GlcN-Tns) and the other breakdown product will be a sulfur-containing carboxylic acid, such as a mercapturic acid. AcCys S-conjugates are termed mercapturic acids, the final excreted product in the mercapturic acid pathway of glutathione-dependent detoxification in mammals (J. L. Stevens, et al., (1989) in Glutathione : Chemical, Bioclzemical, and Medical Aspects- Part B (D. Dolphin, et al.) pp 45-84, John Wiley & Sons).

[0022] It is known that acyl glucosaminyl inositol amidases participate in a pathway of detoxification in bacteria, especially antibiotic-producing bacteria, and that the detoxification pathway is dependent on in vivo production of a protein acyl glucosaminyl inositol amidase by such bacteria. However, pathogenic actinomycetes (that do not produce an antibiotic) also contain a gene encoding an acyl glucosaminyl inositol amidase that becomes activated in the presence of antibiotics administered to a host, for example in treatment of a disease caused by the pathogenic actinomycetes. Thus, the gene (s) encoding the amidases are a family of antibiotic-resistance genes.

[0023] More particularly, it is known that mycothiol (1-D-myo-inosityl-2- (N- acetylcysteinyl) amido-2-deoxy-a-D-glucopyranoside) (MSH) is present in a variety of actinomycetes and plays an essential role in a pathway of detoxification in such bacteria.

Mycothiol is comprised of N-acetylcysteine (AcCys) amide linked to l-D-myo-inosityl-2- amino-2-deoxy-a-D-glucopyranoside (GlcN-Ins) and is the major thiol produced by most actinomycetes. In the mycothiol-dependent detoxification process in actinomycetes, an alkylating agent is converted to an S-conjugate of mycothiol, the latter is cleaved to release a mercapturic acid, and the mercapturic acid is excreted from the cell. A subset of the acyl glucosaminyl inositol amidases are referred to herein as S-conjugate amidases, whose substrate is an S-conjugate containing amide. As used herein, the term"S-conjugate"means that the molecule is a thioether or thioester containing two chemical moieties joined by a sulfur (i. e. ,-S-) moiety. In a preferred embodiment the S-conjugate molecule is derived from mycothiol by the reaction shown in Figure 1, wherein RX is an electrophile and R is an alkyl or alkyloid moiety. However, the acyl glucosaminyl inositol amidases do not require a sulfur-containing amide substrate and instead cleave an GlcN-Ins-containing amide substrate.

[0024] A mycothiol S-conjugate amidase responsible for cleavage of the S-conjugate of mycothiol in mycothiol-producing bacteria has been purified from M. smegmatis and identified as an ortholog of open reading frame Rv1082 in the genome of M. tuberculosis.

The Sanger Centre annotation of this gene places it at nucleic acid residues 1206520 to 1207383 of the M. tuberculosis genome. It codes for a protein of 288 amino acid residues having an identical N-terminal amino acid sequence to that determined for M. smegmatis.

Homologous amidases have been identified in M. leprae and M. avium. The M. leprae homolog (ML2391) is coded by nucleic acid residues 2861602 to 2862474 and is 86% identical to Rv1082 (Sangre Centre). The M. avium homolog is coded by nucleic acid residues 1152499 to 1153362 and is 84% identical to Rv1082 (TIGR-The Institute for Genomic Research). An additional S-conjugate amidase homolog has been identified in the M. bovis genome and is 99% identical to Rv1082 (Sanger Centre). Genes encoding mycothiol S-conjugate amidase appear to be present in all mycobacterial genomes and it seems likely that homologs will be found in other mycothiol-producing actinomycetes.

[0025] In addition, most of the homologous non-mycobacterial proteins are found in actinomycetes that produce mycothiol. It is known that homolog genes encoding S-conjugate amidases are found within antibiotic synthesis operons of the antibiotic producers <BR> <BR> <BR> <BR> Streptomyces lincolfaensis, Amycolatopsis mediterranei, Amycolatopsis orientalis, Streptomyces lavendulae, Streptomyces coelicolor, Streptomyces rochei, and the polyketide erythromycin antibiotic producer Saccharopolyspora erythraea. Two other bacteria, Corynebacterium diphtheria and Deinococcus radiodurans, also encode acyl glucosaminyl inositol amidase homologs. Four of these open reading frames are putative actinomycetes proteins encoded within the lincomycin, erythromycin, mitomycin and the rifamycin antibiotic biosynthetic operons.

[0026] At least four major domains are highly conserved among known bacterial acyl glucosaminyl inositol amidase homologs and three out of the four domains contain conserved histidine residues. These conserved domains are thought to be involved in the amide hydrolysis and binding to glucosaminyl inositol. The histidine residues are though to contribute to binding of a zinc (Zn2+) metal ion in the enzyme. Members of the family of acyl glucosaminyl inositol amidase proteins are highly conserved and share a high degree of identity throughout the amino-terminal half.

[0027] Thus, it is known in the art that acyl glucosaminyl inositol amidases are formed in vivo by bacteria as part of a detoxification pathway, usually in antibiotic-producing bacteria, and most usually in bacteria characterized by intracellular production of mycothiol. An assay for determining amidase activity of such acyl glucosaminyl inositol amidases involves treatment of the amidase producing bacterium with the alkylating agent monobromobimane (mBBr), causing the cellular mycothiol to be converted to its bimane derivative (MSmB) : The latter compound is rapidly cleaved to produce GIcN-Ins and the bimane derivative of N- acetylcysteine (AcCySmB), a mercapturic acid that was rapidly exported from the cells into the medium. The other product of cleavage, GlcN-Ins, is retained in the cell and utilized in the resynthesis of mycothiol.

[0028] A similar in vitro assay utilizing acyl glucosaminyl inositol amidase obtained by purification from a mycothiol-producing bacterium, such as M. smegmatis or M. tuberculosis is readily employed in accordance with the disclosure herein to screen compounds to identify and characterize additional inhibitors that are effective within the compositions and methods of the invention to inhibit amidase activity of the amidases. In an exemplary assay, mycothiol is alkylated with mBBr to produce the stable, fluorescent derivative MSmB, which can be quantified by HPLC using known methods. Mycothiol is easily isolated from M. smegmatis and can be converted quantitatively to MSmB in minutes for use in this assay. Alternatively, recombinant M. tuberculosis MCA can be over-expressed in a host cell, such as E. coli, for use as the enzyme. Exposure of a pure sample of MSmB to an acyl glucosaminyl inositol amidase results in the production of a substantial amount of the breakdown product AcCySmB, indicating that MSmB is cleaved by the acyl glucosaminyl inositol amidase. When the pure sample of MsmB and acyl glucosaminyl inositol amidase is exposed to a cell free extract of an organism that produces an invention naturally occurring inhibitor of the acyl glucosaminyl inositol amidase acyl glucosaminyl inositol amidase the substantial absence of, or reduction in, the amount of the fluorescent mycothiol derivative obtained due to activity of the amidase indicates that the organism from which the extract was prepared naturally produces an inhibitor of acyl glucosaminyl inositol amidase. Samples of the incubation mixture can also be analyzed at intervals for production of the other potential product of MSmB cleavage, GlcN-Ins, as well as for MSmB and AcCySmB. In the absence of an inhibitor, at >50% conversion approximately 1.0 equivalent of MSmB (0.1 nmol) yields 1.00 0.02 equivalent of AcCySmB and 0.80 0. 08 equivalent of GlcN-Ins with the reaction proceeding to 97% conversion of MSmB in 60 minutes at 23°C. Any substantial reduction in this conversion rate indicates inhibitory action of a naturally occurring compound produced by the test organism and present in the test extract.

[0029] Detection of the fluorescent breakdown product AcCySmB is readily made by any method known in the art for detection of fluorescence. A fluorescence detected-HPLC assay for this breakdown product of MsmB is well known (G. L. Newton et al., 2000a, supra) (See also U. S. Patent Application Publication No. 20020045739, which is incorporated herein by reference in its entirety). For acyl glucosaminyl inositol amides which do not contain cysteine a fluorescence detected-HPLC assay for the breakdown product GlcN-Ins is well known (G. L. Newton, et al., 2000b supra).

[0030] mBBr is also known to penetrate cells rapidly and to convert intracellular thiols (e. g. , mycothiol) to their bimane derivatives (Newton, et al. (1995) supra). Thus the test for inhibitory action of a naturally-occurring compound obtained from a cell extract can also be conducted in an in vivo assay to determine whether a test compound is effective as an inhibitor of acyl glucosaminyl inositol amidase activity in vivo by exposing a cultured strain of live mycothiol-producing bacterial cells to a purified test compound and assaying the inhibitory effect of the purified test inhibitor upon the mycothiol production pathway in the mycothiol-producing bacterium. An interruption in the production of mycothiol in the test bacteria causes inhibition of growth and even death. Generally, it is most convenient to conduct such an in vivo assay by culturing the mycothiol-containing bacteria in the presence of the test compound and an antibiotic. Disruption in the mycothiol-producing pathway and in the mycothiol-dependent detoxification pathway in the bacterium caused by inhibitory activity of the test compound on the amidases in the bacterium will interrupt the natural resistance to the antibiotic and other alkylating agents, and the like, provided by mycothiol in the bacterium. Antibiotics useful for this purpose include such antibiotics as cerulenin, erythromycin, exfoliamycin, granaticin, kinamycin, lincomycin, mitomycin, naphthomycins, rifamycins, streptothricins, and vancomycin group antibiotics.

[0031] In accordance with the present invention, there are provided compounds with inhibitory activity against an acyl glucosaminyl inositol amidase, which amidase is produced naturally in actinomycetes and has enzymatic amidase activity for a glucosaminyl inositol (GlcN-Ins)-containing substrate.

[0032] In one embodiment of the invention, the inhibitors of acyl glucosaminyl inositol amidase, preferably mycothiol S-conjugate amidase, are alkaloid compounds of the formula: or a prodrug, pharmaceutically acceptable salt, hydrate, solvate, acid salt hydrate, N- oxide or isomorphic crystalline form thereof; wherein Rl is H or halogen selected from F, Cl, Br, and I ; R is H or OH; R3 is H or halogen selected from F, Cl, Br, and I; R4 is H or CH3 ; R5 is H, F, NH2 ; and R6 is H or CH3 ; provided that when R'is Cl, Br, or I, then R2 is not OH; and provided that when RI is F, then R3 is not F.

[0033] Exemplary compounds include an alkaloid compound, wherein Rl is Br; Ruz is H; R3 is H; R4 is H; Rs is F; and R6 is H. Other exemplary compounds include an alkaloid compound, wherein Rl is Br; Ruz ils H; R3 is H; R4 is CH3 ; R5 is H; and R6 is CH3. Yet additional exemplary compounds include an alkaloid compound, wherein Rl is Br; W is H; R3 is H; R4 is H; RS is NH2 ; and R6 is H.

[0034] In another embodiment, the invention is directed to a therapeutic or prophylactic drug composition for the treatment of an infection, disease, condition, or one or more symptom (s), caused by actinomycetes in a mammalian subject. The composition comprises as an active ingredient a therapeutically effective amount of at least one compound having the formula: or a prodrug, pharmaceutically acceptable salt, hydrate, solvate, acid salt hydrate, N- oxide or isomorphic crystalline form thereof ; wherein Rl is H or halogen selected from F, Cl, Br, and I ; R is H or OH; R3 is H or halogen selected from F, Cl, Br, and I ; R4 is H or CH3 ; Rs is H, F, NH2; and R6 is H or CH3 ; provided that when Rl is Cl, Br, or I, then Ra is not OH; and provided that when Rl is F, then R3 is not F. [0035] In another embodiment, the invention is directed to a method of inhibiting the activity of an acyl glucosaminyl inositol amidase, for example a mycothiol S-conjugate amidase, comprising the step of : administering an effective amount of at least one compound having the formula: or a prodrug, pharmaceutically acceptable salt, hydrate, solvate, acid salt hydrate, N- oxide or isomorphic crystalline form thereof; wherein Rl is H or halogen selected from F, Cl, Br, and I ; R2 is H or OH ; R3 is H or halogen selected from F, Cl, Br, and I; R4 is H or CH3 ; Rs is H, F, NH2 ; and R6 is H or CH3; provided that when Rl is Cl, Br, or I, then R2 is not OH; and provided that when Ri is F, then R3 is not F.

[0036] In yet another embodiment, the invention is directed to a method of treating or preventing an infection, disease, condition, or one or more symptom (s) caused by an actinomycete in a mammalian subject, comprising the step of : administering to said subject a therapeutically effective, non-toxic dose of at least one compound having the formula: or a prodrug, pharmaceutically acceptable salt, hydrate, solvate, acid salt hydrate, N- oxide or isomorphic crystalline form thereof; wherein Rl is H or halogen selected from F, Cl, Br, and I; R2 is H or OH; R3 is H or halogen selected from F, Cl, Br, and I ; R4 is H or CH3 ; is H, F, NH2 ; and R6 is H or CH3; provided that when Rl is Cl, Br, or I, then R2 is not OH; and provided that when Rl is F, then R3 is not F.

[0037] In another embodiment, the invention is directed to a method for decreasing the antibiotic-resistance of pathogenic acyl glucosaminyl inositol amidase-producing bacteria, preferably mycothiol S-conjugate amidase-producing bacteria, comprising the step of : exposing said bacteria to an effective amount of at least one compound having the formula: or a prodrug, pharmaceutically acceptable salt, hydrate, solvate, acid salt hydrate, N- oxide or isomorphic crystalline form thereof; wherein Rl is H or halogen selected from F, Cl, Br, and I ; R2 is H or OH ; R3 is H or halogen selected from F, Cl, Br, and I; R4 is H or CH3 ; Rs is H, F, NH2 ; and R6 is H or CH3 ; provided that when Rl is Cl, Br, or I, then R2 is not OH; and provided that when Rl is F, then R3 is not F.

[0038] In another embodiment, the invention is directed to a method for reducing the virulence in a mammalian subject of a pathogenic acyl glucosaminyl inositol amidase- producing bacteria, preferably a mycothiol S-conjugate amidase-producing bacteria, comprising the step of : administering to said subject an effective amount of at least one compound having the formula : or a prodrug, pharmaceutically acceptable salt, hydrate, solvate, acid salt hydrate, N- oxide or isomorphic crystalline form thereof; wherein Rl is H or halogen selected from F, Cl, Br, and I; R2 is H or OH ; is H or halogen selected from F, Cl, Br, and I ; is H or CH3 ; R5 is H, F, NH2 ; and R6 is H or CH3 ; provided that when Rl is Cl, Br, or I, then R2 is not OH; and provided that when Rl is F, then R3 is not F.

[0039] In another embodiment, the invention is directed to a method of treating an infection, disease, condition, or one or more symptom (s) caused by an actinomycete in a mammalian subject, comprising the steps of : administering to said subject a non-toxic dose of at least one first line antibiotic agent; and administering to said subject a non-toxic dose of at least one compound having the formula: or a prodrug, pharmaceutically acceptable salt, hydrate, solvate, acid salt hydrate, N- oxide or isomorphic crystalline form thereof; wherein Rl is H or halogen selected from F, Cl, Br, and I; R2 is H or OH; R3 is H or halogen selected from F, Cl, Br, and I ; R4isHorCH3 ; Rs is H, F, NH2; and R6 is H or CH3 ; provided that when Rl is Cl, Br, or I, then R2 is not OH; and provided that when Rl is F, then R3 is not F.

[0040] Suitable first-line antibiotic agents include cerulenin, erythromycin, exfoliamycin, granaticin, kinamycin, lincomycin, mitomycin, naphthomycins, rifamycins, streptothricins, and vancomycin group antibiotics. In one exemplary embodiment, the first-line antibiotic agent is isoniazid, rifampin or a combination thereof.

[0041] The compounds, compositions and methods of the invention are useful in treating infections, diseases, conditions, or one or more symptom (s) caused by any an actinomycete, for example mycobacteria and streptomyces, such as M. tuberculosis, M. smegmatis, M. avium, M. leprae, M. bovis BCG, M. intracellulare, M. africanum, M. marinarum, M. chelon.ai, Corynebacterium diphtheria, Actinomycetes israelii and Staphylococcus aureus.

[0042] When administered in treatment of a disease, condition or symptom associated with infection of a mammalian subject with a pathogenic bacteria that produces a native acyl glucosaminyl inositol amidase in conjunction with an antibiotic (i. e. , in combination therapy) the inhibitors of the invention increase the therapeutic effect of the antibiotic, for example by decreasing both virulence and antibiotic resistance of the pathogen.

[0043] In certain methods of the invention the virulence in a mammalian subject of pathogenic acyl glucosaminyl inositol amidase-producing bacteria is reduced by administering to the subject an effective amount of an inhibitor as disclosed herein, wherein the inhibitor has inhibitory activity against acyl glucosaminyl inositol amidase for acyl glucosaminyl inositol amides, wherein administration of the inhibitor to the subject reduces the growth rate of the bacteria in the subject, thereby reducing the virulence of the bacteria in the subject as compared to an untreated subject.

[0044] In yet another embodiment according to the present invention, there are provided methods for decreasing the antibiotic-resistance of pathogenic acyl glucosaminyl inositol amidase-producing bacteria by introducing into the bacteria an inhibitor of acyl glucosaminyl inositol amidase activity. The intracellular presence of the inhibitor decreases activity of the amidase, thereby decreasing the mycothiol content and decreasing the antibiotic-resistance of the bacteria as compared with untreated control bacteria.

[0045] Preferably, the inhibitor of acyl glucosaminyl inositol amidase is a compound that inhibits intracellular production of mycothiol. For example, in one embodiment the candidate compound inhibits intracellular activity of the acyl glucosaminyl inositol amidase.

[0046] In certain methods of the invention the antibiotic-resistance of pathogenic GlcN-Ins- amidase producing bacteria is reduced by introducing into the bacteria an inhibitor of acyl glucosaminyl inositol amidase activity. The intracellular presence of the invention inhibitor decreases activity of the amidase, thereby decreasing the antibiotic-resistance of the bacteria, as compared with untreated control bacteria.

[0047] Preferably, the inhibitor inhibits intracellular production of the amidase. The bacteria may be"exposed to"the compounds of the invention by incubating the bacteria in the presence of the compounds of the invention, by administering the compounds of the invention in an effective amount or concentration to a subject exposed to or infected with the bacteria, such that the bacteria comes in contact with the inhibitor in vivo, or by any means that exposes the bacteria to an effective amount of the inhibitor to substantially impair intracellular production of the amidase. The compounds of the invention are typically specific inhibitors of acyl glucosaminyl inositol amidase and, in particular, mycothiol-S- conjugate amidase. The compounds of the invention may be used as general antibiotics against any actinomycete, including Gram-positive bacteria, such as drug resistant Staphylococcus aureus, and against mycobacteria. The compounds of the invention may additionally be used as co-drugs administered with first-line antituberculars. Because many antibiotic-producing bacteria make mycothiol and use it for detoxification from their own antibiotics in conjunction with mycothiol-S-conjugate amidase, the compounds of the invention may be used to reduce detoxification and improve the yield of antibiotics in industrially-useful antibiotic-producing strains.

[0048] The amidase used to test the inhibitory effect of a putative inhibitor of the invention can be obtained in a number of ways known in the art. For example, as described above, the acyl glucosaminyl inositol amidase can be purified from bacteria that naturally produce such an enzyme, for example a mycothiol S-conjugate amidase, e. g. one capable of hydrolyzing a mycothiol S-conjugate where the S-R group may be an alkyl or alkyloid group to which it is native. Alternatively, the acyl glucosaminyl inositol amidase can be recombinantly produced from genes that encode a naturally occurring protein using methods known in the art.

Alternatively, the mycothiol S-conjugate amidase or acyl glucosaminyl inositol amidase used in the screening methods of the invention may contain structural variations, for example conservative variation of one or more amino acids, compared to the naturally occurring amidase. A"conservative variation"in an amino acid sequence is a sequence that differs from a reference sequence by one or more conservative or non-conservative amino acid substitutions, deletions, or insertions, particularly when such a substitution occurs at a site that is not the active site of the molecule, and provided that the polypeptide essentially retains its functional properties. A conservative amino acid substitution, for example, substitutes one amino acid for another of the same class (e. g., substitution of one hydrophobic amino acid, such as isoleucine, valine, leucine, or methionine, for another, or substitution of one polar amino acid for another, such as substitution of arginine for lysine, glutamic acid for aspartic acid or glutamine for asparagine). One or more amino acids can be deleted, for example, from an amidase polypeptide, resulting in modification of the structure of the polypeptide, without significantly altering its biological activity. For example, carboxyl-terminal amino acids that are not required for amidase activity can be removed.

[0049] Biologically functional viral and plasmid DNA vectors capable of expression and replication in a host useful for recombinant production of the acyl glucosaminyl inositol amidase used to screen for inhibitors of the invention are known in the art. Such vectors are used to incorporate DNA sequences of the invention. In general, expression vectors containing promoter sequences that facilitate the efficient transcription of the inserted prokaryotic genetic sequence are used in connection with the host. The expression vector typically contains an origin of replication, a promoter, and a terminator, as well as specific genes that are capable of providing phenotypic selection of the transformed cells.

[0050] In addition to expression vectors known in the art such as bacterial, yeast and mammalian expression systems, baculovirus vectors may also be used. One advantage to expression of foreign genes in this invertebrate virus expression vector is that it is capable of expression of high levels of recombinant proteins, which are antigenically and functionally similar to their natural counterparts. Baculovirus vectors and the appropriate insect host cells used in conjunction with the vectors will be known to those skilled in the art.

[0051] The term"recombinant expression vector"refers to a plasmid, virus or other vehicle known in the art that has been manipulated by insertion or incorporation of the invention acyl glucosaminyl inositol amidase genetic sequences. Such expression vectors contain a promoter sequence that facilitates the efficient transcription of the inserted genetic sequence of the host. The expression vector typically contains an origin of replication, a promoter, as well as specific genes that allow phenotypic selection of the transformed cells. Vectors suitable for use in the present invention include, but are not limited to the T7-based expression vector for expression in bacteria (Rosenberg, et al., Gene, 56: 125,1987), the pMSXND expression vector for expression in mammalian cells (Lee and Nathans, J. Biol.

Chem., 263: 3521, 1988) and baculovirus-derived vectors for expression in insect cells. The DNA segment can be present in the vector operably linked to regulatory elements, for example, a promoter (e. g., T7, metallothionein I, or polyhedrin promoters).

[0052] The vector may include a phenotypically selectable marker to identify host cells that contain the expression vector. Examples of markers typically used in prokaryotic expression vectors include antibiotic resistance genes for ampicillin (lactamases), tetracycline and chloramphenicol (chloramphenicol acetyltransferase). Examples of such markers typically used in mammalian expression vectors include the gene for adenosine deaminase (ADA), aminoglycoside phosphotransferase (neo, G418), dihydrofolate reductase (DHFR), hygromycin-B-phosphotransferase (HPH), thymidine kinase (TK), and xanthine guanine phosphoribosyltransferse (XGPRT, gpt).

[0053] The isolation and purification of host cell expressed amidase polypeptides may be by any conventional means such as, for example, preparative chromatographic separations and immunological separations such as those involving the use of a monoclonal or polyclonal antibody.

[0054] Transformation of the host cell with the recombinant DNA may be carried out by conventional techniques well known to those skilled in the art. Where the host is prokaryotic, such as E. coli, competent cells which are capable of DNA uptake can be prepared from cells harvested after exponential growth and subsequently treated by electroporation or the CaCl2 method using procedures well known in the art. Alternatively, MgCl2 or RbCl could be used.

[0055] Where the host used is a eukaryote, various methods of DNA transfer can be used.

These include transfection of DNA by calcium phosphate-precipitates, conventional mechanical procedures such as microinjection, insertion of a plasmid encased in liposomes, or the use of virus vectors. Eukaryotic cells can also be cotransformed with DNA sequences encoding the polypeptides of the invention, and a second foreign DNA molecule encoding a selectable phenotype, such as the herpes simplex thymidine kinase gene. Another mothod is to use a eukaryotic viral vector, such as simian virus 40 (SV40) or bovine papilloma virus, to transiently infect or transform eukaryotic cells and express the protein. (Eukaryotic Viral Vectors, Cold Spring Harbor Laboratory, Gluzman ed., 1982). Examples of mammalian host cells include COS, BHK, 293, and CHO cells.

[0056] Eukaryotic host cells may also include yeast. For example, DNA can be expressed in yeast by inserting the DNA into appropriate expression vectors and introducing the product into the host cells. Various shuttle vectors for the expression of foreign genes in yeast have been reported (Heinemann, J. et al., Nature, 340: 205,1989 ; Rose, M. et al., Gene, 60: 237, 1987).

[0057] The recombinant acyl glucosaminyl inositol amidase polypeptide used to screen naturally occurring inhibitors of acyl glucosaminyl inositol amidase for inhibitory activity can also be a fusion protein further comprising a second polypeptide portion having an amino acid sequence from a protein unrelated to the acyl glucosaminyl inositol amidase. Such fusion proteins can be useful, for example, in a two-hybrid assay as is known in the art.

[0058] The practice of certain detailed aspects of the present invention will employ, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art.

Such techniques are explained fully in the literature. See, for example, Molecular Cloning A Laboratory Manual, 2nd Ed. , ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: 1989); DNA Cloning, Volumes I and II (D. N. Glover ed. , 1985); Oligonucleotide Synthesis (M. J. Gait ed., 1984) ; Mullis et al. U. S. Pat. No. 4,683, 195; Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins eds. 1984); Transcription And Translation (B. D. Hames & S. J. Higgins eds. 1984); Culture Of Animal Cells (R. I.

Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); B.

Perbal, A Practical Guide To Molecular Cloning (1984); the treatise, Methods In Enzymology (Academic Press, Inc., N. Y. ) ; Gene Transfer Vectors For Mammalian Cells (J.

H. Miller and M. P. Calos eds., 1987, Cold Spring Harbor Laboratory); Methods In Enzymology, Vols. 154 and 155 (Wu et al. eds. ), Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (D. M. Weir and C. C. Blackwell, eds. , 1986); Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y. , 1986).

[0059] As used herein, the term"nucleic acid"refers to polynucleotides such as deoxyribonucleic acid (DNA), and, where appropriate, ribonucleic acid (RNA). The term should also be understood to include, as equivalents, analogs of either RNA or DNA made from nucleotide analogs, and, as applicable to the embodiment being described, single (sense or antisense) and double-stranded polynucleotides.

[0060] As used herein, the terms"gene,""recombinant gene"and"gene construct"refer to a nucleic acid comprising an open reading frame encoding an invention acyl glucosaminyl inositol amidase, including both exon and (optionally) intron sequences. The term"intron" refers to a DNA sequence present in a given acyl glucosaminyl inositol amidase gene which is not translated into protein and is generally found between exons.

[0061] "Homology"refers to sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing a position in each sequence that may be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same base or amino acid, then the molecules are homologous at that position. A degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences.

[0062] The term"transfection"or"transforming"and grammatical equivalents thereof, refers to the introduction of a nucleic acid, e. g., an expression vector, into a recipient cell by nucleic acid-mediated gene transfer."Transformation", as used herein, refers to a process in which a cell's genotype is changed as a result of the cellular uptake of exogenous DNA or RNA.

[0063]"Cells"or"cell cultures"or"recombinant host cells"or"host cells"are often used interchangeably as will be clear from the context. These terms include the immediate subject cell that expresses the cell-cycle regulatory protein of the present invention, and, of course, the progeny thereof. It is understood that not all progeny are exactly identical to the parental cell, due to chance mutations or difference in environment. However, such altered progeny are included in these terms, so long as the progeny retain the characteristics relevant to those conferred on the originally transformed cell. In the present case, such a characteristic might be the ability to produce a recombinant acyl glucosaminyl inositol amidase polypeptide.

[0064] As used herein, the term"vector"refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. The term"expression vector" includes plasmids, cosmids or phages capable of synthesizing the subject acyl glucosaminyl inositol amidase polypeptide encoded by the respective recombinant gene carried by the vector. Preferred vectors are those capable of autonomous replication and/expression of nucleic acids to which they are linked. In the present specification,"plasmid"and"vector" are used interchangeably as the plasmid is the most commonly used form of vector.

Moreover, the invention is intended to include such other forms of expression vectors which serve equivalent functions and which become known in the art subsequently hereto.

[0065]"Transcriptional regulatory sequence"is a generic term used throughout the specification to refer to DNA sequences, such as initiation signals, enhancers, and promoters, as well as polyadenylation sites, which induce or control transcription of protein coding sequences with which they are operably linked. In preferred embodiments, transcription of a recombinant acyl glucosaminyl inositol amidase gene is under the control of a promoter sequence (or other transcriptional regulatory sequence) that controls the expression of the recombinant gene in a cell-type in which expression is intended. It will also be understood that the recombinant gene can be under the control of transcriptional regulatory sequences which are the same or which are different from those sequences that control transcription of the naturally-occurring form of the regulatory protein.

[0066] As used herein, a"transgenic organism"is any organism, preferably a bacterium, in which one or more of the cells of the organism contain heterologous nucleic acid introduced by way of human intervention, such as by transgenic techniques well known in the art. The nucleic acid is introduced into the cell by way of deliberate genetic manipulation, such as by microinjection or by infection with a recombinant virus or a vector. The term genetic manipulation does not include classical crossbreeding, or in vitro fertilization, but rather is directed to the introduction of a recombinant DNA molecule. This molecule may be integrated within a chromosome, or it may be extrachromosomally replicating DNA. In the typical transgenic organisms described herein, the transgene causes cells to express a recombinant form of the subject acyl glucosaminyl inositol amidase polypeptides.

[0067] The term"isolated"or"purified"as also used herein with respect to invention inhibitors of acyl glucosaminyl inositol amidase activity refers to compounds that are substantially free of cellular material or culture medium when purified from cell extracts of naturally occurring organisms or other chemicals when chemically synthesized. Methods of purifying compounds from cell extracts are well known in the art and are further illustrated in the Examples herein.

[0068] Isolated nucleic acids which differ from the wild type nucleotide sequences known in the art due to degeneracy in the genetic code can also be used to produce acyl glucosaminyl inositol amidases useful in discovering invention inhibitors. For example, a number of amino acids is designated by more than one triplet. Codons that specify the same amino acid, or synonyms (for example, CAU and CAC are synonyms for histidine) may result in"silent"mutations that do not affect the amino acid sequence of the protein.

However, it is expected that DNA sequence polymorphisms that do lead to changes in the amino acid sequences of the subject acyl glucosaminyl inositol amidase polypeptides will exist among prokaryotic cells. One skilled in the art will appreciate that these variations in one or more nucleotides (up to about 3-4% of the nucleotides) of the nucleic acids encoding a particular member of the acyl glucosaminyl inositol amidase polypeptide family may exist among individuals of a given species due to natural allelic variation. Any and all such nucleotide variations and resulting amino acid polymorphisms are within the scope of this invention.

[0069] Fragments of the nucleic acid encoding a biologically active portion of the subject acyl glucosaminyl inositol amidase polypeptides can also be used in assays to screen for discovery of additional examples of the invention inhibitors of acyl glucosaminyl inositol amidase. Such fragments encode a peptide that retains at least a portion of the biological activity of the full-length protein (i. e. , a peptide capable of acyl glucosaminyl inositol amidase activity) as defined herein.

[0070] A nucleic acid encoding a peptide having an activity of a S-conjugate amidase polypeptide may be obtained from mRNA or genomic DNA present in any of a number of antibiotic-producing or pathogenic bacteria described herein, particularly actinomycetes, in accordance with protocols generally known to those skilled in the art. A cDNA encoding an acyl glucosaminyl inositol amidase polypeptide, for example, can be obtained by isolating total mRNA from a bacterial cell. Double stranded cDNAs can then be prepared from the total mRNA, and subsequently inserted into a suitable plasmid or bacteriophage vector using any one of a number of known techniques. A gene encoding an acyl glucosaminyl inositol amidase polypeptide can also be cloned using established polymerase chain reaction techniques in accordance with the nucleotide sequence information provided by the invention.

[0071] Expression vectors may comprise a nucleotide sequence encoding an acyl glucosaminyl inositol amidase polypeptide operably linked to at least one regulatory sequence. Operably linked is intended to mean that the nucleotide sequence is linked to a regulatory sequence in a manner that allows expression of the nucleotide sequence. Regulatory sequences are art-recognized and are selected to direct expression of the peptide having an activity of an acyl glucosaminyl inositol amidase polypeptide. Accordingly, the term regulatory sequence includes promoters, enhancers and other expression control elements. Exemplary regulatory sequences are described in Goeddel; Gene Expression Technology : Methods in Enzymology 185, Academic Press, San Diego, CA (1990). For instance, any of a wide variety of expression control sequences-sequences that control the expression of a DNA sequence when operatively linked to it may be used in these vectors to express DNA sequences encoding the acyl glucosaminyl inositol amidase polypeptides of this invention. Such useful expression control sequences, include, for example, the early and late promoters of SV40, adenovirus or cytomegalovirus immediate early promoter, the lac system, the trp system, the TAC or TRC system, T7 promoter whose expression is directed by T7 RNA polymerase, the major operator and promoter regions of phage lambda, the control regions for fd coat protein, the promoter for 3-phosphoglycerate kinase or other glycolytic enzymes, the promoters of acid phosphatase, e. g., Pho5, the promoters of the yeast. alpha. - mating factors, the polyhedron promoter of the baculovirus system and other sequences known to control the expression of genes of prokaryotic or eukaryotic cells or their viruses, and various combinations thereof. It should be understood that the design of the expression vector may depend on such factors as the choice of the host cell to be transformed and/or the type of protein desired to be expressed. Moreover, the vector's copy number, the ability to control that copy number and the expression of any other proteins encoded by the vector, such as antibiotic markers, should also be considered.

[0072] As will be apparent, such gene constructs can be used to cause expression of the subject acyl glucosaminyl inositol amidase polypeptides in cells propagated in culture, e. g. to produce proteins or peptides, including fusion proteins or peptides, for purification and subsequent use in assays intended to screen for inhibitors of their amidase activity.

[0073] The acyl glucosaminyl inositol amidase polypeptides used to screen for detection of inhibitors of acyl glucosaminyl inositol amidase activity may also be produced by transfection of a host cell with expression vector encoding one of the subject acyl glucosaminyl inositol amidase polypeptide and cultured under appropriate conditions to allow expression of the peptide to occur. The peptide may be secreted and isolated from a mixture of cells and medium containing the peptide. Alternatively, the peptide may be retained cytoplasmically and the cells harvested, lysed and the protein isolated. A cell culture includes host cells, media and other byproducts. Suitable media for cell culture are well known in the art. The peptide can be isolated from cell culture medium, host cells, or both using techniques known in the art for purifying proteins including ion-exchange chromatography, gel filtration chromatography, ultrafiltration, electrophoresis, and immunoaffinity purification with antibodies specific for particular epitopes of the subject acyl glucosaminyl inositol amidase polypeptides.

[0074] As used herein, the term"therapeutically effective amount"is meant to refer to an amount of compound of the present invention that will elicit a desired therapeutic or prophylactic effect or response when administered according to the desired treatment regimen.

[0075] The compounds described herein are preferably administered to mammals in a therapeutically effective amount. As used herein, the term"therapeutically effective amount" is meant to refer to an amount of compound of the present invention that will elicit a desired therapeutic or prophylactic effect or response when administered according to the desired treatment regimen. Dosage may vary depending on the compound, the potency of the compound, the type of disease, and the diseased state of the patient, among other variables.

Dosage amount can be measured by administration of pre-measured dosing means or unit dosages in the form of tablets, capsules, suppositories, powders, emulsions, elixirs, syrups, ointments, creams, or solutions.

[0076] The compounds are preferably combined with a pharmaceutical carrier selected on the basis of the chosen route of administration and standard pharmaceutical practice as described, for example, in Remington's Pharmaceutical Sciences (Mack Publishing Co., Easton, PA, 1980), the disclosures of which are hereby incorporated herein by reference, in their entirety.

[0077] In therapeutic or prophylactic use, the compounds of the invention may be administered by any route that drugs are conventionally administered. Such routes of administration include intraperitoneal, intravenous, intramuscular, subcutaneous, intrathecal, intracheal, intraventricular, oral, buccal, rectal, parenteral, intranasal, intrapulmonary, transdermal or intradermal. Administration may be systemic or localized.

[0078] Compounds described herein may be administered in pure form, combined with other active ingredients, or combined with pharmaceutically acceptable nontoxic excipients or carriers. Oral compositions will generally include an inert diluent carrier or an edible carrier. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. Tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a dispersing agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.

When the dosage unit form is a capsule, it can contain, in addition to material of the above type, a liquid carrier such as a fatty oil. In addition, dosage unit forms can contain various other materials that modify the physical form of the dosage unit, for example, coatings of sugar, shellac, or enteric agents. Further, a syrup may contain, in addition to the active compounds, sucrose as a sweetening agent and certain preservatives, dyes, colorings, and flavorings.

[0079] Alternative preparations for administration include sterile aqueous or nonaqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are dimethylsulfoxide, alcohols, propylene glycol, polyethylene glycol, vegetable oils such as olive oil and injectable organic esters such as ethyl oleate. Aqueous carriers include mixtures of alcohols and water, buffered media, and saline. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers, such as those based on Ringer's dextrose, and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, inert gases, and the like. Preferred methods of administration of the present compounds to mammals include intraperitoneal injection, intramuscular injection, and intravenous infusion. Various liquid formulations are possible for these delivery methods, including saline, alcohol, DMSO, and water based solutions. The concentration of inhibitor may vary according to dose and volume to be delivered and can range from about 1 to about 1000 mg/mL. Other constituents of the liquid formulations can include, preservatives, inorganic salts, acids, bases, buffers, nutrients, vitamins, or other pharmaceuticals such as analgesics.

[0080] Compounds of the present invention also may take the form of any pharmacologically acceptable salt or derivative (for example, prodrug form or conjugate form) that retains inhibitory activity, including for example, isomorphic crystalline forms, N- oxide, hydrates, solvates, acid salt hydrates, metabolites and the like. For example, many amino-containing compounds can be used or prepared as an acid addition salt. Often such salts improve isolation and handling properties of the compound. For example, depending on the reagents, reaction conditions and the like, compounds as described herein can be used or prepared, for example, as their hydrochloride or tessellate salts. Isomorphic crystalline forms, N-oxide, hydrates, solvates, and acid salt hydrates, are also contemplated to be within the scope of the present invention.

[0081] Certain acidic or basic compounds of the present invention may exist as zwitterions.

All forms of the compounds, including free acid, free base and zwitterions, are contemplated to be within the scope of the present invention. It is well known in the art that compounds containing both amino and carboxyl groups often exist in equilibrium with their zwitterionic forms. Thus, any of the compounds described herein throughout that contain, for example, both amino and carboxyl groups, also include reference to their corresponding zwitterions.

[0082] As used herein,"pharmaceutically acceptable"refers to those compounds, materials, compositions, and/or dosage forms that are, within the scope of sound medical judgment, suitable for contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem complications commensurate with a reasonable benefit/risk ratio.

[0083] As used herein, "in combination with,""combination therapy"and"combination products"refer, in certain embodiments, to the concurrent administration to a patient of therapeutic agents (first-line antibiotics, for example) and the compounds of the invention.

When administered in combination, each component may be administered at the same time or sequentially in any order at different points in time. Thus, each component may be administered separately but sufficiently closely in time so as to provide the desired therapeutic effect.

[0084] As used herein, "dosage unit"refers to physically discrete units suited as unitary dosages for the particular individual to be treated. Each unit may contain a predetermined quantity of active compound (s) calculated to produce the desired therapeutic effect (s) in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention may be dictated by (a) the unique characteristics of the active compound (s) and the particular therapeutic effect (s) to be achieved, and (b) the limitations inherent in the art of compounding such active compound (s).

[0085] As used herein,"pharmaceutically acceptable salts"refer to derivatives of the disclosed compounds wherein the parent compound is modified by making acid or base salts thereof. Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids; and the like. The pharmaceutically acceptable salts include the conventional non-toxic salts or the quaternary ammonium salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. For example, such conventional non-toxic salts include those derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric and the like; and the salts prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, sulfanilic, 2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isethionic, and the like. These physiologically acceptable salts are prepared by methods known in the art, e. g., by dissolving the free amine bases with an excess of the acid in aqueous alcohol, or neutralizing a free carboxylic acid with an alkali metal base such as a hydroxide, or with an amine.

[0086] As used herein,"patient"refers to animals, including mammals, preferably humans.

[0087] As used herein, "stereoisomers"refers to compounds that have identical chemical constitution, but differ as regards the arrangement of the atoms or groups in space.

[0088] As used herein, "N-oxide"refers to compounds wherein the basic nitrogen atom of either a heteroaromatic ring or tertiary amine is oxidized to give a quaternary nitrogen bearing a positive formal charge and an attached oxygen atom bearing a negative formal charge.

[0089] When any variable occurs more than one time in any constituent or in any formula, its definition in each occurrence is independent of its definition at every other occurrence.

Combinations of substituents and/or variables are permissible only if such combinations result in stable compounds.

[0090] It is believed the chemical formulas and names used herein correctly and accurately reflect the underlying chemical compounds. However, the nature and value of the present invention does not depend upon the theoretical correctness of these formulae, in whole or in part. Thus it is understood that the formulas used herein, as well as the chemical names attributed to the correspondingly indicated compounds, are not intended to limit the invention in any way, including restricting it to any specific tautomeric form or to any specific optical; or geometric isomer, except where such stereochemistry is clearly defined.

[0091] In any of the above teachings, a compound of the invention may be either a compound of one of the formulae herein described, or a prodrug, pharmaceutically acceptable salt, hydrate, solvate, acid salt hydrate, N-oxide or isomorphic crystalline form thereof.

[0092] The compounds employed in the methods of the present invention may exist in prodrug form. As used herein,"prodrug"is intended to include any covalently bonded carriers which release the active parent drug, for example, as according to compounds of the invention compounds employed in the methods of the present invention in vivo when such prodrug is administered to a mammalian subject. Since prodrugs are known to enhance numerous desirable qualities of pharmaceuticals (e. g. , solubility, bioavailability, manufacturing, etc. ) the compounds employed in the present methods may, if desired, be delivered in prodrug form. Thus, the present invention contemplates methods of delivering prodrugs. Prodrugs of the compounds employed in the present invention may be prepared by modifying functional groups present in the compound in such a way that the modifications are cleaved, either in routine manipulation or ifz vivo, to the parent compound.

[0093] Accordingly, prodrugs include, for example, compounds described herein in which a hydroxy, amino, or carboxy group is bonded to any group that, when the prodrug is administered to a mammalian subject, cleaves to form a free hydroxyl, free amino, or carboxylic acid, respectively. Examples include, but are not limited to, acetate, formate and benzoate derivatives of alcohol and amine functional groups; and alkyl, carbocyclic, aryl, and alkylaryl esters such as methyl, ethyl, propyl, iso-propyl, butyl, isobutyl, sec-butyl, tert-butyl, cyclopropyl, phenyl, benzyl, and phenethyl esters, and the like.

[0094] The compounds employed in the methods of the present invention may be prepared in a number of ways well known to those skilled in the art. The compounds can be synthesized, for example, by the methods described below, or variations thereon as appreciated by the skilled artisan. All processes disclosed in association with the present invention are contemplated to be practiced on any scale, including milligram, gram, multigram, kilogram, multikilogram or commercial industrial scale.

[0095] The dosage of the compounds of the present invention that will be most suitable for prophylaxis or treatment will vary with the form of administration, the particular compound chosen and the physiological characteristics of the particular patient under treatment.

Generally, small dosages may be used initially and, if necessary, increased by small increments until the desired effect under the circumstances is reached. The therapeutic human dosage, based on physiological studies using rats, may generally range from about 0.01 mg to about 100 mg/kg of body weight per day, and all combinations and subcombinations of ranges therein. Alternatively, the therapeutic human dosage may be from about 0.4 mg to about 10 g or higher, and may be administered in several different dosage units from once to several times a day. Generally speaking, oral administration may require higher dosages.

[0096] The compounds of the invention may also be formulated with other optional active ingredients, in addition to the optional pharmaceutical-acceptable carriers. Other active ingredients include, but are not limited to, antibiotics, antivirals, antifungals, anti- inflammatories, including steroidal and non-steroidal anti-inflammatories, anesthetics, and mixtures thereof.

[0097] The present invention will now be illustrated by reference to the following specific, non-limiting examples. Those skilled in the art of organic synthesis may be aware of still other synthetic routes to the invention compounds. The reagents and intermediates used herein are either commercially available or prepared according to standard literature procedures, unless otherwise specified.

EXAMPLES Synthesis of Inhibitor Compounds of the Invention [0098] The inhibitor compounds of the invention were prepared by known combinatorial chemistry techniques using the asymmetric disulfide exchange method disclosed in Nicolaou, et al., J. Chem. Eur. J. 2001,7, 4280; and Nicolaou, et al., Chem. Eur. J. 2001,7, 4296.

INHIBITOR COMPOUNDS 1,2, 3 OF THE INVENTION Ri R2 R3 R4 R5 R6 1 Br H H H F H 2 Br H H CH3 H CH3 3 Br H H H NH2 H Comparative Inhibitor Compounds [0099] The following comparative inhibitor compounds were evaluated and compared to the inhibitor effect of the compounds of the invention: COMPARATIVE INHIBITOR COMPOUND A COMPARATIVE INHIBITOR COMPOUND B COMPARATIVE INHIBITOR COMPOUND C COMPARATIVE INHIBITOR COMPOUND D COMPARATIVE INHIBITOR COMPOUND E COMPARATIVE INHIBITOR COMPOUND F COMPARATIVE INHIBITOR COMPOUND G COMPARATIVE INHIBITOR COMPOUNDS H, l, J, K Ri R2 R3 R4 R5 R6 H F H F H F H I F H F, H NH2 H J Cl OH H H F H K Cl OH H H NH2 H Table 1 COMPARATIVE INHIBITOR COMPOUND COMMON NAME REFERENCE Nicholas, G. M.; Newton, G. L.; Marine natural product Fahey, R. C.; Bewley, C. A. Org. Letters 2001, 3, 1543 B Benharref, A.; Pais, M. J. Nat. Pseudoceratine Marine natural product Prod. 1996, 59, 177 Roll, D. M.; Chang, C. W. J.; C Scheuer, P. J.; Gray, G. A.; Psammaplysin A Shoolery, J. N.; Matsumoto, g. K.; Marine natural product Van Dyne, G. D.; Clardy, J. C. J. Am. Chem. Soc. 1985, 107, 2916 Roll, D. M.; Chang, C. W. J.; Scheuer, P. J.; Gray, G. A.; Psammaplysin B Shoolery, J.N.,; Matsumoto, g. K.; Marine natural product Van Dyne, G. D.; Clardy, J. C. J. Am. Chem. Soc. 1985, 107, 2916 E Litaudon, M ; Guyot, M. Marine natural product Tetrahedron Lett. 1986,27, 4455 Nicholas, G. M.; Newton, G. L.; Fahey, R. C.; Bewley, C. A. Org. Marine natural product Letters 2001, 3, 1543 (1) Quiñoà, E.; Crews, P. G Psanitnaplin A Tetrahedron Lett. 1987,28, 3229; Marine natural product (b) Aranshahi, L; Schmitz, F. J. Org. Chem. 1987, 52, 3584 Enzymatic Assays [0100] The enzymatic activity of the amidases was assayed by quantitation of the bimane derivative of N-acetylcysteine (AcCySmB) produced from the bimane derivative of mycothiol (MSmB), prepared from purified mycothiol (Newton, G. L. , Bewley, C. A., Dwyer, T. J., Horn, R. , Aharonowitz, Y. Choen, G. , Davies, J. , Faulkner, D. J. , and Fahey, R.

C. (1995) Eur. J. Biochem. 230, 821-825). Separation of the various modified thiols was performed by high-pressure liquid chromatography (HPLC).

[0101] A preparation of the mycothiol S-conjugate amidase (30-50% saturated ammonium sulfate fraction chromatographed on Sephadex G-75) was used to study the stoichiometry of the reaction. Reaction was initiated by mixing a sample (9 pL, 7 pg of total protein) of the preparation of the enzyme with 0.9 mL of 50 mM sodium phosphate, pH 7.5, containing 100 1M MSmB. For determination of bimane derivatives of thiols, a sample (70 pL) of reaction mixture was removed, mixed with 4 1L of 5 M methanesulfonic acid, and analyzed by HPLC without dilution. For analysis of GlcN-Ins, a sample (2-8 uL) of the reaction mixture was mixed with enough 1M HEPES chloride, pH 8, to bring the volume to 10 gL and then with 5 uL of acetonitrile and 5 I1L of 10 mM AccQ-Fluor reagent (6-aminoquinolyl-N- hydroxysuccinimidyl carbamate, Waters). The mixtures were incubated for 1 minute at room temperature followed by 10 minutes at 60°C, diluted with 60 L of water, and quantified by HPLC, as described in Anderberg, S. J. , Newton, G. L. , and Fahey, R. C. (1998) J Biol.

Chem. 273,30391-30397.

[0102] The specificity of the amidase for substrate was assessed by measuring the production of GlcN-Ins in most cases. A sample (5 ZL) of 1 mM substrate was mixed with 40 fiL of 3 mM 2-mercaptoethanol, 25 mM HEPES chloride, pH 7.5. The reaction was initiated with 5, uL of purified amidase (50-fold diluted stock, 4.4 pg mL). Triplicate samples were quenched at 0,10, and 30 minutes by mixing each sample with 50 gL of acetonitrile containing 5 mM NEM and incubating at 60°C for 10 minutes. After the samples were cooled on ice, they were clarified by centrifugation for 15 minutes at 14000 g. A sample (15, uL) of the supernatant was modified with AccQ-Fluor for amine analysis in a total reaction volume of 125 uL, as described in Anderberg, S. J. , Newton, G. L. , and Fahey, R. C. (1998) J. Biol.

Chem. 273,30391-30397.

[0103] Fluorescence-detected enzyme inhibition assays were conducted as described above using recombinant MCA that was obtained from a protein G-MCA construct encoding MCA from M. tuberculosis (Nicholas, G. M.; Kovac: P.; Bewley, C. A. J Am. Chem. Soc. 2002, ASAP Article 10. 1021/jaO17891a S0002-7863 (01) 07891-X) and synthetic mycothiol bimane (MSmB).

[0104] MCA inhibition assays were conducted on comparative compounds and the compounds of the invention in the presence of 20 nM natural M. smegmatis MCA or recombinant M. tuberculosis MCA in the presence of 40 mL of 30 mM mycothiol bimane in the presence of 10 mM Tris. HCl, pH7.4. Substrate concentrations ranged from 1 mM to 1 mM. Varied inhibitor concentrations were selected based on measured ICso values for the relevant inhibitor. Assays were set up in 96 well plates by adding 36 mL of the various stock solutions of MSmB, 2 mL of the relevant stock solutions of inhibitors dissolved in either MeOH or 1: 1 H20 : DMSO, and 2 mL of a 0.4 mM solution of MCA, in that order. Plates were incubated at 31°C for 15 minutes, after which time the reactions were quenched immediately with an equal volume of cold 40 mM methanesulfonic acid (4°C). Control reactions in which 2 mL of the relevant solvent were added to the reaction were run in parallel with each inhibitor and the data were normalized to these controls (see below).

Following centrifugation of the 96-well plates (4000 x g, 4°C, 10 minutes), 40 mL of the quenched reaction mixtures were transferred to an unused 96-well plate which was loaded into a refrigerated 96-well plate autosampler for HPLC detection of the extent of cleavage of the fluorescent substrate MSmB. To determine IC50 values, initial velocities were plotted as a function of inhibitor concentration and the data were fit to the equation: Vi/VC = ICso/([I] + IC50) where vs and VC are the initial velocities in the presence of inhibitor or in the control reactions, respectively, and"ICso value"is the median inhibition concentration (concentration that reduces or inhibits MCA by 50%).

[0105] Double reciprocal plots of 1/v versus 1/S, where v is the initial velocity for each reaction condition and S is the substrate concentration, for each data set were made. Non- linear least squares fitting of the inhibition curves for cleavage of MSmB by MCA yielded the IC50 values shown in Table 2.

Table 2 Inhibitor Compound ICgo (uM) COMPARATIVE A 2. 0~0. 2 Marine natural product COMPARATIVE B 10021 Marine natural product COMPARATIVE C 20~11 Marine natural product COMPARATIVE D 26~12 Marine natural product COMPARATIVE E 36~3 Marine natural product COMPARATIVE F 2. 80. 5 Marine natural product COMPARATIVE G 2. 80. 5 Marine natural product COMPARATIVE H 2720640 Synthetic product COMPARATIVE I 450_173 Synthetic product COMPARATIVE J3710 Synthetic product COMPARATIVE K 3511 Synthetic product 1 90~0. 4 2 65_18 3 185t62 [0106] The results in Table 2 demonstrate that the compounds of the invention are potent inhibitors of acyl glucosaminyl inositol amidase, and, in particular, mycothiol-S-conjugate amidase.

[0107] The disclosures of each patent, patent application and publication cited or described in this document are hereby incorporated herein by reference, in their entirety.

[0108] Those skilled in the art will appreciate that numerous changes and modifications can be made to the preferred embodiments of the invention and that such changes and modifications can be made without departing from the spirit of the invention. It is, therefore, intended that the appended claims cover all such equivalent variations as fall within the true spirit and scope of the invention.