Synthesis and crystal structures of 3-hy-droxy-2,4-dimethyl-2H-thio-phen-5-one and 3-hy-droxy-4-methyl-2H-thio-phen-5-one.

The structures of two hy-droxy-thio-phenone derivatives related to the anti-biotic thiol-actomycin are presented. These are the racemic 3-hy-droxy-2,4-dimethyl-2H-thio-phen-5-one, C6H8O2S, and 3-hy-droxy-4-methyl-2H-thio-phen-5-one, C5H6O2S. The main structural feature of both compounds is C(6) hydrogen-bonded chains formed between the OH and C=O groups. In achiral C5H6O2S, these chains propagate only by translation, corresponding to x + 1, y, z + 1. However, in contrast, for racemic C6H8O2S the hydrogen-bonded chains propagate through a -x + , y + , z operation, giving chains lying parallel to the crystallographic b-axis direction that are composed of alternate R and S enanti-omers. The crystals of 3-hydroxy-4-methyl-2H-thiophen-5-one were found to be twinned by a 180° rotation about the reciprocal 001 direction. In the final refinement the twin ratio refined to 0.568 (2):0.432 (2).

Synthesis, antitubercular activity and mechanism of resistance of highly effective thiacetazone analogues.

Defining the pharmacological target(s) of currently used drugs and developing new analogues with greater potency are both important aspects of the search for agents that are effective against drug-sensitive and drug-resistant Mycobacterium tuberculosis. Thiacetazone (TAC) is an anti-tubercular drug that was formerly used in conjunction with isoniazid, but removed from the antitubercular chemotherapeutic arsenal due to toxic side effects. However, several recent studies have linked the mechanisms of action of TAC to mycolic acid metabolism and TAC-derived analogues have shown increased potency against M. tuberculosis. To obtain new insights into the molecular mechanisms of TAC resistance, we isolated and analyzed 10 mutants of M. tuberculosis that were highly resistant to TAC. One strain was found to be mutated in the methyltransferase MmaA4 at Gly101, consistent with its lack of oxygenated mycolic acids. All remaining strains harbored missense mutations in either HadA (at Cys61) or HadC (at Val85, Lys157 or Thr123), which are components of the ß-hydroxyacyl-ACP dehydratase complex that participates in the mycolic acid elongation step. Separately, a library of 31 new TAC analogues was synthesized and evaluated against M. tuberculosis. Two of these compounds, 15 and 16, exhibited minimal inhibitory concentrations 10-fold lower than the parental molecule, and inhibited mycolic acid biosynthesis in a dose-dependent manner. Moreover, overexpression of HadAB HadBC or HadABC in M. tuberculosis led to high level resistance to these compounds, demonstrating that their mode of action is similar to that of TAC. In summary, this study uncovered new mutations associated with TAC resistance and also demonstrated that simple structural optimization of the TAC scaffold was possible and may lead to a new generation of TAC-derived drug candidates for the potential treatment of tuberculosis as mycolic acid inhibitors.

Strategies and challenges involved in the discovery of new chemical entities during early-stage tuberculosis drug discovery.

There is an increasing flow of new antituberculosis chemical entities entering the tuberculosis drug development pipeline. Although this is encouraging, the current number of compounds is too low to meet the demanding criteria required for registration, shorten treatment duration, treat drug-resistant infection, and address pediatric tuberculosis cases. More new chemical entities are needed urgently to supplement the pipeline and ensure that more drugs and regimens enter clinical practice. Most drug discovery projects under way exploit enzyme systems deemed essential in a specific Mycobacterium tuberculosis biosynthetic pathway or develop chemical scaffolds identified by phenotypic screening of compound libraries, specific pharmacophores or chemical clusters, and natural products. Because the development of a compound for treating tuberculosis is even longer than for treating other infection indications, the identification of selective, potent, and safe chemical entities early in the drug development process is essential to ensure that the pipeline is filled with new candidates that have the best chance to reach the clinic.

Benzylguanidines and other galegine analogues inducing weight loss in mice.

Dimethylallylguanidine, also known as galegine, isolated from Galega officinalis, has been shown to have weight reducing properties in vivo. Substitution of the guanidine group with an N-cyano group and replacement of guanidine with amidine, pyrimidine, pyridine, or the imidazole moieties removed the weight reducing properties when evaluated in BALB/c mice. However, retention of the guanidine and replacement of the dimethylallyl group by a series of functionalized benzyl substituents was shown to exhibit, and in some cases significantly improve, the weight reducing properties of these molecules in BALB/c, ob/ob, and diet induced obesity (DIO) mice models. The lead compound identified, across all models, was 1-(4-chlorobenzyl)guanidine hemisulfate, which gave an average daily weight difference (% from time-matched controls; +/- SEM) of -19.7 +/- 1.0, -11.0 +/- 0.7, and -7.3 +/- 0.8 in BALB/c, ob/ob, and DIO models, respectively.

Identification of 2-aminothiazole-4-carboxylate derivatives active against Mycobacterium tuberculosis H37Rv and the beta-ketoacyl-ACP synthase mtFabH.

BACKGROUND: Tuberculosis (TB) is a disease which kills two million people every year and infects approximately over one-third of the world's population. The difficulty in managing tuberculosis is the prolonged treatment duration, the emergence of drug resistance and co-infection with HIV/AIDS. Tuberculosis control requires new drugs that act at novel drug targets to help combat resistant forms of Mycobacterium tuberculosis and reduce treatment duration. METHODOLOGY/PRINCIPAL FINDINGS: Our approach was to modify the naturally occurring and synthetically challenging antibiotic thiolactomycin (TLM) to the more tractable 2-aminothiazole-4-carboxylate scaffold to generate compounds that mimic TLM's novel mode of action. We report here the identification of a series of compounds possessing excellent activity against M. tuberculosis H(37)R(v) and, dissociatively, against the beta-ketoacyl synthase enzyme mtFabH which is targeted by TLM. Specifically, methyl 2-amino-5-benzylthiazole-4-carboxylate was found to inhibit M. tuberculosis H(37)R(v) with an MIC of 0.06 microg/ml (240 nM), but showed no activity against mtFabH, whereas methyl 2-(2-bromoacetamido)-5-(3-chlorophenyl)thiazole-4-carboxylate inhibited mtFabH with an IC(50) of 0.95+/-0.05 microg/ml (2.43+/-0.13 microM) but was not active against the whole cell organism. CONCLUSIONS/SIGNIFICANCE: These findings clearly identify the 2-aminothiazole-4-carboxylate scaffold as a promising new template towards the discovery of a new class of anti-tubercular agents.

Thiacetazone, an antitubercular drug that inhibits cyclopropanation of cell wall mycolic acids in mycobacteria.

BACKGROUND: Mycolic acids are a complex mixture of branched, long-chain fatty acids, representing key components of the highly hydrophobic mycobacterial cell wall. Pathogenic mycobacteria carry mycolic acid sub-types that contain cyclopropane rings. Double bonds at specific sites on mycolic acid precursors are modified by the action of cyclopropane mycolic acid synthases (CMASs). The latter belong to a family of S-adenosyl-methionine-dependent methyl transferases, of which several have been well studied in Mycobacterium tuberculosis, namely, MmaA1 through A4, PcaA and CmaA2. Cyclopropanated mycolic acids are key factors participating in cell envelope permeability, host immunomodulation and persistence of M. tuberculosis. While several antitubercular agents inhibit mycolic acid synthesis, to date, the CMASs have not been shown to be drug targets. METHODOLOGY/PRINCIPLE FINDINGS: We have employed various complementary approaches to show that the antitubercular drug, thiacetazone (TAC), and its chemical analogues, inhibit mycolic acid cyclopropanation. Dramatic changes in the content and ratio of mycolic acids in the vaccine strain Mycobacterium bovis BCG, as well as in the related pathogenic species Mycobacterium marinum were observed after treatment with the drugs. Combination of thin layer chromatography, mass spectrometry and Nuclear Magnetic Resonance (NMR) analyses of mycolic acids purified from drug-treated mycobacteria showed a significant loss of cyclopropanation in both the alpha- and oxygenated mycolate sub-types. Additionally, High-Resolution Magic Angle Spinning (HR-MAS) NMR analyses on whole cells was used to detect cell wall-associated mycolates and to quantify the cyclopropanation status of the cell envelope. Further, overexpression of cmaA2, mmaA2 or pcaA in mycobacteria partially reversed the effects of TAC and its analogue on mycolic acid cyclopropanation, suggesting that the drugs act directly on CMASs. CONCLUSIONS/SIGNIFICANCE: This is a first report on the mechanism of action of TAC, demonstrating the CMASs as its cellular targets in mycobacteria. The implications of this study may be important for the design of alternative strategies for tuberculosis treatment.

Identification of the benzodiazepines as a new class of antileishmanial agent.

The continual increase in drug resistance; the lack of new chemotherapeutic agents; the toxicity of existing agents and the increasing morbidity with HIV co-infection mean the search for new antileishmanial agents has never been more urgent. We have identified the benzodiazepines as a structural class for antileishmanial hit optimisation, and demonstrated that their in vitro activity is comparable with the clinically used drug, sodium stibogluconate, and that the compounds are not toxic to macrophages.

Symmetrical and unsymmetrical analogues of isoxyl; active agents against Mycobacterium tuberculosis.

Symmetrical and unsymmetrical analogues of the antimycobacterial agent isoxyl have been synthesized and tested against Mycobacterium tuberculosis H37Rv and Mycobacterium bovis BCG, some showing an increased bactericidal effect. In particular, compounds 1-(p-n-butylphenyl)-3-(4-propoxy-phenyl) thiourea (10) and 1-(p-n-butylphenyl)-3-(4-n-butoxy-phenyl) thiourea (11) showed an approximate 10-fold increase in in vitro potency compared to isoxyl, paralleled by increased inhibition of mycolic acid biosynthesis in M. bovis BCG. Interestingly, these isoxyl analogues showed relatively poor inhibition of oleate production, suggesting that the modifications have changed the spectrum of biological activity.

Synthesis and properties of methyl 5-(1'R,2'S)-(2-octadecylcycloprop-1-yl)pentanoate and other omega-19 chiral cyclopropane fatty acids and esters related to mycobacterial mycolic acids.

A 23-26-carbon chain length range of omega-19 (1'R,2'S) cyclopropane fatty acids, related to mycobacterial mycolic acids, has been prepared. The key cyclopropyl intermediate, (1'R,2'S)-(Z)-1-formyl-2-octadecylcyclopropane, underwent Wittig chemistry with various reagents to provide vinylic precursors, which were selectively reduced to the corresponding saturated omega-19 cyclopropane fatty acids or esters. The 24-carbon omega-19 cyclopropane ester was made by chain elongation of the 23-carbon ester. Saturated and unsaturated chiral cyclopropane acids and esters were assayed, using wall extracts of Mycobacterium smegmatis; the incorporation of 14C-acetate was used to measure inhibition or stimulation of mycolic acid synthesis. Minor inhibition (2-3%) was shown by the 23- and 24-carbon saturated esters; all the other compounds were stimulants. The most effective (38-55%) stimulators of mycolate synthesis were the unsaturated esters with 23- and 26-carbons and the saturated and unsaturated 25-carbon acids.

Facile synthesis of (Z)-tetracos-5-enoic acid and racemic cis-4-(2-octadecylcyclopropane-1-yl)-butanoic acid.

(Z)-tetracos-5-enoic acid and racemic cis-4-(2-octadecylcyclopropane-1-yl)-butanoic acid have been prepared from 1-eicosene by a new facile route. Periodic acid cleavage of the epoxide of 1-eicosene gave nonadecanal which was condensed with 4-carboxybutyltriphenylphosphonium bromide to give predominately (Z)-tetracos-5-enoic acid. Simmons-Smith type cyclopropanation of (Z)-tetracos-5-enoic acid gave a minor proportion of racemic cis-4-(2-octadecylcyclopropane-1-yl)-butanoic acid accompanied by major amounts of its methyl ester.