A series of pyrazine and quinoxaline derivatives have been synthesized, and their activity against M. tuberculosis (Mtb) and Mycobacterium avium (MAC) are reported. The 4-acetoxybenzyl ester of pyrazinoic acid and 4'-acetoxybenzyl 2-quinoxalinecarboxylate showed excellent activity against Mtb (MIC ranges of less than 1-6.25 microg/mL) but only modest activity against MAC (MICs of 4-32 microg/mL).

The essential cell division protein, FtsZ, from Mycobacterium tuberculosis has been expressed in Escherichia coli and purified. The recombinant protein has GTPase activity typical of tubulin and other FtsZs. FtsZ polymerization was studied using 90°light scattering. The mycobacterial protein reaches maximum polymerization much more slowly (ϳ10 min) than E. coli FtsZ. Depolymerization also occurs slowly, taking 1 h or longer under most conditions. Polymerization requires both Mg 2؉ Bacteria have a variety of genes that are critical for cell division, among them the fts genes (reviewed in references 20, 24, and 26). Cell division occurs at the site of formation of the contractile Z ring, which is composed of a polymer of FtsZ. FtsZ, a 40-kDa protein, is ubiquitous in eubacteria and archaea. Although it has only weak sequence homology to mammalian tubulin, it does contain the tubulin signature motif GGGTGS/TG (7, 9), which is believed to be necessary for the GTPase activity of tubulin. FtsZ polymerizes to form the Z ring in a GTP-dependent manner, analogous to the polymerization of tubulin to form microtubules (4,6,10,21,32). The three-dimensional structures of Methanococcus jannaschii FtsZ and ␣-and ␤-tubulin are quite similar (16,17) and reveal that FtsZ and tubulin form a unique family of GTPases (24).Although the polymerization of tubulin has been studied extensively, understanding FtsZ polymerization has been hampered until recently by the lack of a rapid, easy assay. To date the primary method of examining FtsZ polymerization has relied on centrifugation of the reaction mixture followed by an examination of the pellet or by electron microscopy. Most of the published work has dealt with FtsZ from Escherichia coli (1,6,20). Recently, Mukherjee and Lutkenhaus (22) introduced a light-scattering assay that uses a fluorometer to monitor polymer formation and dissolution. We have used this method to extend our understanding of FtsZ by examining the dynamics of Mycobacterium tuberculosis FtsZ polymerization in vitro. M. tuberculosis FtsZ polymerization is similar to that of E. coli FtsZ in many respects. However, there are some significant differences between the two, with M. tuberculosis FtsZ showing some characteristics more reminiscent of its homolog tubulin than the E. coli protein. This work represents the first study of M. tuberculosis FtsZ, a critical cell division protein for a pathogenic organism of worldwide medical importance. MATERIALS AND METHODSPurification of FtsZ. The M. tuberculosis FtsZ coding sequence was subcloned into the NcoI site of pET15b (Novagen). The resulting plasmid, pJD168, was used to transform E. coli BL21(DE3)/pLysS. Cells were incubated at 32°C in Luria-Bertani (LB) media containing 0.4% glucose for 1 h. Five hundred microliters of transformed cells was added to 250 ml of fresh LB medium containing 0.4% glucose, 100 g of ampicillin/ml, and 34 g of chloramphenicol/ml and incubated overnight at 32°C. The cells were pelleted by centrifugation at room temperature. They were then resusp...

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Development of new antimycobacterial agents for Mycobacterium avium complex (MAC) infections is important particularly for persons coinfected with human immunodeficiency virus. The objectives of this study were to evaluate the in vitro activity of 2,4-diamino-5-methyl-5-deazapteridines (DMDPs) against MAC and to assess their activities against MAC dihydrofolate reductase recombinant enzyme (rDHFR). Seventy-seven DMDP derivatives were evaluated initially for in vitro activity against one to three strains of MAC (NJ168, NJ211, and/or NJ3404). MICs were determined with 10-fold dilutions of drug and a colorimetric (Alamar Blue) microdilution broth assay. MAC rDHFR 50% inhibitory concentrations versus those of human rDHFR were also determined. Substitutions at position 5 of the pteridine moiety included ™CH 3 , ™CH 2 CH 3 , and ™CH 2 OCH 3 groups. Additionally, different substituted and unsubstituted aryl groups were linked at position 6 through a two-atom bridge of either ™CH 2 NH, ™CH 2 N(CH 3 ), ™CH 2 CH 2 , or ™CH 2 S. All but 4 of the 77 derivatives were active against MAC NJ168 at concentrations of <13 g/ml. Depending on the MAC strain used, 81 to 87% had MICs of <1.3 g/ml. Twenty-one derivatives were >100-fold more active against MAC rDHFR than against human rDHFR. In general, selectivity was dependent on the composition of the two-atom bridge at position 6 and the attached aryl group with substitutions at the 2 and 5 positions on the phenyl ring. Using this assessment, a rational synthetic approach was implemented that resulted in a DMDP derivative that had significant intracellular activity against a MAC-infected Mono Mac 6 monocytic cell line. These results demonstrate that it is possible to synthesize pteridine derivatives that have selective activity against MAC.

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Thio analogues of purine, pyridine, and pyrimidine were prepared based on the initial activity screening of several analogues of these heterocycles against Mycobacterium tuberculosis (Mtb). Certain 6-thio-substituted purine analogues described herein showed moderate to good inhibitory activity. In particular, two purine analogues 9-(ethylcarboxymethyl)-6-(decylthio)-9H-purine (20) and 9-(ethylcarboxymethyl)-6-(dodecylthio)-9H-purine (21) exhibited MIC values of 1.56 and 0.78 microg/mL respectively against the Mtb H(37)Rv strain. N(9)-Substitution apparently enhances the antimycobacterial activity in the purine series described herein.

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A series of pyridomorphinans derived from naloxone, oxymorphone, and hydromorphone (7a-k) were synthesized and evaluated for binding affinity at the opioid delta, micro, and kappa receptors in brain membranes using radioligand binding assays and for functional activity in vitro using [(35)S]GTP-gamma-S binding assays in brain tissues and bioassays using guinea pig ileum (GPI) and mouse vas deferens (MVD) smooth muscle preparations. The pyridine ring unsubstituted pyridomorphinans possessing the oxymorphone and hydromorphone framework displayed nearly equal binding affinity at the micro and delta receptors. Their affinities at the kappa site were nearly 10-fold less than their binding affinities at the micro and delta sites. Introduction of aryl substituents at the 5'-position on the pyridine ring improved the binding affinity at the delta site while decreasing the binding affinity at the micro site. Nearly all of the ligands possessing an N-methyl group at the17-position with or without a hydroxyl group at the 14-position of the morphinan moiety displayed agonist activity at the micro receptor with varying potencies and efficacies. In the [(35)S]GTP-gamma-S binding assays, most of these pyridomorphinans were devoid of any significant agonist activity at the delta and kappa receptors but displayed moderate to potent antagonist activity at the delta receptors. In antinociceptive evaluations using the warm-water tail-withdrawal assay in mice, the pyridomorphinans produced analgesic effects with varying potencies and efficacies when administered by the intracerebroventricular route. Among the ligands studied, the hydromorphone-derived 4-chlorophenylpyridomorphinan 7h was identified as a ligand possessing a promising profile of mixed micro agonist/delta antagonist activity in vitro and in vivo. In a repeated administration paradigm in which the standard micro agonist morphine produces significant tolerance, repeated administration of the micro agonist/delta antagonist ligand 7h produced no tolerance. These results indicate that appropriate molecular manipulations of the morphinan templates could provide ligands with mixed micro agonist/delta antagonist profiles and such ligands may have the potential of emerging as novel analgesic drugs devoid of tolerance, dependence, and related side effects.