Cecil D. Kwong

High Throughput Screening for Inhibitors of Mycobacterium tuberculosis H37Rv

There is an urgent need for the discovery and development of new antitubercular agents that target new biochemical pathways and treat drug resistant forms of the disease. One approach to addressing this need is through high-throughput screening of medicinally relevant libraries against the whole bacterium in order to discover a variety of new, active scaffolds that will stimulate new biological research and drug discovery. Through the Tuberculosis Antimicrobial Acquisition and Coordinating Facility (www.taacf.org), a large, medicinally relevant chemical library was screened against M. tuberculosis strain H37Rv. The screening methods and a medicinal chemistry analysis of the results are reported herein.

Antituberculosis Activity of the Molecular Libraries Screening Center Network Library

There is an urgent need for the discovery and development of new antitubercular agents that target novel biochemical pathways and treat drug-resistant forms of the disease. One approach to addressing this need is through high-throughput screening of drug-like small molecule libraries against the whole bacterium in order to identify a variety of new, active scaffolds that will stimulate additional biological research and drug discovery. Through the Molecular Libraries Screening Center Network, the NIAID Tuberculosis Antimicrobial Acquisition and Coordinating Facility tested a 215,110-compound library against Mycobacterium tuberculosis strain H37Rv. A medicinal chemistry survey of the results from the screening campaign is reported herein.

Synthesis of 1,3-thiazine derivatives and their evaluation as potential antimycobacterial agents.

A series of eight 5,6-dihydro-4H-1,3-thiazine derivatives was synthesized by the BF3 x Et2O-catalyzed reaction of selected alpha,beta-unsaturated ketones with thiobenzamide at room temperature. The antimycobacterial activities of these compounds were determined against Mycobacterium tuberculosis H37Rv (ATCC 27294) using the Alamar blue susceptibility assay. Three compounds, 5-hydroxy-3-phenyl-4-aza-2-thiabicyclo[3.3.1]none-3-ene 3a, 4-hydroxy-4-methyl-6-pentyl-2-phenyl-5,6-dihydro-4H-1,3-thiazine 3b, and 4-ethyl-4-hydroxy-2-phenyl-5,6-dihydro-4H-1,3-thiazine 3c exhibited inhibitory activities of 97, 77 and 76%, respectively, at a concentration of 6.25 microg/ml. The actual MIC99 for the most active of these compounds, 3a, was also determined to be >6.25 microg/ml. These results, and especially those for 3a, suggest that 1,3-thiazines are potential lead compounds in the search for new antitubercular agents.

SYNTHESIS AND ANTIVIRAL EVALUATION OF ANALOGS OF ADENOSINE-N1-OXIDE AND 1-(BENZYLOXY)ADENOSINE

The activity of a series of compounds related to adenosine-N1-oxide (1) and 1-(benzyloxy)adenosine (42) against vaccinia virus has been determined both in vitro and in a vaccinia mouse tailpox model. Significant activities have been found both in vitro and in vivo for a number of the synthetic compounds.

In vitro and in vivo antiviral (RNA) evaluation of orotidine 5'-monophosphate decarboxylase inhibitors and analogues including 6-azauridine-5'-(ethyl methoxyalaninyl) phosphate (a 5'-monophosphate prodrug)

A series of 29 pyrimidines comprising analogues of 6-azauridine (e.g. 2- and 4-thio-6-azauridine), 6-substituted uridines (including several known inhibitors of orotidine 5′-monophosphate decarboxylase, ODCase, e.g. pyrazofurin), and 6-azauridine-5′-(ethyl methoxyalaninyl) phosphate (a potential prodrug of 6-AU-5′-MP) were synthesized and evaluated in vitro and in vivo against five RNA viruses: Japanese encephalitis (JE), yellow fever (YF), sandfly fever (SF), Punta Tora (PT) and Venezuelan equine encephalomyelitis (VEE) viruses. 2-Thio-6-azauridine demonstrated the best In vitro activity against all five viruses. However, in vivo activity was not observed in JE-, PT- and VEE-infected mice. The phosphate prodrug of 6-azauridine was significantly more effective than the parent compound in the PT virus mouse model. Optimum in vivo dose/route/schedule was determined for pyrazofurin in PT-virus-infected mice.

Novel substituted pyrimidines as HCV replication (replicase) inhibitors.

Compound 1 was identified as a HCV replication inhibitor from screening/early SAR triage. Potency improvement was achieved via modulation of substituent on the 5-azo linkage. Due to potential toxicological concern, the 5-azo linkage was replaced with 5-alkenyl or 5-alkynyl moiety. Analogs containing the 5-alkynyl linkage were found to be potent inhibitors of HCV replication. Further evaluation identified compounds 53 and 63 with good overall profile, in terms of replicon potency, selectivity and in vivo characteristics. Initial target engagement studies suggest that these novel carbanucleoside-like derivatives may inhibit the HCV replication complex (replicase).

5-Benzothiazole substituted pyrimidine derivatives as HCV replication (replicase) inhibitors

Based on a previously identified HCV replication (replicase) inhibitor 1, SAR efforts were conducted around the pyrimidine core to improve the potency and pharmacokinetic profile of the inhibitors. A benzothiazole moiety was found to be the optimal substituent at the pyrimidine 5-position. Due to potential reactivity concern, the 4-chloro residue was replaced by a methyl group with some loss in potency and enhanced rat in vivo profile. Extensive investigations at the C-2 position resulted in identification of compound 16 that demonstrated very good replicon potency, selectivity and rodent plasma/target organ concentration. Inhibitor 16 also demonstrated good plasma levels and oral bioavailability in dogs, while monkey exposure was rather low. Chemistry optimization towards a practical route to install the benzothiazole moiety resulted in an efficient direct C-H arylation protocol.

Synthesis and SAR of pyridothiazole substituted pyrimidine derived HCV replication inhibitors.

Introduction of a nitrogen atom into the benzene ring of a previously identified HCV replication (replicase) benzothiazole inhibitor 1, resulted in the discovery of the more potent pyridothiazole analogues 3. The potency and PK properties of the compounds were attenuated by the introductions of various functionalities at the R(1), R(2) or R(3) positions of the molecule (compound 3). Inhibitors 38 and 44 displayed excellent potency, selectivity (GAPDH/MTS CC(50)), PK parameters in all species studied, and cross genotype activity.

In vitro and in vivo evaluation of aminopeptidase inhibitors as antimicrosporidial therapies

Microsporidia of the genus Encephulitozoon are emerging obligate intracellular pathogens in immunocompromised hosts. To date, there have been few advances in the treatment of microsporidiosis, and a limited number of drugs have bccn used to treat microsporidial infections with varicd success. Two agents, albendazole and fumagillin, show activity in vitro and in limited clinical applications [2,4,13]. Fumagillin, an angiogenesis inhibitor, has been used topically to treat ocular infections of E. iritestirialis and E. hclleni, howevcr it is systemically toxic to humans at therapeutic doses [8,I3]. In comparison, TNP-470, a derivative of funiagillin, had a relatively low toxicity in an athymic mouse model, and was able to reduce replication of the Encepha/itozoon spp. and Vittufornza coriieae in vitro [3,4,7]. To date, antiinicrosporidial agents have been evaluated primarily on an empirical basis. Several in vitro systems for mcasuring antimicrosporidial activity have been described [ 1,3,4,7]. A variety of animal models describe E/lcep/za[ifozoori spp. infections in squirrel monkeys, rabbits, and a varicty of genetically modified, ininiunosuppressed or immunodeficient mice [17]. The time course for dcvclopment of a fatal B. c.zmiruli infection in athyniic and SClD mice is predictable and reproducible, while infection in immunoconipetent mice i s typically asyniptoiiiatic [6]. Thc E. ctiiiiculi infected athyinic mouse model has been priinarily used for immunological and hostparasite interaction studies, and no wcll-defined model foidrug efficacy studies has bccn described [5]. However, the athymic iiiurine model has been used on a limited basis to investigate the efficacy of TNP-470 and albendazole [3,1 I ] . Proteases are increasingly investigated as targets for therapeutic intervention for infectious diseases through the development of lowtoxicity inhibitors [ 121. Aminopeptidase inhibitors have shown promise in vitro against several protozoan infections [ 14,151. Bcstatin, [(2S,2R),3-aiiiino-2-liydroxy-4-phcnylbtitanoyl]-L-Leucine, a modified dipeptide aiiiinopeptidase inhibitor, is a revcrsible inhibitor with many analogues including a nitrated semisynthetic compound, nitrobestatin, [2S, 3R]-3-ainino-2-hydroxy-4-[-4-nitrophenyl]-butanoyl-L-Leucine. The naturally occurring modified tripeptide, amastatin ([(2S, 2R)]-3aniino-2-hydroxy-5-iiicthylhexanoyl]-Val-Val-Asp-OH), is also a potent protcase inhibitor [IS]. This study evaluated the antiniicrosporidial activity and toxicity of selected aminopeptidase inhibitors in vitro and in vivo using an E. cuniculi infected athyniic mouse model.

Pyridofuran substituted pyrimidine derivatives as HCV replication (replicase) inhibitors

Introduction of nitrogen atom into the benzene ring of a previously identified HCV replication (replicase) benzofuran inhibitor 2, resulted in the discovery of the more potent pyridofuran analogue 5. Subsequent introduction of small alkyl and alkoxy ligands into the pyridine ring resulted in further improvements in replicon potency. Replacement of the 4-chloro moiety on the pyrimidine core with a methyl group, and concomitant monoalkylation of the C-2 amino moiety resulted in the identification of several inhibitors with desirable characteristics. Inhibitor 41, from the monosubstituted pyridofuran and inhibitor 50 from the disubstituted series displayed excellent potency, selectivity (GAPDH/MTS CC(50)) and PK parameters in all species studied, while the selectivity in the thymidine incorporation assay (DNA·CC(50)) was low.