Jeremy L. Clark

Inhibitors of the IMPDH enzyme as potential anti-bovine viral diarrhoea virus agents

Ribavirin and mycophenolic acid (MPA) are known inhibitors of the IMPDH enzyme (E.C. 1.1.1.205). This enzyme catalyzes the conversion of inosine monophosphate to xanthine monophosphate, leading eventually to a decrease in the intracellular level of GTP and dGTP. The antiviral effect against bovine viral diarrhoea virus (BVDV) of 15 analogues related to MPA was determined. MDBK cells were infected with the cytopathic strain of BVDV in presence or absence of test compounds. Viral RNA was extracted from the cell supernatant fluids and quantified by RT-PCR. Ribavirin showed a potent antiviral effect against BVDV with 90% effective concentration (EC90) of 4 μM. MPA along with several analogues, including both its corresponding aldehyde and alcohol, and modifications in the length of the side chain (C2- and C4-derivatives) were tested. We have identified previously unreported IMPDH inhibitors that have potent anti-BVDV activity, namely: C6-MPAlc (5), C6-MPA-Me (7), C4-MPAlc (8), C4-MPA (10) and C2-MAD (20). Most of these compounds inhibited the IMPDH enzyme in the nanomolar range (4–800 nM) in cell-free assays. Some compounds, such as mizoribine, which is a potent inhibitor of IMPDH in vitro (enzyme 50% inhibitory concentration IC50=4 nM), had no detectable anti-BVDV activity up to 100 μM. The compounds were essentially non-toxic to a confluent monolayer of MDBK cells. However, in exponentially growing cells, they showed minimal toxicity at 100 μM over a 24 h period, but the toxicity was more pronounced after 3 days [50% cytotoxic concentration (CC50) value ranged from 5 to 30 μM].

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.

Synthesis of 2‐Deoxy‐2‐Fluoro‐2‐C‐Methyl‐D‐Ribofuranoses

The synthesis of methyl 3,5‐di‐O‐benzoyl‐2‐deoxy‐2‐fluoro‐2‐C‐methyl‐β‐D‐ribofuranoside and the conversion to the corresponding 1‐O‐acetyl‐3,5‐di‐O‐benzoyl‐2‐deoxy‐2‐fluoro‐2‐C‐methyl‐D‐ribofuranose and 1,3,5‐tri‐O‐benzoyl‐2‐deoxy‐2‐fluoro‐2‐C‐methyl‐D‐ribofuranose is reported. The key synthetic step is the fluorination of the tertiary center of methyl 3,5‐di‐O‐benzyl‐2‐C‐methyl‐β‐D‐arabinofuranoside to provide methyl 3,5‐di‐O‐benzyl‐2‐deoxy‐2‐fluoro‐2‐C‐methyl‐β‐D‐ribofuranoside.

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.

Synthesis and biological evaluation of (3'S)-3'-deoxy-3'-fluoro-3'-C-ethynylcytidine.

The novel pyrimidine nucleoside, (3 ′S)-3 ′-deoxy-3 ′-fluoro-3 ′-C-ethynylcytidine (1) was synthesized from cytidine in seven steps. The key step in the synthesis was the introduction of the tertiary fluorine at the 3 ′-position. Compound 1 was evaluated in vitro against several RNA viruses.