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A series of macrocyclic compounds containing a cyclic constraint in the P2–P4 linker region have been discovered and shown to exhibit excellent HCV NS3/4a genotype 3a and genotype 1b R155 K, A156T, A156 V, and D168 V mutant activity while maintaining high rat liver exposure. The effect of the constraint is most dramatic against gt 1b A156 mutants where ∼20-fold improvements in potency are achieved by introduction of a variety of ring systems into the P2–P4 linker.

Previous reports from our laboratories described potent tripeptide thrombin inhibitors which incorporate heterocycle-substituted chlorophenyl groups in the P1 position. Using these as lead compounds for further optimization, we identified sites of metabolism and designed analogs with 4-fluoroproline in P2 and cyclopropane-containing side chains in P3 as an approach to reducing metabolism and improving their oral pharmacokinetic performance. The large (300-fold) difference in potency between analogs containing (4 R )- and (4 S )-4-fluoroproline was rationalized by analyzing inhibitor–enzyme interactions in crystal structures of related compounds and by molecular modeling which indicated that the more potent (4 R )-4-fluoroproline isomer stabilizes a proline ring conformation that is preferred for binding to the enzyme. An optimal compound from this work, 41 , exhibits high potency in a coagulation assay in human plasma (2×APTT = 190 nM), excellent selectivity versus the digestive enzyme trypsin ( K i = 3300 nM), and excellent oral bioavailability in dogs with moderate clearance ( F = 100%, CL = 12 mL/min/kg).

Thrombin-inhibitor X-ray crystal structures, in combination with the installation of binding elements optimized within the pyrazinone series of thrombin inhibitors, were utilized to transform a weak triazolopyrimidine lead into a series of potent oxazolopyridines. A modification intended to attenuate plasma protein binding (i.e., conversion of the P3 pyridine to a piperidine) conferred significant factor Xa activity to this series. Ultimately, these dual thrombin/factor Xa inhibitors demonstrated excellent in vitro and in vivo anticoagulant efficacy.

In this study, we have demonstrated that the critical hydrogen bonding motif of the established 3-aminopyrazinone thrombin inhibitors can be effectively mimicked by a 2-aminopyridine N-oxide. As this peptidomimetic core is more resistant toward oxidative metabolism, it also overcomes the metabolic liability associated with the pyrazinones. An optimization study of the P(1) benzylamide delivered the potent thrombin inhibitor 21 (K(i) = 3.2 nM, 2xaPTT = 360 nM), which exhibited good plasma levels and half-life after oral dosing in the dog (C(max) = 2.6 microM, t(1/2) = 4.5 h).

Removal of the β-ketoamide functionality from L-370,518 (Ki = 0.09 nM) provided a 5 nM Ki inhibitor of thrombin: L-371,912. Comparison of the enzyme-inhibitor crystal structures suggests a possible explanation for the relatively small change in affinity for thrombin. L-371,912 is selective for thrombin over related serine proteases and is efficacious in an animal model of arterial thrombosis.

A systematic investigation was undertaken to determine the role of the P1' sidechain in a series of hydroxyethylene isostere based inhibitors of HIV-1 protease. Substitution and homologation of the benzyl P1' side chain of the Phe-Phe isostere based pseudo peptides 1 (L-682,679) and 2 (L-685,434) with various heteroalkyl groups leads to a series of extremely potent inhibitors of the enzyme. Several examples of the most potent inhibitors were very effective in an ex vivo cell based viral spread assay using human H9 T-lymphocytes and the IIIb isolate of HIV-1. Compound 19 is 120 times more potent than 1 and 16 times more potent than 2 in inhibiting the spread of infection in this assay.

A series of 10-hydroxy-7,8-dihydropyrazino[1′,2′:1,5]pyrrolo[2,3- d ]pyridazine-1,9(2 H ,6 H )-diones was synthesized and tested for their inhibition of HIV-1 replication in cell culture. Structure–activity studies indicated that high antiviral potency against wild-type virus as well as viruses containing integrase mutations that confer resistance to three different structural classes of integrase inhibitors could be achieved by incorporation of small aliphatic groups at certain positions on the core template. An optimal compound from this study, 16 , inhibits integrase strand-transfer activity with an IC 50 value of ⩽10 nM, inhibits HIV-1 replication in cell culture with an IC 95 value of 35 nM in the presence of 50% normal human serum, and displays modest pharmacokinetic properties in rats (iv t 1/2 = 5.3 h, F = 17%).

A series of potent novel 8-hydroxy-3,4-dihydropyrrolo[1,2-a]pyrazine-1(2H)-one HIV-1 integrase inhibitors was identified. These compounds inhibited the strand transfer process of HIV-1 integrase and viral replication in cells. Compound 12 is active against replication of HIV-1 in cell culture with a CIC(95) of 0.31microM. Further SAR exploration led to the preparation of pseudosymmetrical tricyclic pyrrolopyrazine inhibitors 23 and 24 with further improvement in antiviral activity.

Starting from a 2-amino-6-methylpyridine P1 group and following a strategy of enlarging it whilst reducing its polarity, we have developed a series of potent, moderately basic azaindoles which are intrinsically much more selective for thrombin versus trypsin. Certain pyrazinone acetamide azaindole derivatives have pharmacokinetic parameters after oral administration to dogs, or efficacy in vitro, comparable to an optimized pyrazinone acetamide 2-amino-6-methylpyridine derivative.

Several hydrogenated derivatives of the potent NMDA antagonist 1 have been prepared and evaluated as competitive inhibitors of [ 3 H]-1 binding. These compounds were also tested for their ability to act as noncompetitive antagonists of NMDA in vitro. These studies indicate that two aromatic rings are not strictly required for high-affinity binding or NMDA antagonism

6-Chloromethylbenzo[a]pyrene (6-CMBP) labeled with 13C in the chloromethyl group was used as a model for those carcinogens which form essentially free carbocations. Using 13C-NMR to identify products, the selectivity with which this electrophile modifies nucleosides was investigated. At pH 7, guanosine and deoxyguanosine are the most nucleophilic nucleosides toward the carbocation generated by solvolysis of 6-CMBP. Attack at N-7 predominates over attack at N-2. At higher pH, the nucleophilicity of guanosine and deoxyguanosines increases markedly. In addition, the site of modification changes to N-1 with secondary modification at O-6. The pH dependence of the rate of this reaction implicates a group with pK-value approx. 8.7 which was assigned to the hydrogen on N-1. The presence of a methyl group on the N-7 position of guanosine lowers this pK-value to approx. 7.2. Consequently, N7-methylguanosine shows the high nucleophilicity at physiological pH that guanosine has at high pH. These observations lead to the suggestion of a one base: two-site model for chemical carcinogenesis.

On 1n,π*-excitation, the title compound 2 undergoes a photoinduced intramolecular [4 + 2]-cycloaddition affording the tetracyclic enol ether 3 as the only product in 79% yield. The assigned structure of 3 was confirmed by its conversion to the p-nitrobenzoate 6 whose structure was determined by X-ray analysis.