Structure-based design, synthesis, biological evaluation and X-ray structural studies of fluorine containing HIV-1 protease inhibitors are described. The synthesis of both enantiomers of the gem-difluoro-bis-THF ligands was carried out in a stereoselective manner using a Reformatskii-Claisen reaction as the key step. Optically active ligands HIV-1LAI were converted to protease inhibitors. Two of these inhibitors (3 and 4) exhibited HIV-1 protease inhibitory Ki’s in picomolar range. Both inhibitors showed very potent antiviral activity with EC50 values of 0.8 nM and 3.1 nM respectively against the laboratory strain HIV-1LAI. Both inhibitors exhibited improved lipophilicity profiles compared to darunavir. Also, both inhibitors showed much improved blood-brain-barrier permeability in an in vitro model. A high resolution X-ray structure of inhibitor 4-bound HIV-1 protease was determined. The X-ray structure revealed that fluoro ligand makes extensive interactions with the HIV-1 protease S2 subsite, including hydrogen-bonding interactions with the protease backbone atoms. Also, both fluorine atoms on the bis-THF ligand formed strong interactions with the flap Gly48 carbonyl oxygen.
GRL-02031 (1) is an HIV-1 protease (PR) inhibitor containing a novel P1′ (R)-aminomethyl-2-pyrrolidinone group. Crystal structures at resolutions of 1.25 to 1.55 Å were analyzed for complexes of 1 with the PR containing major drug resistant mutations, PRI47V, PRL76V, PRV82A and PRN88D. Mutations of I47V and V82A alter residues in the inhibitor-binding site, while L76V and N88D are distal mutations having no direct contact with the inhibitor. Substitution of a smaller amino acid in PRI47V and PRL76V, and the altered charge of PRN88D are associated with significant local structural changes compared to the wild-type PRWT, while substitution of alanine in PRV82A increases the size of the S1′ subsite. The P1′ pyrrolidinone group of 1 accommodates to these local changes by assuming two different conformations. Overall, the conformation and interactions of 1 with PR mutants resemble those of PRWT with similar inhibition constants in good agreement with the antiviral potency on multidrug resistant HIV-1.
An extremely drug resistant mutant of HIV-1 protease (PR) bearing 20 mutations (PR20) has been studied with two potent antiviral investigational inhibitors. GRL-5010A and GRL-4410A were designed to introduce hydrogen bond interactions with the flexible flaps of the PR by incorporating gem-difluorines and alkoxy, respectively, at the C4 position of the bis-THF of darunavir. PR20 provides an excellent model for high level resistance, since clinical inhibitors are >1000-fold less active on PR20 than on wild-type enzyme. GRL-5010A and GRL-4410A show inhibition constants of 4.3 ± 7.0 and 1.7 ± 1.8 nM, respectively, for PR20, compared to the binding affinity of 41 ± 1 nM measured for darunavir. Crystal structures of PR20 in complexes with the two inhibitors confirmed the new hydrogen bond interactions with Gly 48 in the flap of the enzyme. The two new compounds are more effective than darunavir in inhibiting mature PR20 and show promise for further development of antiviral agents targeting highly resistant PR mutants.
We describe the design, synthesis and biological evaluation of a series of novel HIV-1 protease inhibitors bearing isophthalamide derivatives as the P2–P3 ligands. We have investigated a range of acyclic and heterocyclic amides as the extended P2–P3 ligands. These inhibitors displayed good to excellent HIV-1 protease inhibitory activity. Also, a number of inhibitors showed very good antiviral activity in MT cells. Compound 5n has shown an enzyme Ki of 0.17 nM and antiviral IC50 of 14 nM. An X-ray crystal structure of inhibitor 5o-bound to HIV-1 protease was determined at 1.11 Å resolution. This structure revealed important molecular insight into the inhibitor-HIV-1 protease interactions in the active site.
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