Picomolar inhibitors of HIV reverse transcriptase featuring bicyclic replacement of a cyanovinylphenyl group

Abstract

Members of the catechol diether class are highly potent non-nucleoside inhibitors of HIV-1 reverse transcriptase (NNRTIs). The most active compounds yield EC50 values below 0.5 nM in assays using human T-cells infected by wild-type HIV-1. However, these compounds such as rilpivirine, the most recently FDA-approved NNRTI, bear a cyanovinylphenyl (CVP) group. This is an uncommon substructure in drugs that gives reactivity concerns. In the present work, computer simulations were used to design bicyclic replacements for the CVP group. The predicted viability of a 2-cyanoindolizinyl alternative was confirmed experimentally and provided compounds with 0.4 nM activity against the wild-type virus. The compounds also performed well with EC50 values of 10 nM against the challenging HIV-1 variant that contains the Lys103Asn/Tyr181Cys double mutation in the RT enzyme. Indolyl and benzofuranyl analogues were also investigated; the most potent compounds in these cases have EC50 values toward wild-type HIV-1 near 10 nM and high-nanomolar activities toward the double-variant. The structural expectations from the modeling were much enhanced by obtaining an X-ray crystal structure at 2.88 Å resolution for the complex of the parent 2-cyanoindolizine 10b and HIV-1 RT. The aqueous solubilities of the most potent indolizine analogues were also measured to be ~40 μg/mL, which is similar to that for the approved drug efavirenz and ~1000-fold greater than for rilpivirine.

Figure 1

Modeled compounds where Ar is the uracilylethoxyphenyl substructure of 5 or 6 with R’ = Cl. The number in parentheses is the computed relative free energy of binding (kcal/mol) with HIV-RT from the FEP calculations; the statistical uncertainty in the results is ±0.5 kcal/mol.

Figure 2

Illustration of a configuration from an MC simulation of the indole 7l bound to wild type HIV-1 reverse transcriptase. Carbon atoms of 7l are rendered in yellow. All water molecules and many residues in front of the ligand have been removed for clarity.

Figure 3

Stereo view of the crystal structure for 10b complexed with HIV-RT. Numerous contacts in the binding site are apparent and consistent with those in the crystal structures for 3 and 4 and in the modeled structure in Figure 2. The PDB code is 4MFB.

Figure 4

A space-filling rendering made from the crystal structure of 10b bound to HV-RT. Carbon atoms of 10b are in yellow. Notable points are the hydrogen bond between a uracil oxygen atom and the backbone NH of Lys103, the aryl-aryl contacts with Tyr181 and Trp229, and the orientation of Lys103 including the contact of the C_β atom with the inhibitor.

Figure 5

Two views comparing the crystal structures for 3 and 10b. In A, the difference in conformation of the uracilylethoxy side chain is apparent, while B illustrates that the cyano group of 3 extends farther into the tunnel region and closer to Lys223.

Scheme 1. Synthesis of the 2-Cyanoindoles

Reagents: (a) K₂CO₃, DMF, 100–120 °C, 12 h; (b) methyl azidoacetate, −40 °C, NaOMe, MeOH, 12 h; (c) xylene, reflux, 4 h; (d) LAH,THF, 0 °C, 0.5 h; (e) MnO₂, DCM, rt, 24 h; (f) NH₂ OSO₃ H, MeOH, reflux, 0.5 h; (g) Cu(OAc)₂, CH₃ CN, reflux, 1 h; (h) 4-DMAP, Boc₂O, THF, rt, 12 h; (i) H₂ /Pd/C, MeOH, rt, 0.5 h; (j) 3-benzoyl,1-bromoethyluracil, K₂CO₃, DMF, 60 °C, 2 h; (k) TBAF, THF, reflux, 24 h; (l) NH₃, MeOH, rt, 12 h.

Scheme 2. Synthesis of the 2-Cyanoindolizines

Reagents: (a) DABCO, neat, 2 h; (b) Ac₂O, 100 °C, 2 h →140 °C, 16 h; (c) CuI, Cs₂CO₃, 2,2,6,6-tetramethyl-3,5-heptanedione, dioxane, 120 °C, 18 h; (d) BBr₃, DCM, −78 °C → 0 °C, 3 h; (e) 3-benzoyl,1-bromoethyluracil, Cs₂CO₃ or K₂CO₃, DMF, 60 °C, 3 h; (f) NH₄OH, DCM, 16 h.

Scheme 3. Synthesis of the 2-Cyanobenzofurans

Reagents: (a) PPh₃, CBr₄, DCM, 0°C → RT, 2 h; (b) CuI, K₂CO₃, 80 °C, 4 h; (c) NaCN, DMSO, 100 °C, 24 h; (d) CuI, Cs₂CO₃N,N-dimethylglycine hydrochloride, dioxane, 90 °C, 18 h; (e) BBr₃, DCM, −78 °C → 0 °C, 3 h; (f) 3-benzoyl,1-bromoethyluracil, Cs₂CO₃ or K₂CO₃, DMF, 60 °C, 3 h; (g) NH₄OH, DCM, 16 h.