Substituent effects on P2-cyclopentyltetrahydrofuranyl urethanes: Design, synthesis, and X-ray studies of potent HIV-1 protease inhibitors

Abstract

The design, synthesis, and biological evaluation of novel C3-substituted cyclopentyltetrahydrofuranyl (Cp-THF)-derived HIV-1 protease inhibitors are described. Various C3-functional groups on the Cp-THF ligand were investigated in order to maximize the ligand-binding site interactions in the flap region of the protease. Inhibitors 3c and 3d have displayed the most potent enzyme inhibitory and antiviral activity. Both inhibitors have maintained impressive activity against a panel of multidrug resistant HIV-1 variants. A high-resolution X-ray crystal structure of 3c-bound HIV-1 protease revealed a number of important molecular insights into the ligand-binding site interactions.

HIV-1 protease inhibitors continue to be a critical component of frontline therapy in the treatment of HIV patients.^1-3 Our continuing studies on the structure-based design of inhibitors targeting the protein backbone led to the discovery of a variety of novel HIV-1 protease inhibitors (PIs) with broad-spectrum activity against multidrug-resistant HIV-1 variants.^4-8 We recently reported various C3-functionalized cyclopentyltetrahydrofuran (Cp-THF)-derived P2-ligands designed to specifically interact with the flap Gly48 amide NH in the S2-subsite of the HIV-1 protease.⁹ One of these inhibitors, 2 (Fig. 1), containing a 3-(R)-hydroxy group on the Cp-THF core displayed exceptionally potent enzyme inhibitory (K_i = 5 pM) and antiviral activity (IC₅₀ = 2.9 nM). This inhibitor also exhibited potent activity against a panel of multidrug-resistant HIV-1 variants. The X-ray crystal structure of 2-bound HIV protease revealed an extensive hydrogen-bonding network with the enzyme backbone.⁹ Of particular interest, the 3-(R)-hydroxy group of the Cp-THF ligand was involved in an interesting water-mediated interaction with the backbone NH amide bond of Gly48. This specific interaction was not present in inhibitor 1. These additional interactions observed with 2 may have contributed toward its impressive drug resistance profile. ⁹

Figure 1.

Structure of protease inhibitors 1,2, 3c–d.

Based upon the 2-bound X-ray crystal structure of HIV-1 protease, and given the significant gain in antiviral activity observed with the addition of C3-polar substituents on the Cp-THF P2 ligand, we subsequently speculated that N-substituted functionalities, particularly N-acyl, N-carbamate or N-sulfonyl derivatives could function as both a hydrogen-bonding donor and acceptor. The NH proton could conceivably form an effective hydrogen bond with the proximal Gly48 carbonyl while amide or urethane carbonyl oxygen may form an additional interaction with the protease backbone. Indeed, our previous exploration of such hydrogen bond donor and acceptor functionalities on P2-ligand frameworks led to remarkably potent HIV-1 protease inhibitors with broad-spectrum antiviral activity. ^4-9 Herein, we report the design, synthesis and biological evaluation of a series of HIV-1 protease inhibitors with C-3 substituted Cp-THF as the P2-ligand. A number of inhibitors exhibited exceptionally potent antiviral activity against a panel of multidrug-resistant HIV-1 variants. A protein-ligand X-ray crystal structure also provided important molecular insight into the ligand-binding site interactions.

The synthesis of ligands containing various N-substituents with either stereochemistry at C3 was accomplished starting from our previously reported optically active ketone intermediate 4¹⁰ as shown in Scheme 1. Ketone 4 was converted to methyloxime derivatives 5 in 96% yield. Reduction of 5 with a mixture of Pd/C and Raney-Ni under hydrogen pressure (80 psi) provided the corresponding amine as a 3:1 diastereomeric mixture.¹¹ The amine mixture was reacted with Ac₂O in the presence of Et₃N and a catalytic amount of DMAP to yield a mixture of isomeric TBS-protected amide intermediates in 80% yield. Treatment of the respective amides with TBAF in THF and chromatographic separation furnished diastereomerically pure ligands 6a and 6b in excellent yield. Similarly, the amine mixture was treated with methyl chloroformate and pyridine, or mesyl chloride and Et₃N to afford the corresponding carbamates and sulfonamides. Treatment of the respective crude mixtures with TBAF in THF followed by chromatographic separation afforded diastereoisomeric N-carbamate ligands 6c and 6d, and N-mesyl ligands 6e and 6f, respectively. The assignment of stereochemistry on the ligands was carried out by NOE or NOESY experiments of the corresponding mixed activated carbonates 8a, 8c, and 8e. To probe the importance of the free NH, we have synthesized ligand 6g containing a 3-(R)-O-dimethylaminocarbamate group. This was synthesized in three consecutive steps starting from our previously reported optically active alcohol 7.⁹ Treatment of 7 with 4-nitrophenyl chloroformate in the presence of pyridine furnished the corresponding mixed activated carbonate. The resulting carbonate was reacted with a bubbling stream of Me₂NH gas to provide the corresponding TBS-protected ligand. Removal of the TBS-group with TBAF furnished ligand 6g in excellent yield.

Scheme 1.

Synthesis of C3-substituted ligands 6a–g.

For the synthesis of HIV protease inhibitors, all ligand alcohols 6a–g were reacted with 4-nitrophenyl chloroformate in the presence of pyridine in CH₂Cl₂ to furnish the corresponding mixed activated carbonates 8a–g (Scheme 2).^9,10 The activated carbonates were then reacted with previously reported hydroxyethylamine isostere 9⁹ in the presence of Et₃N in THF/CH₃CN for 2–4 days to give corresponding inhibitors 3a–g.

Scheme 2.

Synthesis of protease inhibitors 3a–g.

Inhibitors 3a–g were initially tested in enzyme inhibitory assays using the method developed by Toth and Marshall,¹² and then evaluated for in vitro antiviral assays. Results are shown in Table 1. All inhibitors displayed impressive inhibitory potency and high antiviral activity. Inhibitors 3a and 3b that, contain a 3-(S)- or 3-(R)-N-acetyl substituent on the Cp-THF ligand, exhibited similar potency (7.4 and 7.5 pM, respectively). Interestingly, the stereochemistry at C3 seemed to have little effect. Inhibitor 3c with a 3-(S)-N-methoxycarbonyl displayed the most impressive enzyme and antiviral potency (K_i = 1.8 pM and IC₅₀ = 1.6 nM). The isomeric inhibitor 3d provided slightly lower potency. Inhibitors 3e and 3f that contain a 3-N-mesyl on the Cp-THF ligand, showed a substantial reduction in activity probably due to the increase in steric bulk created by the N-mesyl group. Inhibitor 3g that contains an O-substituted dimethylaminocarbamate in place of N-substituted carbamate at C3 provided a good antiviral activity, similar to that of 3d.

Table 1.

Structures and potency of inhibitors 3a–g^a

Entry	Inhibitor structure	Ki (pM)	IC50 (nM)b
1		7.4	25
2		7.5	31
3		1.8	1.6
4		4.0	4.6
5		32	28
6		180	50
7		20	4.7

^aValues are the mean value of at least two experiments.

^bHuman T-lymphoid (MT-2) cells (2 × 10³) were exposed to 100 TCID₅₀ of HIV-1_LAI and cultured in the presence of each PI, and IC₅₀ values were determined using the MTT assay. The IC₅₀ values of amprenavir (APV), saquinavir (SQV), indinavir (IDV), and darunavir (DRV) were 0.03, 0.015, 0.03, and 0.003 μM, respectively.

Inhibitors 3c and 3d, were further evaluated against a panel of multidrug-resistant (MDR) HIV-1 variants and their antiviral activities were compared to clinically available PI, darunavir (DRV).^6,13 Results are shown in Table 2. All inhibitors exhibited low nanomolar EC₅₀ values against the wild-type HIV-1_ERS104pre laboratory strain, isolated from a drug-naïve patient.¹³ Inhibitor 3d had the most potent activity (EC₅₀ = 3 nM) similar to that of DRV. When tested against a panel of multidrug-resistant HIV-1 strains, the EC₅₀ of 3d remained in the low nanomolar value range (15–24 nM) and its fold-changes in activity were similar to those observed with DRV.^6,13 Interestingly, inhibitor 3c, with the opposite (S) stereochemistry at C3, displayed slightly lower antiviral activities against all viral strains compared to 3d. However, the fold-changes in EC₅₀ for 3c remained low (<3) against all MDR HIV-1 viruses. The fold-changes contrasted with those of 3d and even DRV, for which the respective EC₅₀’s increased by a factor of at least three against the MDR viruses examined.¹⁴

Table 2.

Comparison of the antiviral activity of 3c, 3d, and DRV against multidrug-resistant HIV-1 variants

Virusa	EC50 (μM) ±SDs, (fold-change)b
	3c	3d	DRV
HIV-1ERS104pre (wt)	0.029 ± 0.002	0.003 ± 0.001	0.004 ± 0.001
HIV-1MDR/B (X4)	0.075 ± 0.011 (3)	0.018 ± 0.003 (6)	0.019 ± 0.006 (5)
HIV-1MDR/C (X4)	0.030 ± 0.006 (1)	0.015 ± 0.005 (5)	0.011 ± 0.003 (3)
HIV-1MDR/G (X4)	0.039 ± 0.001 (1)	0.020 ± 0.005 (7)	0.011 ± 0.002 (3)
HIV-1MDR/TM (X4)	0.074 ± 0.006 (3)	0.024 ± 0.004 (8)	0.028 ± 0.001 (7)

^aAmino acid substitutions identified in the protease-encoding region of HIV-1_ERS104pre, HIV-1_MDR/B, HIV-1_MDR/C, HIV-1_MDR/G, HIV-1_MDR/TM compared to the consensus type B sequence cited from the Los Alamos database include L63P in HIV-1_ERS104pre; L10I, K14R, L33I, M36I, M46I, F53I, K55R, I62V, L63P, A71V, G73S, V82A, L90M, I93L in HIV-1_MDR/B; L10I, I15V, K20R, L24I, M36I, M46L, I54V, I62V, L63P, K70Q, V82A, and L89M in HIV-1_MDR/C; L10I, V11I, T12E, I15V, L19I, R41K, M46L, L63P, A71T, V82A, and L90M in HIV-1_MDR/G; L10I, K14R, R41K, M46L, I54V, L63P, A71V, V82A, L90M, I93L in HIV-1_MDR/TM. HIV-1_ERS104pre served as a source of wild-type HIV-1.

^bEC₅₀ values were determined by using PHA-PBMs as target cells and the inhibition of p24 Gag protein production for each drug was used as an endpoint. The numbers in parentheses represent the fold-change in EC₅₀ values for each isolate compared to the EC₅₀ values for the wild-type HIV-1_ERS104pre. All assays were conducted in duplicate, and the data shown represent mean values (±1 standard deviations) derived from the results of two or three independent experiments. PHA-PBMs were derived from a single donor in each independent experiment. DRV (darunavir).

In order to gain molecular insights on the ligand-binding site interactions responsible for the potent activity and excellent resistance profile of 3c, we have determined the X-ray crystal structure of the HIV wild-type protease complexed with 3c (Fig. 2).¹⁵ The structure was refined to an R-factor of 14.9% and a resolution of 1.23 Å. The inhibitor is bound to the protease dimer in two orientations related by a 180° rotation, with a 50/50 relative occupancy. The protease backbone structure showed a very low rms deviation of 0.11 Å for all Cα carbons compared to the protease complexes of 2⁹ or DRV.²¹ As shown in Figure 2, the inhibitor interactions in the protease binding site extend from S2 to S2′ protease subsites and consist of a series of strong hydrogen bonds and weaker C–H⋯O and C–H⋯π interactions similar to those previously described for DRV,²¹ or inhibitor 2.⁹ The Cp-THF cyclic oxygen forms a strong hydrogen bond with the backbone amide NH of Asp29 in the protease S2-binding site, similar to that previously observed with other Cp-THF-based inhibitors 1⁵ and 2.⁹ Critical differences, however, are observed with the additional interactions that the 3-(S)-N-methoxycarbonyl amino substituent on the Cp-THF makes throughout the S2–S3 subsites. As shown in Figure 2, the carbamate NH forms a strong hydrogen bond with the Gly48 backbone carbonyl. The carbamate carbonyl is observed to interact with the Arg8′ guanidine side chain through a conserved water molecule. The methyl of the methoxy group then fits within the S3-hydrophobic pocket. The C3-N-methoxycarbonyl amino group creates a network of tight hydrogen bonds that literally links the protease flap region to the S2–S3 subsites’ dimer interface. These new interactions and enthalpic nature of the additional hydrogen bonding created by the P2-ligand may certainly exert an enhanced anchoring effect of the inhibitor into the S2-subsite and further stabilize the closed conformation of the protease–ligand complex.

Figure 2.

Stereoview of the X-ray structure of inhibitor 3c (green)-bound HIV-1 protease (PDB code: 4DFG). All strong hydrogen bonding interactions are shown as dotted lines.

In conclusion, we have reported the structure-based design of a series of highly potent HIV-1 protease inhibitors incorporating C3-substituted cyclopentyltetrahydrofuranyl urethanes as P2-ligands. Various C3-N-substituents were investigated in order to create multiple interactions in the S2-subsite of the protease and specifically with the protease flap region. Inhibitors 3c and 3d displayed remarkable inhibitory potency and antiviral activity. When tested against a panel of MDR HIV-1 strains, inhibitor 3d, with a 3-(R)-methoxycarbonyl on the Cp-THF ligand, provided the most impressive EC₅₀s and fold-changes in activity which are comparable to those observed with clinically available DRV. Isomeric inhibitor 3c displayed lower antiviral activity. However, it exhibited strikingly low fold-changes of antiviral activity when tested against MDR HIV-1 viruses. An X-ray crystal structure of the protease–3c complex was determined at a 1.23 Å resolution. Inhibitor 3c made extensive interactions throughout the protease binding site. The complex network of hydrogen-bonding interactions created by the N-methyl carbamate substituent in addition to those created by the isostere in the protease active site may account for the impressive antiviral activity and superb resistance profile observed with inhibitor 3c. Further designs along this line and ligand optimization are currently underway.