A series of novel HIV-1 protease inhibitors based on the (hydroxyethylamino)-sulfonamide isostere incorporating substituted phenyls and benzheterocycle derivatives bearing rich hydrogen bonding acceptors as P2 ligands were synthesized. Prolonged chain linking the benzhereocycle to the carbonyl group resulted in partial loss of binding affinities. Introduction of a small alkyl substituent with appropriate size to the -CH2- of P1-P2 linkage as a side chain resulted in improved inhibitory potency, and in this study, isopropyl was the best side chain. Replacement of the isobutyl substituent at P1′group with phenyl substituent decreased the inhibitory potency. One of the most potent inhibitor, compound 23 showing high affinity to HIV-1 protease with an IC50 value of 5 nm, also exhibited good anti-SIV activity (EC50 = 0.8 μm) with low toxicity (TC50 > 100 μm). The flexible docking of inhibitor 23 to HIV-1 protease active site rationalized the interactions with protease.
Inhibition of human immunodeficiency virus type-1 protease (HIV-1 PR) results in the production of immature, non-infectious virus particles, which provides a promising therapeutic target for anti-HIV treatment (1,2). Nowadays, HIV-1 protease inhibitors (PIs) constitute one of the essential components of the highly active antiretroviral therapy (HAART) (3,4). Up to date, 10 PIs have been approved by FDA, and a number of PIs are in advanced clinical trials (5). Most of those inhibitors are developed based on the peptidomimetic structures that mimic the transition state for the cleavage of the enzyme’s natural substrate (6,7). A better understanding of HIV-1 protease-ligand cocrystal structures and molecular modeling, and the interactions between HIV-1 protease and inhibitors will facilitate the discovery of novel potent PIs.
Most efforts on developing new PIs have been focused on designing various P2–P3 and P2′groups, which were expected to possess high affinity to HIV-1 PR (8–12). Based on a rationale of mimicking its substrate for PR, several ligands with high affinity were discovered, some of which are shown in Figure 1. All of the P2 groups of HIV-1 PIs harbor a carbonyl group for linkage to P1 group, thus providing a hydrogen bonding with the water molecule that bridges inhibitors to the flaps of the enzyme (11,12). It has also been revealed that introduction of those P2 groups will largely improve hydrophobic and hydrogen bonding interactions with the enzyme active site, resulting in substantial increase of inhibitory potency. The oxygen of 3(S)-tetrahydrofuryloxy (THF)substituent at P2 group of amprenavir (APV in Figure 2), one of the approved PIs, is involved in weak interaction with the Asp-29 and Asp-30 backbone amides (13). Structurally similar to APV, Darunavir (DRV in Figure 2) that replaces 3(S)- THF substituent of amprenavir with bis-THF has showed much superiority over protease inhibitory activity, antiviral potency, drug resistance, and pharmacokinetic properties. Structural analysis has revealed that DRV closely contacts with the main chain of the protease active amino acids (Asp-29 and Asp-30), and it has one extra hydrogen bond formed with the protein backbone in the S2 pocket (14–16).
These SAR studies and the information on the interaction of protease and inhibitors have enabled us to seek for novel PIs. We designed the skeleton 1 and 2 based on the (R)-(hydroxyethylamino) sulfonamide isostere presented in APV and DRV in Figure 2. Various substituted phenyls and benzheterocycles P2 ligands bearing abundant hydrogen bonding acceptors were introduced to expect effective hydrogen bond to Asp-29 and Asp-30 and to fill well in the hydrophobic pocket of S2 active sites. To find out the optimal length between P1 and P2 groups, various linkages with different lengths between the benzhereocycle and the carbonyl group were designed. Different alkyl substituents were also introduced to P1-P2 linker to study the steric effect and hydrophobic interaction with amino acids residues in the S1-S2. We also replaced the isobutyl substituent at P1′group with benzyl substituent, which enhanced the symmetry characteristics of the inhibitors. The acetamide substituent was designed to allow for additional interactions with side chains of S2′subsites and to maintain the critical hydrogen bonding to Asp-30. Herein, we report our preliminary findings in developing novel protease inhibitors.
Methods and Materials
Chemical synthetic procedure
The syntheses of compounds 8a-c are summarized in Scheme 1. The protected phenylalanine 3 was converted to the chloromethyl ketone 4 by treating the diazoketone intermediate with hydrochloric solution in ether (10). Chelation-controlled reduction of ketone by LiAlH(OtBu)3 in ethanol at −78 °C gave a greater degree of diastereoselectivity to the erythro chlorohydrin (17). The final product was obtained by crystallization in ethyl acetate/hexane. Treatment of amino chlorohydrin with anhydrous potassium carbonate in methanol gave the corresponding amino epoxide 5. Terminal opening of the epoxide 5 yielded the intermediates 6a-b in nearly quantitatively yield. The primary amines 8a-c were obtained in a two-step sequence by first reaction of 6a or 6b with 4-substituted benzenesulfonyl chloride and subsequent removal of Cbz group with Pd/C and ammonium formate.
The coupling step for these compounds was performed using HBTU as a catalyst in the presence of NMM under N2 atmosphere to generate our target inhibitors (18; Scheme 1).
Scheme 2 illustrates the procedure for the synthesis of acid 11. 2-Benzoxazolinone 9 was prepared from o-aminophenol following a described method (17). Reaction of 9 and ethyl bromoacetate in benzene in the presence of NaH and subsequent hydrolysis of ester 10 by 4 N HCl in dioxane gave the acid 11 in overall 40% yield. All the other acids were obtained commercially or prepared by the following methods: The N, N-phthaloyl-amino acids were obtained according to previously described procedure (19,20). The N-saccharinyl acetic acid was prepared from sodium saccharin and chloroacetic acid (21).
Inhibition data for HIV-1 protease were obtained as follows. Recombinant HIV-1 protease was expressed from Escherichia coli and purified as previously described (4). Distinct inhibitor concentrations were dissolved in 4.0 μL of DMSO and incubated with 1.0 μL of HIV-1 protease (2 μm) in 200 μL of assay buffer (0.1 m MES, 0.2 m NaCl, 5 mm EDTA, pH5.5) at 4 °C for 30 min. Then, the peptide substrate (Lys-Ala-Arg-Val-Leu-Phe(NO2)-Glu-Ala-Met, purchased from AC-SCIENTIFIC Inc, Xi'an City, China) dissolved in 4.0 μL of DMSO was added into the system to a final concentration of 500 nm. The hydrolysis of the substrate resulted in a linear increase in UV absorbance at 310 nm over a 30-min period The IC50 was defined as the concentration required to inhibit the absorbance by 50% (22). One of the currently marketed drugs, Indinavir (IDV), was used as the positive control.
Cell-based anti-SIV assay
The toxicity of the compounds was measured in a 96-well microplate containing 1 × 105 CEM cells/mL per well and containing appropriate dilutions of the tested compounds. After 5 days of incubation at 37 °C in 5% CO2 containing humidified air, the cytotoxicity of the agents was determined by MTT assay. The TC50 value was defined as the concentration that elicits cytotoxicity in 50% of CEM cells. Inhibition of SIV was measured in CEM174 cell cultures infected with 100 TCID50 of SIV per well, and the other conditions was the same as in toxicity assay. CEM giant (SIV-induced syncytium) cell formation was examined microscopically. The EC50 was defined as the compound concentration required to protect cells against the cytopathogenicity of SIV by 50%. Every compound was tested twice. IDV was used as the positive control at a concentration of 10 μm.
To rationalize these observations, a modeling study was performed, using Program AutoDock 3.0 for docking inhibitors on the HIV-1 protease active site (23,24). The crystal structure of HIV PR in complex with its inhibitor 1 was retrieved from Brookhaven Protein Data Bank (entry code 1HPV). Ligands and solvents removed from the PDB file and 3D grids (40 Å × 40 Å × 40 Å, with 0.375 Å grid spacing) were calculated using AutoGrid algorithm. Ligand structures were constructed and set up for flexible docking using AutoDockTools, with 12 ∼15 rotatable bonds for the compounds. The Lamarckian genetic algorithm was applied to calculate the interactions between molecules and HIV PR, following a protocol of 30 independent runs per ligand with an initial population of 50 randomly placed individuals, a maximum number of 1.5 × 106 energy evaluations and a docking runs of 270 000. The probability of performing local searches on an individual in the population was 0.06, using a maximum of 300 iterations per local search. The predicted binding free energy of inhibitor (ΔG in Table 1) was used to evaluate their binding affinity to HIV-1 PR.
Table 1. Structure, inhibitory potencies, toxicities, and antiviral activities of inhibitors with various P2–P3, P1′ and P2′ groups
Results and Discussion
Table 1 showed the activity results of the title compounds. Inhibitors 12a and 15a (IC50: 8 nm and 20 nm, EC50: 1.0 μm and 2.0 μm) with benzheterocycle as P2 ligand displayed higher inhibitory activities than 21a and 24 with aryl group. And the docking energy also showed that the 12a and 15a had lower ΔG than 21a and 24. We suspected that a hydrogen bond might be formed between the N atom in the aryl ring and the PR, which reduced the docking energy and resulted a better activity. However, when we added another two carbonyl groups into the aryl ring, the compounds 15-17 compared with 12-14 did not get better inhibitory activities as we expected. Also, the replacement of one carbonyl of phthaloyl with sulfone group gave inhibitors 22 with similar potency to 15a, indicating that the addition of an oxygen atom did not increase the inhibitory activity. These results indicated that the additional hetero-atoms in R2 group could not form hydrogen bonds with the protease, but increased the polarity of R2 group which led to higher docking energy and reduced the IC50 and EC50 values. On the other hand, the compounds 15-17 and 22 had lower cellular toxicities than 12-14, so we suspected that the higher polarity might affect their entrancing through the cell membrane, which could reduce their toxicities but also depressed their antiviral activities at the same time. Compounds 21a and 21b with naphthyl group as R2 exhibited lower activities, which might be because the naphthyl group was too bulky for the S2-S3 subsets and unfavorable their interactions with the residues was weakened as the docking energy was higher. And according to the lower polarity of the R2 group, the compounds 21a and 21b showed lower toxicities, which was consistent with our suspect earlier. The higher toxicity of inhibitors 12b-14b and 21b suggested that replacement of acetamide with bromide resulted in the enhancement of toxicity.
Inhibitor 23 exhibited significant ability to inhibit enzyme activity, with an IC50 value of 5 nm benefiting from the incorporation of 2-benzoxalinone as P2–P3 ligand. The most active compound 23 showed the lowest docking energy (−13.82 Kcal/mol). The top-ranked docked conformation of inhibitor 23 is superimposed with the experimentally determined binding structure of Vx-478 (amprenavir, 1hpv). As seen from this model, it appeared that 2-benzoxazolinone carbonyl oxygen resided within an effective hydrogen bonding distance to Asp29 and Asp30 (3.3 Å) in the S2 region of the active site. Additionally, the phenyl ring interacted with the amino acids residues (Ile47) within the S2 pockets. The hydrogen bonding interaction between acetamide carbonyl and Asp29′ could be formed (distance 3.3 Å). All these interactions contributed to the high inhibitory potency of inhibitor 23.
To explore the optimal length between the P2 group and the carbonyl group, we designed several inhibitors with different linkages between benzheterocycle and the carbonyl group. Compared with 12a, inhibitor 13a and 14a showed attenuated inhibitory activities with 4-fold and 7.5-fold decrease in IC50, respectively. Similarly, the inhibitory potency of 16a and 17a was reduced when compared with that of 15a (IC50 =20 nm). These reduced inhibition data might result from the increased length linking the ring to the carbonyl group, such change presumably rendered the aromatic portion not fill well in the S2 pocket and interfered with the interface between the inhibitor and residues in the protease active site.
We also attempted to introduce a methyl substituent to inhibitor 15a at the -CH2- of P1-P2 linkage to yield inhibitor 18a. The introduction of methyl substituent in the linkage resulted in a 1.5-fold improvement in potency (IC50 = 12 nm). The increased inhibitory potency might be because of the additional lipophilic interactions with the residues within S1-S2 subsites by adding methyl substituent. And 15a and 18a showed equivalent ability to SIV-infected cells with moderate EC50 value (2.0 μm), which meant the increased bulk did not impress their permeance into the cells. To extend this observation, larger alkyl substituents, isopropyl substituent, and isobutyl substituent were introduced to the -CH2- of P1-P2 linkage to produce 19a and 20a. However, decreased inhibitory potency were observed, which might be because the larger alkyl group within S2 pocket affected the critical hydrogen bond to Asp 29.
In addition to the screening of P2-P3 ligands, attempts to change P1′ and P2′ ligands were conducted. Replacement of acetamide group of 12a-21a with bromide produced 12b-21b, which resulted in more or less decrease in inhibitory potency except 13b and 21b. The hydrogen bonding (acetamide CO to Asp29 NH), contributing to the higher affinity to HIV-1 protease, might be sacrificed by this replacement to result in decrease in potency. Although 13b and 21b showed higher IC50 values, they still exhibited better ΔG and EC50 results than 13a and 21a, respectively, so we suspected that there was some other factors might affect their enzymatic inhibitory activities, which we were studying now. To match the C2-symmetric feature of HIV-1 protease, benzyl was attempted to replace isobutyl to enhance the symmetry of inhibitors (12c-20c). However, the unexpected decreases in potency were observed for these replacements. This observation indicated that the smaller isobutyl was the more preferable ligand for HIV-1 protease S1′ pocket.
Conclusions and Future Directions
A series of novel HIV-1 protease inhibitors based on the (hydroxyethylamino)-sulfonamide isostere incorporating substituted phenyls and benzheterocycle derivatives bearing rich hydrogen bonding acceptors as P2 ligands were synthesized. Prolonged chain linking the benzheterocycle to the carbonyl group resulted in partial loss of binding affinities. Introduction of a small alkyl substituent with appropriate size to the -CH2- of P1-P2 linkage as a side chain resulted in improved inhibitory potency, and in this study, isopropyl was the best side chain. Replacement of the isobutyl substituent at P1′group with phenyl substituent decreased the inhibitory potency. One of the most potent inhibitors, compound 23 showing high affinity to HIV-1 protease with an IC50 value of 5 nm, also exhibited good anti-SIV activity (EC50 =0.8 μm) with low toxicity (TC50 > 100 μm). The flexible docking of inhibitor 23 to HIV-1 protease active site rationalized the interactions with protease. Further design and chemical modifications of these inhibitors utilizing structure-based drug design strategies are currently underway.
We are grateful for the financial support from the National Natural Science Foundation of China (NO. 30670415). We also acknowledge Prof. Arthur J. Olson of the Scripps Research Institute in La Jolla, CA for his provision of the AutoDock 3.0 program.