Design, synthesis and preliminary evaluation of peptidomimetic inhibitors of HIV aspartic protease with an epoxyalcohol core

Two peptidomimetic inhibitors based on a novel epoxyalcohol core were designed to target the epoxide ring at the catalytic aspartates of HIV-protease for irreversible inhibition of the enzyme. The inhibitors were synthesized with a multi-step approach which includes Horner-Emmons olefination of a phenylalanine-derived phosphono ketone, stereoselective reduction of the resulting trans -enones to allylic alcohols and syn -epoxidation of the latters. The epoxyalcohols thus obtained were assayed for their ability to inhibit HIV-PR and were shown to inhibit the protease with IC 50 values of 39 and 150 µ M, respectively. This confirms that the designed epoxides are recognised with fairly good affinity by the enzyme’s active site, a pre-requisite for selective irreversible inhibition


Introduction
The human immunodeficiency virus (HIV) is the causative agent of the acquired immunodeficiency sindrome (AIDS); in the last decade much effort has been devoted to the development of efficient chemotherapics that can control the progress of the viral infection. 1,2][3][4][5] HIV-PR is an aspartyl protease whose active form is a symmetric homodimer of a chain consisting of 99-residues.The two subunits are correlated by a C 2 axis running across the binding site, and each monomer contributes a catalytic aspartyl residue. 6,7The peptide substrate is recognized in an extended conformation 8 and the aspartate diad is responsible for the catalytic acceleration through a general acid / general base mechanism. 9HIV-PR inhibitors used in the clinical practice are peptidomimetics that reversibly inhibit the enzyme by competing with the substrate for the active site.[3][4]9 Mutations at, or near, the binding site may decrease the enzyme's affinity for an inhibitor, without significantly altering its activity, leading to functional mutant proteases that are resistant to reversible inhibitors.An alternative approach to the inactivation of HIV-PR is thus based on irreversible inhibitors; these should be less sensitive to mutations because a reduced affinity will lower the equilibrium concentration of the enzyme-inhibitor complex and slow down, but not necessarily stop, the reaction between the inhibitor and the enzyme leading to its inactivation.
Targeting the active site of aspartyl proteases for irreversible inhibition requires a potent electrophile, which must be able to alkylate the carboxylate group notwithstanding its inherent low nucleophilicity.At the same time the electrophile must be sufficiently selective not to interact with other potential targets.The combination of these two contrasting properties makes the design of efficient irreversible inhibitors a challenging problem.
It was shown some years ago that 3-(4-nitro)phenoxy-1,2-epoxypropane (EPNP, 1), a general inhibitor of aspartyl proteases, irreversibly inactivates HIV-PR by alkylating the active site Asp25 (K inact = 11mM). 15,16Not surprisingly, however, this activated epoxide is not selective and its reaction with DNA has been recently demonstrated. 17In 1994, Ortiz de Montellano and coworkers reported the irreversible inhibition of HIV-PR by epoxides and α,β-unsaturated ketones structurally derived from haloperidol. 18The same group later showed that only the haloperidol derived epoxides selectively alkylate one of the active-site aspartate residues, while reaction of the protease with the more reactive enones or ynones resulted in alkylation of cystein residues (Cys67 and Cys95) and the N-terminal proline in both subunits. 19Haloperidol derived epoxides include 2 (K inact = 521 µM) 16 and the simplified inhibitor 3 (K inact = 65 µM). 19ased on these reports, a number of designed inhibitors have been described in which the epoxide is incorporated in a peptide or peptidomimetic structure in order to improve the inhibitor's binding affinity; [20][21][22][23][24] some examples (4-6) are reported in Figure 1.A mechanism for the alkylation of the catalytic aspartate, involving hydrogen-bond assisted ring opening of the bound epoxide has also been proposed. 25,268][29] Herein we describe the design, synthesis and preliminary evaluation of HIV-PR inhibitors based on a novel epoxyalcohol motif.

Results and Discussion
Design.Structural studies of complexes of HIV-PR with peptide substrates and peptidomimetic inhibitors have allowed the conclusion that in the extended conformation in which the substrate is recognized by the protease, essential binding interactions are established between three residues on each side of the scissile bond and corresponding subsites of the enzyme. 8,9,13ccording to standard nomenclature, the residues are designated P3, P2, P1, P1', P2', P3' while the corresponding subsites are referred to as S3--S3'.

S2
Efficient peptidomimetic inhibitors can be obtained by replacing the scissile peptide bond P1-P1' with a non-scissile dipeptide isostere Ψ[P1-P1'] and optimizing the P3--P3' residues for binding to the corresponding subsites. 9,13,14Hydroxyethylene and dihydroxyethylene dipeptide isosteres 30 are widely used in the design of aspartic protease inhibitors as the enzyme-inhibitor complex is stabilized by interactions between the catalytic aspartates and the hydroxy groups of the isostere, which are believed to mimic the tetrahedral transition state for amide hydrolysis. 9,13e thus decided to incorporate a hydroxy group in the design of our inhibitors, whose function is to bind the active site carboxylates and hold the epoxide in the correct position for irreversible alkylation (Figure 2).Epoxyalcohol inhibitors 7 and 8 (Figure 3) were designed starting from the structure of hexameric inhibitor PV, based on a dihydroxyethylene isostere, which inhibits HIV-PR with an IC 50 of 15 nM. 28This C 2 -symmetric inhibitor has Phe in P1 and P1', an arrangement that has been shown to be most effective for HIV-PR inhibition, 9,13,14 Val in P2, P2' and is capped with phenoxyacetic acid (Poa) in both P3 and P3'.The latter residue has a low molecular weight and reduced peptide character while preserving hydrophobic interactions with the S3, S3' subsites.In 7 an epoxide ring is grafted on the P3--P1 portion of PV so as to generate an epoxyalcohol with the same configuration as in the central diol of PV.An aminomethyl group on the trans epoxide ring allows the introduction of a second Poa residue in a position intermediate between P1' and P2'; inhibitor 7 is thus expected to interact with the S3, S2, S1 and S1'/S2' subsites of the protease.While hexameric pseudopeptides are optimal for binding to the protease's active site, we have shown that pentameric and even tetrameric structures based on hydroxyethylene 29 and dihydroxyethylene isosteres 28 still provide sufficient binding interactions to inhibit the protease with IC 50 values in the nM to µM range.Since low molecular weight and reduced peptide character are important for the inhibitor's pharmacological properties, we decided to synthesize also the simplified epoxyalcohol The next steps (Scheme 2) follow the general approach previously described 32 for the synthesis of diaminodiol dipeptide isosteres.Thus, Horner-Emmons olefination of phosphonoketone 15a with the aldehyde 11, under the conditions described by Mikolajczik, 33 gave the E enone 16a in 78 % yield (Scheme 2).The E geometry of the double bond was confirmed by the 16 Hz coupling constant between the vinyl protons in the 1 H NMR spectrum of 16a.Similarly, the N-Boc enone 16b, was obtained from 11 and the corresponding Bocprotected phosphonoketone 15b. 32The enones 16 were then reduced with sodium borohydride in methanol, at 0 °C, giving the allylic alcohols 17a,b as 3 : 1 mixtures of diastereoisomers.Earlier work had allowed to establish that, in the NMR of allylic alcohols closely related to 17, 32,34 the H-4 vinylic proton of the S,R isomer (Scheme 2) always resonates at higher field than the corresponding proton of the S,S-isomer.A similar situation has also been observed in other allylic alcohols derived from phenylalanine. 35From the chemical shift of this diagnostic proton it was thus possible to assign the S,R and S,S configurations to the major and minor isomers of alcohols 17a,b, respectively.
The stereoselectivity of the reduction of enones 16 is consistent with chelation control by the metal or by the boron atom which might bind the carbamate nitrogen as suggested by Hoffmann et al. 36 (Figure 4a).Alternatively, the same result could be explained with a Felkin-Anh model in which NHBoc is the medium group and benzyl is the large group 32 (Figure 4b).Based on calculations of the volume of the two groups, it has been recently suggested that NHBoc is larger than benzyl; 35 however, the bulky t-butyl group of NHBoc is four bonds away from the reaction center, to which it is connected by a linear, and relatively small, array of atoms.Thus the effectiveness of NHBoc in shielding the carbonyl from attack by the nucleophile may be overestimated by these calculations.Although the stereoselectivity of the reduction was not high, the diastereoisomers could be separated and obtained in pure form by column chromatography.The enantiomeric purity of alcohols (S,R)-17a,b was assessed from the 1 H and 13 C NMR spectra of the corresponding Mosher's esters, in which only one set of signals was observed, indicating that no epimerization had occurred at the carbon atom adjacent to the carbonyl up to this stage in the synthesis.The N-Boc alcohol (S,R)-17b was deprotected and the phenoxyacetyl valine residue was introduced by reaction with the activated ester 13 giving alcohol 17c (Scheme 2).The final step is the epoxidation of allylic alcohols 17a,c to give the required epoxides 8 and 7.
It is well known that peracid epoxidation of acyclic allylic alcohols is controlled by hydrogen bonding between the peracid and the hydroxy group of the substrate, leading to syn-epoxyalcohols 37 and high syn stereoselectivity was found in the epoxidation of a series of allylic alcohols structurally related to 17; 32,38 on this basis the syn configuration shown in scheme 2 was assigned to epoxyalcohols 7 and 8.
Preliminary evaluation of the biological activity.Epoxyalcohols 7 and 8 were preliminarly assayed for their ability to competitively inhibit recombinant, wild-type HIV-PR using a standard activity test with a fluorogenic substrate. 28IC 50 values for 7, 8, for the reference inhibitor PV and for epoxyalcohol 18, which was available from a previous investigation, 32 are reported in Table 1.Data in the Table indicate that, on going from hexameric dihydroxyethylene inhibitor PV to tetrameric epoxyalcohol 7, inhibition potency decreases by three orders of magnitude.This is not surprising considering that 7 can only interact with four of the six protease's subsites involved in substrate binding, as discussed earlier in the section on design.On the other hand, the IC 50 of 7 is comparable with that of other tetrameric inhibitors based on hydroxyethylene 29 and dihydroxyethylene 28 isosteres, indicating that the more rigid trans-epoxide core of 7 does not interfere with binding to the protease's active site.Furthermore, very high affinity for the active site is not strictly required as 7 was designed as irreversible inhibitor.Removing the valine residue in P2 leads to a further increase in IC 50 , as observed for 8; an accurate evaluation of the inhibitory potency could not be obtained for this compound because of its poor solubility.Replacement of the Poa groups of 8 with Boc (in P2) and phenyl (P1'/P2') as in 18, considerably decreases the inhibitor's activity, confirming the ability of this residue to provide strong interactions with the protease. 39

Conclusions
A stereoselective approach originally developed for the synthesis of hydroxyethylene and dihydroxyethylene dipeptide isosteres has now been extended to the synthesis of HIV-PR inhibitors based on an epoxyalcohol core.Epoxyalcohols 7 and 8, designed to irreversibly inhibit HIV-PR by alkylating the catalytic carboxylate groups, were synthesized by this approach in approximately 10% yield overall, starting from phenylalanine.Both epoxyalcohols are recognized by HIV-PR leading to inhibition with IC 50 values in the µM range.As recognition by the binding site is a prerequisite for selective reaction of the epoxide with the active site carboxylates, the ability of epoxyalcohols 7 and 8 to competitively inhibit HIV-PR makes them good candidates for selective irreversible inhibition of the enzyme.Epoxyalcohol 7 is particularly promising in this respect with an IC 50 value comparable to that of other tetrameric inhibitors of HIV-PR and a molecular weight only slightly exceeding the limit of 500 postulated by Lipinski's "rule of five". 40A preliminary experiment indeed indicates that time-dependent inhibition is slowly taking place when HIV-PR is incubated with a given amount of epoxyalcohol 7.

Experimental Section
General Procedures.Moisture-sensitive reactions were carried out in oven-dried vessels under a positive argon pressure.Tetrahydrofuran was distilled from sodium-benzophenone prior to use.Flash column chromatography was performed on Merck silica gel 60 (230-400 mesh); Merck silica gel 60 F254 coated plastic sheets (0.25 mm) were used for TLC and developed with iodine and/or permanganate.Melting points were determined with a Büchi 510 open capillary apparatus and are uncorrected.Optical rotations were measured at 589 nm in methanol with a Perkin-Elmer 261 polarimeter fitted with a 10 cm cell.IR spectra were recorded as Nujol mulls, unless otherwise noted, on a JASCO 200 FT/IR spectrophotometer. 1 H NMR spectra (400 MHz) and 13 C NMR spectra (100.4MHz) were recorded for CDCl 3 solutions containing tetramethylsilane as an internal standard, on a Jeol EX400 spectrometer.Electrospray ionization mass spectra (ESI-MS) were obtained on a SCIEX Perkin-Elmer API1 spectrometer and electron impact mass spectra (EI-MS) were obtained on a VG 7070 spectrometer at the interdepartmental centre for mass spectrometry of the University of Trieste.CHN analyses were obtained on a Carlo Erba 1106 elemental analyzer.Diastereomeric ratios were measured by NMR.

N-(2-Oxoethyl)-2-phenoxyacetamide (11).
A solution of the crude acetal 10 (2.20 g, 8.23 mmol) in CH Cl (100 mL) was vigorously stirred with 40 mL of 20% aqueous TFA until 10 could not be detected at the TLC (3-4 days).The organic phase was separated, extracted with saturated aqueous NaHCO , water and sat.brine (50 mL each) and dried over anhydrous Na SO .Evaporation of the solvent gave a residue which was triturated with ether giving the solid amide 11 (1.31 g, 82%), m.p. 88-90 °C.(12).L-valine (2.1 g, 18 mmol) in 0.7 M aqueous NaOH (25 mL) was added dropwise to a solution of ester 9 in 50 mL dioxane and the reaction mixture was kept at room temperature for 1 h.The solution was acidified to pH 2 with conc.HCl and the acid 12 precipitated upon cooling (3.31

Figure 2 .
Figure 2. Proposed mechanism of inactivation of HIV-PR by epoxyalcohol inhibitors.

Table 1 .
IC 50 values for HIV-PR inhibition a From ref.28.