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Design and Synthesis of a Covalently Linked HIV-1 Protease Dimer Analog and Peptidomimetic Inhibitors Abstract

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Design and Synthesis of a Covalently Linked HIV-1 Protease Dimer Analog and Peptidomimetic Inhibitors Abstract Design and Synthesis of a Covalently Linked HIV-1 Protease Dimer Analog and Peptidomimetic Inhibitors Yoshiaki Kiso Department of Medicinal Chemistry, Kyoto Pharmaceutical University, Abstract: The HIV-1 protease analogs were synthesized by the solid-phase method and the dimer analog covalently linked by a disulfide bridge was constructed using the thioether chemical ligation method. The HIV-1 protease analogs effectively cleaved the Tyr-Phe-type substrate, but had weak affinity to the Tyr-Pro-type substrate. Consequently, the molecular recognition of the analogs differs from the wild-type enzyme. Based on the substrate transition-state mimetic concept, allophenylnorstatine-containing HIV protease inhibitors were designed and synthesized. Among them, a dipeptide-based HIV protease inhibitor, KNI-764, exhibited potent antiviral activities, low cytotoxicity and good pharmacokinetic properties. Also, KNI-764 showed strong inhibition against both wild-type HIV-1 protease and synthetic HIV protease analogs, whereas saquinavir, ritonavir, and indinavir weakly inhibited the synthetic protease analogs. This study suggests that the small-sized dipeptide HIV protease inhibitor, KNI-764, is a good candidate for anti-HIV drugs 1. Introduction Protection/deprotection steps and amide-forming reactions are important in peptide synthesis. We have developed new methods and reagents such as reductive acidolysis (ref. 1), 2-(benzotriazole-1-y1)- oxy-1,3-dimethylimidazolidinium hexafluorophosphate (BOI) (ref. 2), and 2-chloro-1,3-dimethylimi- dazolidinium hexafluorophosphate (CIP) (Fig.1) (ref. 3). These new methods are successfully applied to the syntheses of biologically important peptides. The modern synthetic methods (ref. 4) of peptide chemistry have greatly contributed to the fields important for human being, such as AIDS research. Fig. 1. Structure of BOI and CIP. 896 ( 32 ) J . Synth . Org . Chem . , Jpn . Fig. 2. The overlapping gag/pol reading frame of HIV-1, the polyprotein translation products Pr55gag and Pr160gag-pol, and the proteolytic cleavage sites. (gag, group- specific antigen; pol, polymerase; TF, transframe protein; PR, protease; RT, reverse transcriptase; RN, ribonuclease H; IN, integrase.). The human immunodeficiency virus type-1 (HIV-1) codes for a virus-specific aspartic protease responsible for processing the gag and gag-pol polyproteins and for the proliferation of the retrovirus (Fig. 2). The HIV-1 protease, a 99 amino acid residues peptide, functions in homodimeric form which is associated non-covalently. The HIV-1 protease is a potential target for the development of anti-AIDS agents (ref. 5). Mam- malian aspartic proteases such as renin and pepsin have two characteristic Asp-Thr-Gly sequences at the active center of the enzymes and both side-chain carboxyl groups are important in the catalysis of the peptide bond cleavage. In contrast, the retroviral protease has only one Asp-Thr-Gly sequence and functions as a homodimer. The HIV-1 protease can recognize Phe-Pro and Tyr-Pro sequences as the retrovirus-specific cleavage site, but mammalian aspartic proteases do not have such specificity. These features provided a basis for the rational design of selective HIV protease-targeted drugs for the treat- ment of AIDS and related complex. 2. Synthesis of HIV-1 Protease Analogs HIV-1 protease is formed from two identical 99 amino acid peptides (Table 1) by means of hydro- philic and hydrophobic interactions, not by a disulfide bond. Kent et al. (ref. 6) synthesized an HIV-1 protease analog, in which the two Cys residues were replaced by the isosteric L-a-amino-n-butyric acid, whereas we replaced the two Cys residues by a natural amino acid, L-alanine. The [Ala67'95]derivative (1) of HIV-1 protease (NY-5 isolate) was synthesized by the solid-phase method based on the Boc-Bzl strategy using PAM resin (ref. 7). Each Boc-amino acid derivative was incorporated by the efficient Vol . 56, No .11 (November 1998) ( 33 ) 897 Table 1. Amino acid sequences of HIV proteases and analogs . method of the solid-phase peptide synthesis (SPPS) (Fig. 3)(refs 2, 4) , which consists of Nƒ¿-selective deprotection by dilute methanesulfonic acid, in situ neutralization, and rapid coupling reaction using BOI reagent. The final deprotection was performed by the HF method, and the product was purified by the usual method (ref. 6). The chemically synthesized [Ala67'95]-HIV- 1 protease had sufficient cleaving effect on the synthetic nonapeptide substrate, Ac-Arg-Ala-Ser-Gln-Asn-Tyr-Pro-Val-Val-NH2 (pH 5 .75, 37•Ž)(ref. 8). We examined the protease inhibitory activities of the peptide compounds containing the hydroxymethylcarbonyl (HMC) structure by the assay system using these enzyme analogs and the syn- thetic nonapeptide substrate. Fig. 3. Coupling pathway of efficient SPPS employing in situ neutralization . DIEA = diisopropylethylamine. 898 ( 34 ) J. Synth. Org. Chem., Jpn . Fig. 4. Synthesis of N-terminal fragment of HIV-1protease analog . MSA = methanesulfonic acid, DCM = dichioromethane, DOX = dioxane, HBTU = 2- (1H-benzotriazole-1 -yI)-oxy-1 ,1 ,3,3-tetramethyluronium hexafluorophosphate, DMF = dimethylformamide, DIEA = diisopropyl ethylamine. 2.1. Thioester Linkage Chemical Ligation Furthermore, we synthesized [Tyr6,42,Nle36,46, (NHCH2COSCH2C0)51-52, Ala67,95]HIV-1 protease (2) (NY-5 isolate) using the thioester linkage chemical ligation method (ref. 9). The N-terminal portion was synthesized as shown in Figure 4, whereas the C-terminal portion was synthesized as shown in Figure 5 by the efficient Boc strategy of SPPS stated above. The chemical ligation reaction of [Tyr6,42, Nle36,46]-HIV-1 protease (1-50)-Gly-SH and BrCH2C0-[Ala67,95]-HIV-1 protease (53-99) was carried out Fig. 5. Synthesis of C-terminal fragment of HIV-1 protease analog. DIPCDI = diisopropylcarbodiimide, HOBt = N1-hydroxybenzotriazole. Vol.56, No.11 (November 1998) ( 35 ) 899 in pH 4.0 medium. The MALDI-TOF/MS value (m/z 10694.9) of the final product purified by high- performance liquid chromatography (HPLC) agreed well with the theoretical value. After folding, the ligated compound was found to exhibit comparable enzymatic activity to the recombinant wild-type HIV-1 protease using the synthetic nonapeptide substrate. 2.2. Thioether Linkage Chemical Ligation Englebretsen et al. (ref. 10) reported a synthesis of an HIV-1 protease analog employing thioether forming ligation which was a reaction of peptide-mercaptoethylamide with bromoacetyl-segment. In the synthesis, they prepared the mercaptoamide segment using a thiol-releasing resin by acid treatments. However, this strategy sometimes incurred trouble due to the released thiol group. We have investigated various linkages releasing the thiol function by acid treatment, which contained substituted benzyl thioethers. However, large peptide segments could not be obtained in good purity using these linkages. We suspected that the results were due to the thiol involving side reactions and attempted to protect the thiol group during the acid treatment. In order to obtain the thiol segment without difficulty, we employed a new strategy using a multi- detachable linker to avoid contact of thiol with strong acids (ref. 11). An aminoethyldithio-2-isobutyric acid (AEDI), which can connect the mercaptoamide peptide to solid support by a disulfide bond, is stable under the conditions during Fmoc-based SPPS and acid treatments. We introduced AEDI on an acid-labile resin and used it as a multi-detachable linker. In this strategy, the peptide containing a masked thiol moiety was cleaved from the resin by acid deprotection, and then the thiol was generated by reduc- tion. A mercaptoethylamide peptide segment, HIV-1 protease (1-50)-NHCH2CH2SH, was prepared start- ing from an Fmoc-AEDI-O-Clt-resin (Fig. 6). The SPPS was achieved by ABI 431A synthesizer with standard DCC-HOBt protocol, and then the peptide segment containing the linker moiety was cleaved by HF-dimethyl sulfide-m-cresol (3:6:1)(0•Ž, 1h). The treatment of the resultant with dithiothreitol (DTT) in 6M guanidine•HC1 (pH8) gave the desired product in good purity. The purification by RP- Fig. 6. Synthetic scheme for HIV-1protease analog (3) using thioether forming ligation. Clt: 2-chiorotrityl 900 ( 36 ) J . Synth . Org . Chem . , Jpn HPLC gave a homogeneous product in 29.9% overall yield, which was characterized by amino acid analysis of the hydrolyzate and MALDI-TOF MS. Another segment, BrCH2C0-[Ala67'95]HIV-1 protease (52-99) was also prepared by the usual SPPS, deprotection by HF-m-cresol and purification on RP-HPLC (8.0%). The ligation of the above two segments was carried out in 6M guanidine•HBr, 200mM Tris buffer (pH8.5) at r.t. for 5h with vigorous stirring. The product was purified on Hiload 16/60 superdex 75pg in 8M urea and then gel-filtered to remove urea. The desired product, [NHCH2CH2SCH2CO51-52, Ala67,95]- HIV-1 protease (3) was obtained in 50% yield and characterized by MALDI-TOF MS (m/z: 10759.2•}0.1%, calcd: 10758.6). After folding, the synthetic protease analog possessed enzymatic activity, though it showed a slight difference in the recognition of substrates. The synthetic protease had comparable enzymatic activity to the wild-type enzyme using a substrate, Lys-Ala-Arg-Val-Tyr*Phe(NO2)-Glu-Ala-Nle-NH2. The Km values of the synthetic and wild-type proteases were 22.1 and 18.3 .tM, respectively. On the other hand, the protease analog had larger Km (23.5 mM) than the wild-type protease (6.46 mM) to Ser-Gln- Asn-Tyr*Pro-Ile-Val substrate. The non-peptide region at the flap of the synthetic protease may affect the recognition of the substrate. 2.3. A Covalently-linked HIV-1 Protease Dimer Analog Interestingly, single-chain tethered dimers of HIV-1 proteases, artificial peptides having the same topology as pepsin-like typical aspartic proteases, also show similar enzymatic activity and are more stable than the natural enzyme (ref. 12). We synthesized an HIV-1 protease dimer analog which consists of two identical peptide chains covalently linked by a disulfide bridge (Fig.
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