WO1991008221A1

WO1991008221A1 - Peptide inhibitors of hiv protease

Info

Publication number: WO1991008221A1
Application number: PCT/US1990/007059
Authority: WO
Inventors: Daniel H. Rich; Garland R. Marshall; Jeremy Green; Chongqing Sun
Original assignee: Wisconsin Alumni Research Foundation
Priority date: 1989-12-04
Filing date: 1990-12-03
Publication date: 1991-06-13

Abstract

The invention provides HIV protease inhibitors which are potential drugs for HIV infected patients or cells. Preferably, peptides are provided that have an amino alcohol core that mimics a Phe-Pro and/or Leu-Pro binding site for the protease. The core is preferably ringed by at least three amino acids. In one form, an N-terminal cap is positioned adjacent to an Asn residue.

Description

PEPTIDE INHIBITORS OF HIV PROTEASE

This invention was made with U.S. government support awarded by the National Institutes Of Health (NIH) Grant Nos: GM40092, DK20100, AI27302 The U.S. government has certain rights in this invention.

Technical Field

This invention relates to uses of a modified peptide as an inhibitor of HIV protease. More particularly, it relates to incorporating a ino-alcohol variants of the protease's preferred binding site in a special peptide backbone to preferentially bind and occupy the protease.

Background Art

An important problem facing medical science is the development of drugs for those already infected by HIV. Inhibition of viral proteases as an approach to antiviral therapy has been demonstrated with another virus by Korant et al. , 32 J. Cell. Biochem. 91-95 (1986), who showed that an inhibitor of cysteine proteases, cystatin, inhibited replication of poliovirus in tissue culture. Also, Oroszlan, Abstract AIDS Symposium (June 1987) demonstrated inhibition by pepstatin of processing of gag-pol poly protein by purified HIV protease in vitro and in tissue culture in the presence of DMSO. The disclosure of the above articles and of all other articles referred to herein are incorporated by reference as if fully set forth herein.

The goals of any drug to be used in humans are that the drug should have high potency, high specificity, few side effects, stability, and reasonable costs of manufacture. Unfortunately, to date no drug for AIDS inhibition treatment has satisfactorily met all of these criteria.

Disclosure Of The Invention

We identify herein peptide variants which preferentially bind to HIV protease so as to inhibit its activity. In one embodiment, the present invention provides a peptide having the moiety:

where R_ is selected from the group consisting of isobutyl, benzyl, cyclohexylmethyl, and other arylalkyl or alkyl moieties with less than twelve carbons; and where R2 and R3 each has at least one amino acid residue, and at least one of R2 or R3 has at least two amino acid residues.

In another aspect, R2 is also linked to the following moiety:

0

I aryl residue - c -

Preferably, R2 and R3 are residues of one or more of the twenty amino acids commonly found in proteins or one of the other naturally occurring amino acids. See A. Lenhniger, Biochemistry, p.69-72 (1970). In an especially preferred form, R2 contains an asparagine residue. Also, Rl is preferably a side chain found on a naturally occurring amino acid.

The objects of the invention therefore include providing compositions of the above kind that are relatively inexpensive to produce and will inhibit HIV protease. These and still other objects and advantages of the present invention will be apparent from the description which follows. The following embodiments do not represent the full scope of the invention. Rather, the invention may be employed in other embodiments.

Reference is therefore to be made to the claims herein for interpreting the scope of the present invention.

Brief Description Of The Drawings

Fig. 1 depicts the synthesis of half of a peptide of the leucine amino alcohol type;

Fig. 2 depicts the synthesis of the other half of the molecule;

Fig. 3 depicts the synthesis of three final products;

Fig. 4 is similar to Fig. 1, but depicts phenyl variants;

Fig. 5 is similar to Fig. 3, but depicts phenyl variants; Fig. 6 depicts another system for protecting one end of the final product;

Fig. 7 depicts the synthesis of one-half of another peptide; Fig. 8 depicts the sythesis of two variants of the other half of the peptide;

Fig. 9 depicts two coupling reactions; and

Fig. 10 depicts two N-terminal caps.

Best Modes For Carrying Out The Invention

General Procedures

In the discussion below, the following abbreviations are used: AcOH - acetic acid; Boc - t-butoxycarbonyl; BuOH - butanol; DCM - dichloromethane; DMF - N,N-dimethyl- formamide; DMSO - dimethylsulphoxide; EDCI - N-ethyl-N'- (3-dimethylaminopropyl)carbodiimide; HOBt - 1-hydroxyben- zotriazole; MeOH - methanol; NMM - N-methylmorpholine; Z - benzyloxycarbonyl. A: Isobutyl chloroformate mediated coupling reactions:

To a cold (-25°C) solution of Nα-protected amino acid (100 mmol) in dry dichloromethane (200-400 rtiL) is added N- methylmorpholine (100 mmol) followed by careful addition of isobutyl chloroformate (100 mmol) maintaining the temperature of the solution between -15 and -25°C. The solution is stirred at -25°C for a further 20 minutes, then a solution of amino acid or peptide ester hydrohalide or toluenesulphonate salt (100 mmol) in DMF and/or DCM is added followed by addition of NMM (100 mmol). The reaction is stirred for 1 hour at -20°C and for a further 1-3 hours at room temperature. The solvent is removed in vacuo and the residue taken up in ethyl acetate and washed with saturated NaHCθ3 (x2), 5% citric acid solution, saturated NaHCθ3, water and brine, and dried over MgSθ4. Evaporation of the solvent yields a solid which is recrys- tallized from a suitable solvent combination. B: EDCI/HOBt mediated peptide couplings:

Boc amino acid and HOBt (1.5 equivalent) are dissolved in dry DMF (2-5 mL per mmol) and cooled to 0°C. EDCI (1.1 equivalent) is added and the solution stirred for 30-40 minutes. A solution of amino acid or peptide, ester or chloromethylketone, amine hydrochloride (0.9 equivalent) in DMF (1-5 mL per mmol) is added, followed by NMM (0.9 equivalent). The reaction is stirred for 1- 2 hours at 0°C and overnight at room temperature. The reaction is worked up by direct precipitation of the crude product from a concentrated DMF solution by careful addition of a large volume of 60% saturated NaHCθ3. The precipitate is collected by filtration over a hardened filter paper (Whatman No. 50 or 54), and the solid washed thoroughly with saturated NaHCθ3 (200-500 mL), water (200- 500 mL), 5% citric acid solution (200-500 ml), and water (200-500 mL) . The product is dried in vacuo, redissolved in DMF, filtered through a glass wool plug and reprecipitated by addition of water. Again the product is filtered, washed with water, and dried. C: Cleavage of Boc protecting group with 4N HC1 in dioxane:

The Boc-protected peptide or amino acid derivative is dissolved in 4N HC1 in dioxane (Pierce) (6-40 equivalents) and stirred at room temperature for 30-60 minutes, the course of reaction being monitored by thin layer chromatograph . Once complete, the solution is evaporated to dryness at <30°C and the residue evaporated from anhydrous ether (x3) to remove traces of HC1. The crude product is dried in vacuo in a desiccator over NaOH pellets overnight and is used without further purification or characterization. D: Preparation of amino acid diazomethyl ketones:

Nα-Protected amino acid (50 mmol) is dissolved in dry THF (100 mL) and cooled to -25°C. NMM (50 mmol) is added, followed by isobutylchloroformate (50 mmol), with the temperature being maintained at -25°C during addition.

After stirring for 5 minutes anhydrous ether (100 mL) is added while simultaneously cooling the solution to -70°C. The cold solution is quickly filtered under a blanket of N2 and the cold filtrate treated with a solution containing approximately 70 mmol of diazomethane in ether (250 ml). The reaction is allowed to gradually warm up to room temperature and excess diazomethane is removed by purging the solution with a stream of N2 for 15- 30 minutes. The solution is evaporated to dryness and the residue taken up in ether (200 mL) , and washed with saturated NaHC03 and brine, and dried over MgS04> The product may be crystallized from ether-hexane or hexane. E: Conversion of diazomethylketones to halomethylketones: The amino acid diazomethylketone is dissolved in anhydrous dioxane. After slow addition of the first equivalent of HCl in dioxane is complete (slow addition is necessitated by the rapid evolution of nitrogen), a further 6-10 equivalents is added and the cooling bath removed. The reaction is stirred at room temperature for 1 hour and the product precipitated by addition of ether. The product is filtered, washed with copious amounts of ether, and dried in vacuo over NaOH pellets. Prolonged storage of these compounds results in significant colouration and decomposition. F: Acetylation of peptides:

A solution of the peptide amine hydrochloride in dry DMF (10-20 mL/g) is cooled to 0°C in an ice-water bath and neutralized with triethylamine (2.1-2.5 equivalents). A solution of acetyl chloride in DCM (0.3-1.8 M) is then added dropwise and the reaction left to stir for 1 hour at 0°C, and for a further 1 hour at room temperature. The solution is then concentrated to a small volume under high vacuum, and water is added to precipitate the acetylated peptide, which is collected by filtration, and washed with water, before drying in vacuo over NaOH pellets. G: Preparation of Aminoketones:

N -Protected peptide chloromethylketone derivatives (1.0 mmol) are dissolved in DMF (4-7 mL per mmol) and Nal (1-1 mmol) added and the mixture stirred for 15 minutes.

The peptide ester toluenesulphonate salt (1.1 mmol) in DMF (2-5 mL per mmol) is added followed by N HC03 (2.1 mmol) and the reaction left to stir for 12-18 hours at room temperature. Upon complete reaction, the solution is diluted with ethyl acetate (50-70 ml), and washed with water (IN KHSO4 ^may also be used, though the potential solubility of an amine-containing peptide must be considered) . The aqueous layer is extracted with ethyl acetate (x2) and the combined extracts washed with water and brine before drying over Na2S04- The product thus obtained is of good purity, though silica gel chromatography carried out to remove trace impurities contributes to epimerisation at the chiral centre to the ketone carbonyl. H. Reduction of aminoketones to aminoalcohols:

The crude aminoketone (1.0 mmol) is taken up in anhydrous MeOH (10 mL) , cooled to 0°C and treated with NaBH4 (2.5 equiv. ) in a single portion. The course of reaction is monitored by thin layer chromatography, and addition of extra NaBH4 is occasionally necessary. Upon completion of reduction, the solution is treated with ethyl acetate (50 mL) and water (50 mL) . The aqueous phase is extracted with ethyl acetate (x2) and the combined extracts washed with water and brine, and dried over Na2S04. The product is purified by silica gel chromatography using a gradient of methanol in chloroform for optimal separations.

I. Hydroqenolytic deprotection of final products:

The amino alcohol containing peptides (50 μmol) are dissolved in 90% acetic acid (3.5 mL) and 20% palladium hydroxide on charcoal (Pearlman's catalyst) is added under an 2 atmosphere. Deprotection is achieved by passage of H2 as a steady stream through the solution for 3-4 hours. The catalyst is removed by filtration through pre-washed and swollen Celite, and the filtrate concentrated in vacuo to approximately 1 mL total volume. The solution is diluted with water (20 ml), washed with chloroform (x3), and re-concentrated to 5-8 mL total volume. The solution is filtered through a glass wool plug, and lyophilized to yield the final product peptide as a fine colourless powder.

Examples

1.1 (3S)-t-Butoxycarbonylamino-l-diazo-5-methyl-2- hexanone, (BocLeuCHN₂) . Compound 1.1 was prepared according to general procedure D from Boc leucine monohy- drate (13.89 g, 55.7 mmol). The product was crystallized from ether-hexane.

1.2 (3S)-Amino-l-chloro-5-methyl-2-hexanone hydrochloride, (Cl-H₂+LeuCH₂Cl) . Compound 1.2 was prepared from Compound 1.1 (5.46 g, 21.4 mmol) in ether (21 mL), and a solution of 4N HCl in dioxane (25 mL, 100 mmol) as described in general procedure E.

1.3 (3S)-(N-t-Butoxycarbonylasparaqinyl)amino-l- chloro-5-methyl-2-hexanone, (BocAsnLeuCH₂Cl) . Compound 1.3 was prepared from Boc asparagine (3.45 g, 14.9 mmol) and Compound 1.2 (2.70 g, 13.5 mmol) according to general procedure B. 1.4 (3S)-(N-t-butoxycarbonylleucinylas- paraqinyl)amino-l-chloro-5-methyl-2-hexanone,

(BocLeuAsnLeuCH₂Cl) . Compound 1.4 was prepared by deprotection of Compound 1.3 (5.78 g , 15.3 mmol) according to general procedure C, and coupling with Boc leucine monohydrate (4.16 g, 16.7 mmol) according to general procedure B.

1.5 (3S)-N-Acetylleucinylasparaqinyl)amino-1-chloro- 5-methyl-2-hexanone, (AcLeuAsnLeuCH₂Cl) . Compound 1.5 was prepared by deprotection of Compound 1.4 (0.99 g,

2.02 mmol) according to general procedure C, followed by acetylation by general procedure F.

1.6 (3S)-N-t-Butoxycarbonyl-O-benzylserinylleucinyl- asparaginyl)-amino-l-chloro-5-methyl-2-hexanone, (BocSer(Bzl)LeuAsnLeuCH₂Cl) . Compound 1.6 was prepared by deprotection of Compound 1.4 (1.14 g, 2.32 mmol) according to general procedure C, and coupling with Boc serine benzyl ether (0.77 g, 2.61 mmol) according to general procedure B. 1.7 (3S)-N-Acetyl-O-benzylserinylleucinylas- paraqinyl)-amino-l-chloro-5-methyl-2-hexanone, (AcSer(Bzl)LeuAsnLeuCH∑Cl) . Compound 1.7 was prepared by deprotection of Compound 1.6 (643 g, 0.96 mmol) according to general procedure C, followed by acetylation according to general procedure F.

2.1 Nα-t-Butoxycarbonylisoleucylvaline methyl ester, (BocIleValOMe) . Compound 2.1 was prepared from valine methyl ester hydrochloride (19.99 g, 119 mmol) and Boc isoleucine hemihydrate (26.05 g, 108 mmol) according to general procedure A, and recrystallized from ethyl acetate-hexane.

2.2 N-Benzyloxycarbonylprolylisoleucylvaline methyl ester, (ZProIleValOMe) . Compound 2.2 was prepared from ^• Compound 2.1 (14.00 g, 40.6 mmol) by deprotection accord- ing to general procedure C, followed by coupling with Z- proline (10.09 g, 40.5 mmol) according to general procedure A. The product was recrystallized from ethyl acetate-hexane.

2.3 Prolylisoleucyl aline methyl ester tosylate, (TosO-H₂+ProIleValOMe) . Compound 2.3 (5.11 g, 10.7 mmol) was deprotected by catalytic hydrogenation as follows. The protected tripeptide was dissolved in methanol (130 mL) together with p-toluenesulphonic acid (2.17 g, 11.4 mmol), and 10% palladium on charcoal (0.5 g) was added under an N2 atmosphere. Hydrogen gas was bubbled through the mixture for 16 hours. Then the solution was filtered through celite to remove the catalyst, and evaporated to dryness to reveal the tripeptide as a foam. The excess p-toluenesulphonic acid was removed by trituration with ether and the tripeptide salt stored sealed under N2 as a hygroscopic glass.

3.1 (2RS,3S)-3-(N-Acetylleucinylasparaqinyl)-amino- 2-hydroxy-5-methyl-l-(N-prolylisoleucylvaline methyl ester)-hexane, (AcLeuAsnLeu*(CH0HCH₂N)ProlleValOMe) . Compound 3.1 was prepared from Compound 1.5 (133 mg, 308 μmol) and Compound 2.3 (235 mg, 457 μmol) in DMF

(4 mL) as described in general procedure G. The crude product was reduced without further purification using NaBH4 (26 mg, 679 μmol) as described in general procedure H. The crude product was purified by chromatography on silica gel, using a gradient of 5-11% MeOH in CHCI3.

3.2 (2RS,3S)-3-(N-Acetyl-O-benzylserinylleucinylas- paraginyl)-amino-2-hydroxy-5-methyl-l-(N-prolylisoleucyl- valine methyl ester)-hexane, (AcSer(Bzl)LeuAsnLeu* (CHOHCH₂N)ProIleValOMe) . Compound 3.2 was prepared from Compound 1.7 (160 mg, 262 μmol) and Compound 2.3 (201 g, 392 μmol)in DMF (4.5 mL) as described in general procedure G. The crude product was reduced without further purification as described in general procedure H, and purified by chromatography on silica gel, using a gradient of 5-11% MeOH in CHCI3. 3.3 (2RS,3S)-3-(N-Acetylserinylleucinylasparaginyl)- amino-2-hydroxy-5-methyl-l-(N-prolylisoleucylvaline methyl ester)-hexane, (AcSerLeuAsnLeu* (CHOHCH₂N) ProIleValOMe.AcOH) . Compound 3.2 was prepared from Compound 3.2 (46 mg, 50.5 μmol) by hydrogenation over palladium hydroxide on charcoal (16 mg) in 90% acetic acid as described in general procedure I.

4.1 (3S)-t-Butoxycarbonylamino-l-diazo-4-phenyl-2- butaπone, (BocPheCHN₂) . Compound 4.1 was prepared according to general procedure D from Boc phenylalanine (13.28 g, 50.0 mmol). The product was crystallized from ether-hexane.

4.2 (3S)-Amino-l-chloro-4-phenyl-2-butanone hydrochloride, (Cl-H₂+PheCH₂Cl) . Compound 4.2 was prepared from Compound 4.1 (10.01 g, 34.6 mmol) in dioxane (25 mL) , and a solution of 4N HCl in dioxane (50 mL, 200 mmol) as described in general procedure E.

4.3 (3S)-(N-t-Butoxycarbonylasparaginyl)amino-1- chloro-4-phenyl-2-butanone, (BocAsnPheCH₂Cl) . Compound 4.3 was prepared from Compound 4.2 (7.04 g, 30.1 mmol) and Boc asparagine (7.66 g, 33.0 mmol) in DMF (100 mL) , as described in general procedure B.

4.4 (3S)-N-t-Butoxycarbonylleucinylas- paraginyl)amino-l-chloro-4-phenyl-2-butanone, (BocLeuAsnPheCH₂Cl) . Compound 4.4 was prepared by deprotection of Compound 4.3 (9.74 g, 23.7 mmol) according to general procedure C, followed by coupling with Boc leucine (6.55 g, 26.3 mmol) (monohydrate dried together with HOBt hydrate by repeated azeotropic removal of water from DMF-toluene solution) in DMF (70 mL) , as described in general procedure B.

4.5 (3S)-(N-Acetylleucinylasparaginyl)amino-l- chloro-4-phenyl-2-butanone, (AcLeuAsnPheCH₂Cl) . Compound 4.5 was prepared by deprotection of Compound 4.4 (213 mg, 0.41 mmol) according to general procedure C, and subsequently acetylated according to general procedure F. 4.6 (3S N-t-Butoxycarbonyl-O- benzylserinylleucinylasparaginyl)-amino-l-chloro-4-phenyl- 2-butanone, (BocSer(Bzl)LeuAsnPheCH2Cl) . Compound 4.6 was prepared by deprotection of Compound 4.4 according to general procedure C, followed by coupling with Boc serine benzyl ether (0.99 g, 1.89 mmol) in DMF (11 mL) , as described in general procedure B.

4.7 (3S)-(N-Acetyl-0-benzylserinylleucinylas- paraginyl)-amino-l-chloro-4-phenyl-2-butanone, (AcSer(Bzl)LeuAsnPheCH₂Cl) . Compound 4.7 was prepared from Compound 4.6 (674 mg, 0.96 mmol), by deprotection according to general procedure C, and acetylation accord¬ ing to general procedure F.

5.1 (2RS,3S)-3-(N-Acetylleucinylasparaqinyl)-amino- 2-hydroxy-4-phenyl-l-(N-prolylisoleucylvaline methyl ester)-butane, AcLeuAsnPhe*(CH0HCH₂N)ProIleValOMe. Compound 5.1 was prepared from Compound 4.5 (149 mg, 319 μmol) and Compound 2.3 (247 mg, 481 μmol) in DMF (4 mL) as described in general procedure G. The crude product was reduced without further purification using

NaBH4 (29 mg, 767 μmol) as described in general procedure H. The crude product was purified by chromatography on silica gel, using a gradient of 5-11% MeOH in CHCI3.

5.2 (2RS,3S)-3-(N-Acetyl-O-benzylserinylleucinylas- paraginyl)-amino-2-hydroxy-4-phenyl-l-(N-prolylisoleucyl- valine methyl ester)-butane, AcSer(Bzl)LeuAsnPhe* (CHOHCH₂N)ProIleValOMe. Compound 5.2 was prepared from Compound 4.7 (188 mg, 292 μmol) and Compound 2.3 (223 mg, 435 μmol) in DMF (4.5 mL) as described in general proce- dure G. The crude product was reduced without further purification using NaBH4 (28 mg, 733 μmol) as described in general procedure H. The crude product from aqueous work up was dried by azeotropic removal of water to prevent precipitation and purified by chromatography on silica gel, using a gradient of 5-11% MeOH in CHCI3. 5.3 (2RS,3S)-3-(N-Acetylserinylleucinylasparaqinyl)- amino-2-hydroxy-4-phenyl-l-(N-prolylisoleucylvaline methyl ester)-butane, (AcSerLeuAsnPhe*

(CHOHCH₂N)ProIleValOMe.AcOH) . Compound 5.3 was prepared from Compound 5.2 (36 mg, 37.8 μmol) by hydrogenation over palladium hydroxide on charcoal (14 mg) in 90% acetic acid as described in general procedure I.

6.1 (2RS,3S)-3(N-r2-aminobenzoyl1-leucinyl- asparaqinyl)-amino-2-hydroxy-4-phenyl-l-(N-prolyl- isoleucyl-valyl methyl ester)-butane. (Abz-Leu-Asn-

Phe*(CH0HCH2)Pro-Ile-Val-0Me) . A BOC protected variant of Compound 3.1, Compound 3.4, (102 mg) was deprotected with 4N HCl in dioxane (4 ml) as described in general procedure C, and subsequently coupled with 2-aminobenzoic acid (29 mg) using EDC1 (42 mg) and HOBt (40 mg) as described in general procedure B (2-aminobenzoic acid was not pre- activated) . The product was precipitated from DMF by addition of 60% saturated NaHCθ3. The precipitate was filtered and the solid washed thoroughly with saturated NaHCθ3 and water. The product was dried in vacuo, and purified by preparative thin layer chromatography on 0.5 mm plates using 10% MeOH in CHCI3 as eluent.

7.1 (3S)-(N-Benzyloxycarbonylasparaginyl)amino-1- chloro-4-phenyl-2-butanone. (ZAsnPheCH₂Cl) . Compound 7.1 was prepared from Compound 4.2 (0.78g, 3.35mmol) and Z- asparagine (0.94g, 3.53mmol) in DMF (20ml) and DCM (15ml), as described in general procedure B.

8.1 N-t-Butoxycarbonylisoleucylphenylalanine methyl ester. (BocIlePheOMe) . Compound 8.1 was prepared from phenylalanine methyl ester hydrochloride (1.72g. 8.00mmol) and Boc-isoleucine hemihydrate (1.93g, δ.OOmmol) according to general procedure A, and recrystallized from ethyl acetate-hexane.

8.2 N-t-Butoxycarbonylprolylisoleucylphenylalanine methyl ester. (BocProIlePheOMe) . Compound 8.2 was prepared from Compound 8.1 (l.OOg, 2.55mmol) by deprotection according to general procedure C followed by coupling with Boc-proline (0.576g, 2.675mmol) as follows: Boc-proline and HOBt (0.568g, 4.20mmol) were dissolved in DCM (15ml) and cooled to 0°C. EDC1 (0.513g, 2.675mmol) was added and the solution stirred for 30 min. The deprotected Compound 8.1 in DMF (3ml) was added, followed by NMM (0.31ml, 2.80mmol). The reaction was stirred for 10-12 hours at room temperature. The reaction mixture was diluted with ethyl acetate (50ml), washed with saturated NaHC03 (15ml), 5% citric acid solution (15ml), brine

(25ml) and dried over gSθ . The concentrated product was purified by silica gel chromatography with 50% ethyl acetate in hexane.

8.3 N-t-Butoxycarbonylisoleucyl-3-(2- naphthyl)alanine methyl ester (BocIle-(2-Nap)AlaOMe) .

Compound 8.3 was prepared from 3-(2-naphtyl)alanine methyl ester hydrochloride (0.616g, 2.32mmol) and Boc-isoleucine hemihydrate (0.614g, 2.55mmol) according to the procedure described for Compound 8.2. 8.4 N-t-Butoxycarbonylprolylisoleucyl-3-(2- naphthyl)alanine methyl ester. (BocProIle-(2-Nap)AlaOMe) . Compound 8.4 was prepared from Compound 8.3 (0.75g, 1.69mmol) by deprotection according to general procedure C, followed by coupling with Boc-proline (0.39g, 1.78mmol) according to the procedure described for Compound 8.2. 9.1 (2RS, 3S)-3-(N-Benzyloxycarbonylasparaqinyl)- amino-2-hydroxy-4-phenyl-l-(N-prolylisoleucylphenylalanine methyl ester)-butane. (ZAsnPhe*(CH0HCH₂N)ProIlePheOMe) . Compound 9.1 was prepared from Compound 8.2 (0.31g, 0.633mmol) by deprotection according to general procedure C, followed by the reaction with Compound 7.1 (0.254g,0.57mmol) in DMF (7ml) as described in general procedure G. The crude product was reduced without further purification using NaBH4 (54mg, 1.425mmol) as described in general procedure H. The crude product was purified by silica gel chromatography with 3% MeOH in CHCI3 .

9♦2 2Rs,3S)-3-(N-Benzyloxycarbonylasparaqinyl)- amino-2-hydroxy-4-phenyl-l-(N-prolylisoleucyl-3-f2- naphthyl1alanine methyl ester)-butane. (ZAsnPhe*(CHQHCH₂N)Prolle-(2-Nap)AlaQMe) . Compound 9.2 was prepared from Compound 8.4 (0.28g, 0.517mmol) by deprotection according to general procedure C, followed by the reaction with Compound 7.1 (0.208g, 0.466mmol) in DMF (6ml) as described in general procedure G. The crude product was reduced without further purification using

NaBH4 (44mg, 1.165mmol) as described in general procedure H. The crude product was purified by silica gel chromatography with 3% MeOH in CHCI3.

10.1 (2RS, 3S)-3-(N-2-Quinolylcarbonylasparaqinyl)- amino-2-hydroxy-4-phenyl-l-(N-prolylisoleucylphenylalanine methyl ester)-butane. (QuAsnPhe*(CHOHCH₂N)ProIlePheOMe) . Compound 9.1 (43.6mg, 0.054mmol) was deprotected by catalytic hydrogenation as described for Compound 2.3, using palladium hydroxide on carbon (lOmg) and p- toluenesulphonic acid (10.5mg, 0.054mmpl), followed by coupling with 2-quinolinecarboxylic acid (10.5mg, 0.059mmol) as follows. 2-Quinolinecarboxylic acid and HOBt (11.7mg, 0.086mmol) were dissolved in DCM (4ml) and cooled to 0°C EDCI (11.5mg) was added and the solution stirred for 30 min. The deprotected Compound 9.1 in DMF

(3ml) was added, followed by NMM (0.007ml, O.Oδmmol). The reaction was stirred for 10-12 hours at room temperature. The reaction mixture was diluted with DCM (20ml), washed with saturated NaHC03 (5ml), water (10ml), brine (10ml) and dried over MgSθ4. the concentrated product was purified by silica gel chromatography with 3% MeOH in CHCI3.

10.2 (2RS, 3S)-3-(N-2-Quinolylcarbonylasparaginyl)- ^• amino-2-hydroxy-4-phenyl-l-(N-prolylisoleucly-3-r2- naphthyl]alanine methyl ester) - butane.

(QuAsnPhe*(CH0HCH₂N)Prolle-(2-Nap)AlaQMe. Compound 9.2 (140mg, 0.16mmol) was deprotected by catalytic hydrogenation as described for Compound 2.3, using palladium hydroxide on carbon (40mg) and p- toluenesulphonic acid (32mg, O.lδmmol), followed by coupling with 2-quinolinecarboxylic acid (31mg, 0.18mmol) as described for Compound 10.1. The crude product was purified by silica gel chromatography with 3% MeOH in CHCI3.

Other Variants

It should be appreciated that many other peptides of this type can readily be synthesized using analogous procedures. One builds out from a core which is the ketone precursor of the amino-alcohol. The leucine variant or phenyl variant are preferred, but at R^ various other naturally occurring or synthetic variant amino acid side chains (preferably low aryl or low alkyl groups) can be chosen (such as cyclohexyl methyl). Thereafter, one links a series of amino acid residues on one side of the core to build half the peptide. During these reactions, the free nitrogen can be protected. Separately, one then builds the other half of the peptide. Thereafter, one then couples the two halfs together and creates the alcohol.

Preferably, the final product has an Asn moiety in it. Also, the N-terminal end can have the nitrogen protected (e.g. with a group such as acetyl, t-butoxy- carbonyl, benzyloxycarbony1, aminobenzoyl, benzoyl or very low alkyl (or C1-C5)). At the other end of the moiety, various groups such as OH or low alkyl, low aryl, esters or amides can be used.

Substitutions for hydrogen on the carbons on the proline ring and at the carbon linking the proline ring to the alcohol carbon are also within the scope of the invention. In an especially preferred form, an N-terminal cap contains an aryl residue. However, many other higher and lower carbon containing aryl, arylalkyl, and alkyl moieties should also work as N-terminal caps. For example, the claim 7 end terminal caps could have a nitrogen substitute for a carbon on the aryl ring, and/or the hydrogens on the rings replaced by various substitutes.

It will be appreciated that the above description deals only with the preferred embodiments of the inven¬ tion. A number of other modifications and changes are intended to be within the scope of the invention. The claims should therefore be looked to in judging the full scope of the invention.

Inhibition

In addition to testing on live animals, inhibition potential can be tested by the ability to inhibit retro- viral proteases with synthetic substrates and with a murine sarcoma virus Pr65gag in the assay of Katoh et al. , 329 Nature 654-656 (1987). The inhibitory potency of each analog is preferably determined by using an assay analogous to that reported by J. Schneider et al. 54 Cell. 363 (1988). Briefly, synthetic HIV protease and the inhibitor are preincubated for 10 minutes in the buffer solution, and then a substrate, e.g. Ac-Thr-Ile-Met-Met-

Gln-Arg-NH2, is added. The reaction mixture is maintained at 25°C. At fixed periods of time, the reaction is stopped by the addition of 10% TFA, and the formation of product monitored by analysis by HPLC. Examples of claimed compounds (final form) have been tested and shown IC50 values of lnM or smaller. Industrial Applicability

The present invention provides a means to inhibit the ability of HIV protease to act. This should permit further understanding as to the mechanism of the enzyme and thus the virus. It also should provide potential drugs.

Claims

ClaimsWe claim:

1. A peptide having the moiety:

where Ri is selected from a group consisting of isobutyl, benzyl, cyclohexylmethyl, and other arylalkyl or alkyl moieties with less than twelve carbons; where R2 and R3 each has at least one amino acid residue; and

where at least one of R2 or R3 has at least two amino acid residues.

2. The peptide of claim 1, wherein R^ is a side chain from a naturally occurring amino acid.

3. The peptide of claim 2, wherein R2 has a residue of asparagine.

4. The peptide of claim 3, wherein opposite ends of the peptide both have protecting groups.

5. The peptide of claim 4, wherein a 2- aminobenzoic acid protecting moiety is one of the protecting groups.

6. The peptide of claim 1, wherein R2 is also linked to a moiety:

0 aryl residue - C II -

7. The peptide of claim 6, wherein, the aryl residue is selected from the group consisting of:

and

O. CO