KR0161138B1 - Irreversible hiv protease inhibitors with anti-aids effect, process for the preparation thereof and compositions containing the same - Google Patents

Abstract

The present invention relates to a novel compound useful as an inhibitor of HIV protease, a method for preparing the same, and a composition comprising the same. Alternatively, solvates can be usefully used as agents for the treatment or prevention of HIV or HIV infection because of their high inhibitory effect and low cytotoxicity as irreversible inhibitors of HIV proteases.

Wherein R ¹ , R ² and R ³ are as defined in the specification.

Description

Irreversible HIV Protease Inhibitor with Anti-Aids (AIDS) Effect, Method of Preparation and Composition Comprising the Same

The present invention relates to a human immunodeficiency virus (HIV) protease inhibitory compound, a method for preparing the same, and more particularly to a novel cis-epoxy compound that irreversibly inhibits HIV protease and its preparation. A method and a composition for the treatment and prevention of acquired immune deficiency syndrome (AIDS, hereinafter referred to as AIDS) caused by HIV infection, including the compound as an active ingredient.

HIV is a retrovirus that causes AIDS and AIDS syndrome. Retroviruses contain RNA as a genetic information material, which infects the host and creates double-stranded DNA from viral RNA by reverse transcriptase. This double-stranded DNA is integrated into the host's chromosome by integrase, making it possible not only to infect new viral RNA, but also to produce viral proteins using the host's enzymatic mechanism. The resulting polyprotein must be modified to form a new virus, which is made by an enzyme in the host or an enzyme in the virus. One of the most important of these enzymes is the protease, an enzyme that breaks down polyproteins into structural proteins and enzymes necessary for virus formation.

Of the retrovirus proteases, HIV proteases have been the most intensive study subjects. HIV does not synthesize each of the proteins from the mRNA to make the bark proteins and the enzymes that are needed, but rather the long form of the multiprotein gag-protein (p55) or gag-pol protein (the bark proteins and enzymes are combined together). p165) is first produced, and then the polyprotein is broken down by proteases inside it into enzymes necessary for virus formation, such as shell proteins, reverse transcriptase and integrase. Inhibiting the protease responsible for this degradation process stops the replication of the virus. Protease inhibitors are based on this principle.

Mutation experiments have reported that viruses without protease function are not infectious [Kohl et al., Proc. Nat. Acad. Sci., USA, 85, 4686-4960 (1988); and Peng et al., J. Virol., 63, 2550 (1989). Thus, inhibitors that inhibit the function of HIV proteases have been suggested as potential therapeutic agents for diseases caused by HIV infection.

The HIV protease consists of 99 amino acids and its structure was determined by X-ray structural determination [Navia, et al., Nature 337, 615-620 (1989); Wlodawer, et al., Science, 245, 616-621 (1989); and Miller, et al., Science, 246, 1149-1152 (1989). The HIV protease contains two identical monomers as dimers, each of which has a molecular weight of 10793. Such HIV proteases are typical aspartic proteases with an arrangement of aspartate-threonine-glycine at the reaction site.

The development of HIV protease inhibitors follows the trend of developing inhibitors of other types of aspartic proteases (particularly Lenin). The basic approach is to develop a compound similar to the enzyme's transition state (TSA) to increase the affinity of the enzyme, thus enhancing its binding to the enzyme. HIV protease inhibitors developed in this way have been disclosed in several documents (Roberts, et al., Science, 248, 358 (1990); European Patent Publications 0337714; 0346847; 356223; 352000; 357332; 362002; and 361341; Bone et al., JASC, 113, 9382 (1991). However, all of the known HIV protease inhibitors are reversible inhibitors that increase the affinity of the enzyme, which is insufficient to be used as an actual therapeutic agent.

Accordingly, the present inventors have developed and applied an irreversible inhibitor incorporating a cis-epoxide structure different from the transition state in order to obtain a stronger inhibitory effect than the reversible inhibitor mimicking the transition state as described above (1993). Korean Patent Application No. 93-10811 dated June 14), the present invention introduces an alkylamine other than L-amino acid in the cis-epoxide structure at the C-terminus and at the N-terminus is prepared from penicylamine. The introduction of the β-methanesulfonyl-L-valinyl amide derivatives has led to the development of novel (4R, 3S) -cis epoxide compounds with more potent irreversible HIV virus growth inhibition.

Accordingly, it is an object of the present invention to provide novel compounds with improved human immunodeficiency virus (HIV) inhibitory activity and methods for their preparation.

It is another object of the present invention to provide a composition useful for the prevention or treatment of acquired immunodeficiency syndrome or HIV infection, comprising the compound as an active ingredient.

In order to achieve the above object, the present invention provides a compound having a cis-epoxide structure of the general formula (I) and a pharmaceutically acceptable salt, hydrate or solvate thereof:

Wherein R ¹ is a straight, branched or cyclic alkyl radical having 1 to 8 carbon atoms, straight, branched or cyclic alkoxy having 1 to 8 carbon atoms; Lower alkoxy substituted with aryl; Or heterocycle, and R ² and R ³ are independently of each other lower alkyl, lower alkyl substituted by aromatic radicals, cyclic alkyl having 3 to 8 carbon atoms, lower alkyl substituted by cyclic alkyl radicals or aromatic radicals.

Hereinafter, the present invention will be described in more detail.

As used herein, lower alkyl refers to straight or branched chain alkyl of 1 to 4 carbon atoms including methyl, ethyl, isopropyl, isobutyl, t-butyl.

In addition, heterocycle means a triangular to hexagonal compound including 1 to 3 heteroatoms selected from oxygen, nitrogen, and sulfur atoms, and the nitrogen or sulfur atoms may be in oxidized form. Heterocycles of the present invention also include bicycles in which the aforementioned heterocycles are combined with benzene, cyclohexane or other heterocycles. Representative heterocycles include pyrrole, pyrrolinyl, pyrrolidinyl, pyrazoyl, pyrazolinyl, pyrazolidinyl, imidazoline, imidazolinyl, imidazolidinyl, pyridyl, piperidinyl, pyrazinyl, pi Ferrazinyl, pyrimidinyl, pyridazinyl, oxazolyl, oxazolinyl, oxazolidinyl, isoxazolyl, isoxazolinyl, isoxazolidinyl, morpholinyl, thiazolyl, thiazolidinyl, isothiazolyl , Isothiazolidinyl, indolyl, quinolinyl, tetrahydroquinolinyl, isoquinolinyl, benzimidazolyl, benzothiazoylyl, benzoxazoyl, benzoperfuranyl, furyl, dihydrofuranyl , Tetrahydrofuranyl, dioxanyl, dioxolinyl, thienyl, benzotianyl, benzotyranyl and the like.

Abbreviations for amino acids as used herein are in accordance with the International Union of Pure and Applies Chemistry-International Union of Biochemistry (IUPAC-IUB) Joint Conference on Biochemical Nomenclature for Amino Acids and Peptides (Eur. J. Biochem. 158). , 9-31 (1984).

The compounds according to the invention may also have asymmetric carbon centers and may exist as racemates, racemic compounds, diastereomeric mixtures and individual diastereomers, all of which are forms of isomers.

Preferred compounds of the invention are compounds of formula (I) wherein R ₁ is 3-thiophene, R ₂ is isopropyl and R ₃ is benzyl.

Specific examples of the representative compound of the present invention are as follows.

Compound 1: [(5S)-[[(N-methoxyacetyl) -β-methanesulfonyl-L-valinyl] amino]-(4R, 3S) -epoxy-6-phenyl-1-hexanoyl]- [(2S)-[1-phenyl-3-methyl] butyl] amide

Compound 2: [(5S)-[[(N-isopropyloxyacetyl) -β-methanesulfonyl-L-valinyl] amino]-(4R, 3S) -epoxy-6-phenyl-1-hexanoyl] -[(2S)-[1-phenyl-3-methyl] butyl] amide

Compound 3: [(5S)-[[((N-Isovaleroyl) -β-methanesulfonyl-L-valinyl] amino]-(4R, 3S) -epoxy-6-phenyl-1-hexanoyl] -[(2S)-[1-phenyl-3-methyl] butyl] amide

Compound 4: [(5S)-[[(N-3-thiophenacetyl) -β-methanesulfonyl-L-valinyl] amino]-(4R, 3S) -epoxy-6-phenyl-1-hexanoyl ]-[(2S)-[1-phenyl-3-methyl] butyl] amide

Compound 5: [(5S)-[[(N-3-furanacetyl) -β-methanesulfonyl-L-valinyl] amino]-(4R, 3S) -epoxy-6-phenyl-1-hexanoyl] -[(2S)-[1-phenyl-3-methyl] butyl] amide

Compounds of formula (I) according to the invention can be prepared as shown in Scheme 1 below.

Wherein R ¹ , R ² and R ³ are as defined above, Is an amino group protecting group.

According to Scheme 1, first, a compound of Formula (II) is reacted with a compound of Formula (III) and then oxidized with mCPBA (m-chloroperbenzoic acid, metachloroperoxybenzoic acid) to give a general formula (IV). To a compound. From this, the benzyloxycarbonyl protecting group is removed, the compound of formula (V) is coupled, and the oxidation reaction is carried out as above to obtain a compound of formula (VI). From this the benzyloxycarbonyl protecting group is removed and R ¹ -CH ₂ COOH is coupled to give the desired compound of formula (I).

The coupling reaction includes, for example, dicyclohexyl carbodiimide, 3-ethyl-3 '-(dimethylamino) -propylcarbodiimide (3-ethyl-3'-(dimethylamino) -propylcarbodiimide, EDC), bis- (2-oxo-3-oxazolidinyl) -phosphinic chloride, diphenylphosphoryl azide and the like can be used, but are not limited thereto. The carboxylic acid used in the process may be converted to an acid halide or other active ester derivative followed by a coupling reaction. The acid halide derivatives include acid chlorides, and active ester derivatives are used to activate carboxylic acid groups to form amide bonds by coupling reactions with amines or to form ester bonds by coupling reactions with alcohols. Commonly used ones include, for example, alkoxycarbonyl halides such as methoxycarbonyl chloride, isobutoxycarbonyl chloride, and the like, carboxylic acid anhydrides derived from coupling reagents, N-hydroxybenzotriazole derived esters, N-hydroxy phthalimide derived ester, N-hydroxysuccinimide derived ester, N-hydroxy-5-norbornene-2 ', 3'-dicarboxamide derived ester 2,4,5- Trichlorophenol derived esters and the like, but are not limited thereto.

Deprotection reactions, for example benzyloxycarbonyl groups, can be removed by conventional methods, for example by reaction under hydrogen pressure in the presence of a Pd / C catalyst.

Compounds of formula (II) in Scheme 1 may be prepared as shown in Scheme 2 below.

Wherein X is a halogen atom, for example Br or I, Is an amino group protecting group.

Scheme 2 is described by Keinan et al., Tetrahedron, 47, 4631-4638 (1991); and Corey Shimaji, J. Am. Chem. Soc., 105, 1662-1664 (1983) shows a process for producing cis-β, γ-olefin acid based on the method described in the above. A compound of the general formula (II) was obtained by subjecting the compound of the general formula (VII) to vitig reaction to obtain a compound of the general formula (VIII), which was then stirred by heating to a reflux temperature of the solvent in t-butanol under an acid catalyst. Can be prepared. Here, the acid treatment of the orthoester of the general formula (VIII) according to the method described in the paper to form a compound of the general formula (IX) and then base treatment to remove the protective group α in β, γ-olefin acid In the present invention, the otroester of general formula (VIII) is dissolved in t-butanol in the presence of an acid catalyst because the yield of the target compound is not only very low, but also difficult to separate. By heating and stirring at the reflux temperature of the solvent, β, γ-olefin acid compound (II) which was not converted into α, β-olefin acid was obtained in 80 to 90% yield.

Functionalized yamins that provide a group of formula (III) in Scheme 1 are described in Weinreb et al., Tetrahedhon Lett., 22, 3815 (1981), as shown in Scheme 3 below. N, O-dimethylamide prepared by the conventional method may be prepared by Grignard reaction, followed by a bitig reaction and a hydrogenation reaction.

In addition, the compound of the general formula (V) used in Scheme 1 may methylate a thiol group in the presence of a base from L-penicylamine and then protect the amine with a benzyloxycarbonyl group, as shown in Scheme 4 below. It can manufacture by.

Since the compound of general formula (I) of the present invention has HIV protease inhibitory activity, it may be used as an agent for treating or preventing HIV or HIV infection. For this purpose, an amount of 5 to 30 mg / kg body weight per day may be administered to the host at one time or several times, and the dosage level for a particular patient may be determined by the specific compound to be used, weight, age, sex, health condition, diet, It may vary depending on the time of administration, the method of administration, the rate of excretion, the drug mixture and the severity of the disease.

The compounds of the invention can also be administered orally or parenterally as desired. Capsules, tablets, pills, powders and granules may be used as oral dosage forms. In view of compound properties, capsules are particularly preferred, and tablets are preferably enteric preparations. When prepared in an oral dosage form, the active compound of the present invention may include at least one inert diluent such as sucrose, lactose or starch, and at least one lubricant such as magnesium stearate. When prepared in injectable preparations can be prepared according to the known art, for example in combination with sterile injectable aqueous or oily suspensions and with suitable dispersing, wetting or suspending agents. Examples of acceptable solvents include, for example, polyethylene glycol , Ethylene glycol, polypropylene glycol, ethanol and the like.

On the other hand, the compound of formula (I) of the present invention may be administered in parallel with one or more other AIDS treatments, immunomodulators and the like, if any agent useful for the treatment and prevention of HIV infection, any other than the above-mentioned formulations may be used Can be.

Hereinafter, the present invention will be described in more detail with reference to the following Preparation Examples and Examples. However, the following examples are only for the understanding of the preparation method of the novel compounds according to the present invention, but the present invention is not limited thereto.

[Production Example 1]

Preparation of (S) -2-amino-3-methyl-1-phenyl butane

[(1-1) Preparation of L-N-benzyloxycarbonyl-N'-methoxy-N'-methyl-phenylalanine amide]

25 g (0.084 mol) of N-benzyloxycarbonyl-L-phenylalanine and 18.4 ml (0.167 mol) of N-methylformoline were dissolved in 500 ml of dichloromethane and then 10.8 ml (83.6 mmol) at -15 ° C. Isobutyl chloroformate was added. After 15 minutes, 1.2 equivalents of N, O-dimethylhydroxylamine was added at -15 ° C and stirred at the same temperature for 1 hour and then at room temperature for 3 hours. The organic layer was washed twice with 500 ml of saturated sodium chloride solution, dried over anhydrous MgSO ₄ , passed through celite, and the organic solvent was removed to obtain 26.7 g (yield 93%) of the title compound.

¹ H NMR (CDCl ₃ ) δ 2.87 (m, 1H), 3.05 (m, 1H), 3.17 (s, 3H), 3.66 (s, 3H), 4.96 (m, 1H), 5.07 (s, 2H), 5.23 (d, 1 H), 7.12-7.34 (m, 10 H)

[(1-2) Preparation of (S) -3-[(N-benzyloxycarbonyl) amino] -1-methyl-4-phenyl-2-butanone]

6.84 g (0.02 mol) of the compound prepared in (1-1) was dissolved in 60 ml of tetrahydrofuran, and 30 ml (0.09 mol) of 3M methyl magnesium bromide ether solution was added at 0 ° C., followed by stirring at room temperature for 3 hours. It was. The reaction was terminated by addition of water at 0 ° C., the solvent was distilled off under reduced pressure, diluted with 300 ml of ether, and washed three times with 200 ml of ammonium chloride solution. Subsequently, the mixture was dried over anhydrous MgSO ₄ , distilled under reduced pressure, and subjected to column chromatography (hexane: ethyl acetate = 8: 2) to obtain 5.11 g (yield 86%) of the title compound.

¹ H NMR (CLCl ₃ ) δ 2.13 (s, 3H), 3.04 (m, 2H), 4.57 (m, 1H), 5.08 (s, 2H), 5.17 (d, 1H), 7.12-7.34 (m, 10H )

[(1-3) Preparation of (S) -3-[(N-benzyloxycarbonyl) amino] -2-methyl-4-phenyl-1-butene]

8.57 g (0.024 mole) of methyltriphenylphosphonium bromide was dissolved in 60 mL of anhydrous toluene and 44 mL (0.022 mole) of 0.5M potassium bis (trimethylsilyl) amide toluene solution was added at -20 ° C and then at the same temperature. Stir for 1 hour. To the resulting solution, a solution obtained by dissolving 5.94 g (0.02 mol) of the compound prepared in the above (1-2) in 50 ml of anhydrous toluene was added at -20 ° C for 10 minutes and then stirred at the same temperature for 1 hour. The reaction was terminated by addition of water, the solvent was distilled off under reduced pressure, and then diluted with 200 ml of dichloromethane and washed with 1N hydrochloric acid. After drying over anhydrous MgSO ₄ and distillation under reduced pressure to remove an organic solvent, column chromatography (hexane: ethyl acetate = 9: 1) was carried out to obtain 5.25 g (yield 89%) of the title compound.

¹ H NMR (CLCl ₃ ) δ 1.79 (s, 3H), 2.84 (m, 2H), 4.31 (m, 1H), 4.52 (bs, 1H), 4.81 (d, 2H), 5.07 (s, 2H), 7.13-7.35 (m, 10H)

[(1-4) Preparation of (S) -2-amino-3-methyl-1-phenyl butane]

2.95 g (0.01 mol) of the compound prepared in (1-3) was dissolved in 30 ml of methanol, 200 mg of 10% Pd / C was added, followed by stirring for 4 hours under 1 atmosphere of hydrogen. Subsequently, the inorganic material was removed by passing through celite, and the organic solvent was distilled off under reduced pressure to quantitatively obtain 1.63 g of the title compound.

¹ H NMR (CLCl ₃ ) δ 0.94 (m, 6H), 1.11 (bs, 2H), 1.65 (m, 1H), 2.39 (m, 1H), 2.82 (m, 2H), 7.13-7.32 (m, 5H )

[α] _D = -37.4 (C = 0.12, dichloromethane)

e. e. = -37.4 / -38.1 = 98%

[Production Example 2]

Preparation of N-benzyloxycarbonyl-S-methyl-penicylamine

8.9 g (0.06 mol) of L-penicylamine was added to 120 ml of dioxane and 40 ml of water, cooled to 0 ° C, and 20 ml of 6N aqueous sodium hydroxide solution was added and dissolved. 9.24 g (0.066 mol) of methane iodide was added to the resulting solution, and the flask was capped and then reacted at 0 ° C. for 3 hours and then at room temperature for 2 hours. The reaction was cooled to 0 ° C., 15 ml of 6N sodium hydroxide and 10.20 g (0.09 mol) of benzylchloroformate were added, followed by stirring at 0 ° C. for 1 hour and then at room temperature for 2 hours to terminate the reaction. After distilling off the solvent under reduced pressure, 20 ml of water and ether were added to remove unreacted benzylchloroformate, and then the organic layer was removed. 300 ml of ethyl acetate was added to the aqueous layer, and the pH was adjusted to 3 or less with 6N hydrochloric acid. The organic layer was separated, dried over anhydrous MgSO ₄ , and the solvent was distilled off under reduced pressure to obtain 14.25 g (yield 80%) of the title compound.

¹ H NMR (CDCl ₃ ) δ 1.2 (s, 6H), 2.1 (s, 3H), 4.3 (d, 1H), 5.1 (s, 2H), 7.1 (m, 5H)

[Manufacture example 3]

Preparation of (S) -5-[(N-benzyloxycarbonyl) amino] -6-phenyl-hex-3- (cis) -ene-1-carbonyl acid

[(3-1) 5-L- (N-benzyloxycarbonyl) amino-6-phenyl-hex-3- (cis) -enyl-4'-methyl-2 ', 6', 7'-trioxa -Bicyclo- [2 ', 2', 2 ']-production of oxetane]

1- (2-triphenylphosphonium-methyl) -4'-methyl-2 ', 6', synthesized according to the method described in Keinan et al., Tetrahedron, 26, 4631-4638 (1991), 60.89 g (0.12 mol) of 7'-trioxa-bicyclo- [2 ', 2', 2 ']-oxetane bromide was dissolved in 400 ml of tetrahydrofuran and stirred at -78 ° C. Subsequently, 220 ml (0.11 mol) of 0.5 M potassium hexamethyl disilazane solution was cooled at -78 ° C for 1 hour, and then slowly added to the solution for 20 minutes, and at -78 ° C for 1 hour, and then at room temperature for 1 hour. After stirring, water was added to terminate the reaction. After removing the solvent, it was dissolved in ethyl acetate and washed with saturated NaHCO ₃ solution and water. The organic layer was dried over anhydrous MgSO ₄ , distilled under reduced pressure, and subjected to column chromatography (hexane: ethyl acetate: triethylamine = 70: 30: 5) to obtain 36.5 g (yield 84%) of the title compound.

¹ H NMR (CDCl ₃ ) δ 0.8 (s, 3H), 2.2-3.0 (m, 4H), 3.9 (s, 6H), 4.6 (m, 1H), 4.8 (br, 1H), 5.05 (s, 2H ), 5.4-5.6 (m, 2H), 7.1-7.5 (m, 10H)

[(3-2) Preparation of (S) -5-[(N-benzyloxycarbonyl) amino] -6-phenyl-hex-3- (cis) -ene-1-carbonyl acid]

2.5 g (6 mmol) of the compound prepared in (3-1) was dissolved in a mixed solution of t-butanol and water containing less than 1% concentrated hydrochloric acid and stirred at a reflux temperature of the reaction solvent for about 20 hours. The reaction solvent was removed by distillation under reduced pressure, and the pH was adjusted to 9 or more by adding K ₂ CO ₃ solution, and then washed with ethyl acetate. The pH of the aqueous layer was adjusted to 2 with HCl, extracted with ethyl acetate, dried over MgSO ₄ , and the organic solvent was removed to yield 1.62 g (yield 80%) of the title compound.

¹ H NMR (CDCl ₃ ) δ 2.7-3.3 (m, 4H), 4.6 (m, 1H), 4.8 (br, 1H), 5.05 (s, 2H), 5.4 (t, 1H), 5.6 (m, 1H ), 7.1-7.5 (m, 10 H)

MS (FAB, m / e) 340 (M + l), [α] _D = +25.2 (C = 0.50, methanol)

Example 1

[(5S)-[[N-isovaleroyl) -β-methanesulfonyl-L-valinyl] amino]-(4R, 3S) -epoxy-6-phenyl-1-hexanoyl]-[(2S )-[1-phenyl-3-methyl] butyl] amide (compound 3) preparation]

[(1-1) [(5S)-(N-benzyloxycarbonyl) amino] -3- (cis) -ene-6-phenyl-1-hexanoyl]-[(2S)-[1-phenyl- Preparation of 3-methyl] butyl] amide]

0.339 g (1 mmol) of the compound prepared in Preparation Example 3 was mixed with 1.5 equivalents of EDC, 1-hydroxy-benzotriazol hydrate (HOBT), and triethylamine, respectively. After dissolving in dimethylformamide, 0.179 g (1 mmol) of the product of Preparation Example 1 was added at 0 ° C. and stirred at room temperature for 12 hours. The solvent was distilled off under reduced pressure, dissolved in ethyl acetate, and washed with 1N hydrochloric acid and a saturated solution of NaHCO ₃ . The organic layer was dried over anhydrous MgSO ₄ , distilled under reduced pressure to remove the solvent, and column chromatography (ethyl acetate: hexane = 1: 1) was carried out to obtain 0.44 g (yield 87.5%) of the title compound.

¹ H NMR (CDCl ₃ ) δ 1.01 (m, 6H), 1.78 (m, 1H), 2.42-3.25 (m, 6H), 4.05 (m, 1H), 4.58 (m, 1H), 4.95 (bs, 1H ), 5.08 (s, 2H), 5.35 (m, 1H), 5.58 (m, 1H), 7.09-7.41 (m, 16H)

[(1-2) [(5S)-(N-benzyloxycarbonyl) amino]-(4R, 3S) -epoxy-6-phenyl-1-hexanoyl]-[(2S)-[1-phenyl- Preparation of 3-methyl] butyl] amide]

339 mg (0.7 mmol) of the compound prepared in (1-1) was dissolved in 20 ml of dichloromethane, and 3 equivalents of metachloroperoxybenzoic acid was added thereto, followed by stirring at room temperature for 24 hours. 30 ml of 10% Na ₂ S ₂ O ₃ solution was added thereto, stirred for 30 minutes, and the organic layer was washed with saturated NaHCO ₃ solution. The organic layer was dried over anhydrous MgSO ₄ and the organic solvent was removed to give 287 mg (yield 82%) of the title compound.

¹ H NMR (CDCl ₃ ) δ 0.83-0.99 (m, 6H), 1.77 (m, 1H), 2.61-3.24 (m, 8H), 3.74 (m, 1H), 4.11 (m, 1H), 4.95 (d , 1H), 5.10 (s, 2H), 7.21-7.50 (m, 15H)

[(1-3) [(5S)-[[(N-benzyloxycarbonyl) -β-methanesulfonyl-L-valinyl] amino]-(4R, 3S) -epoxy-6-phenyl-1- Hexanoyl]-[(2S)-[1-phenyl-3-methyl] butyl] amide]

2.50 g (5 mmol) of the compound prepared in (1-2) was dissolved in 50 ml of methanol, mixed with 10% by weight of 10% Pd / C, and stirred for 3 hours under hydrogen balloon conditions. The reaction solution was passed through celite to remove the inorganic catalyst, and then the reaction solvent was removed by distillation under reduced pressure. 1.49 g (5 mmol) of the product of Preparation Example 2 and 1.5 equivalents of EDC, HOBT and triethylamine were respectively dissolved in 50 ml of dimethylformamide, and the amine obtained above was added at 0 ° C. and stirred at room temperature for 3 hours. It was. The solvent was distilled off under reduced pressure, dissolved in dichloromethane and washed with saturated NaHCO ₃ solution. The organic layer was dried over anhydrous MgSO ₄ , distilled under reduced pressure to remove the solvent, and then 20 ml of dichloromethane and 5 equivalents of metacloperoxybenzoic acid were added and stirred at room temperature for 2 hours. 200 ml of 10% Na ₂ S ₂ O ₃ solution was added thereto, stirred for 30 minutes, and the organic layer was washed with saturated NaHCO ₃ solution. The organic layer was dried over anhydrous MgSO ₄ , the organic solvent was removed, and column chromatography (hexane: ethyl acetate = 3: 7) was carried out to obtain 1.80 g (yield 52%) of the title compound.

¹ H NMR (CDCl ₃ ) δ 0.86-0.95 (m, 6H), 1.54-2.21 (m, 9H), 2.69-3.42 (m, 7H), 4.05-4.17 (m, 2H), 4.52 (d, 1H) , 5.11 (s, 2H), 7.18-7.55 (m, 15H)

FAB MS: 678 (M + 1)

[(1-4) [(5S)-[[(N-isovaleroyl) -β-methanesulfonyl-L-valinyl] amino]-(4R, 3S) -epoxy-6-phenyl-1- Preparation of hexanoyl]-[(2S)-[1-phenyl-3-methyl] butyl] amide]

339 mg (0.5 mmol) of the compound prepared in (1-3) was dissolved in 20 ml of methanol, mixed with 10 wt% of 10% Pd / C, and stirred for 3 hours under hydrogen-balloon conditions. The reaction solution was passed through celite to remove the inorganic catalyst, and then the reaction solvent was removed by distillation under reduced pressure. 51 mg (0.5 mmol) of isovaleric acid and 1.5 equivalents of EDC, HOBT and triethylamine were respectively dissolved in 10 ml of dimethylformamide, and the amine obtained above was added at 0 ° C. and stirred at room temperature for 3 hours. The solvent was distilled off under reduced pressure, dissolved in ethyl acetate, and washed with saturated NaHCO ₃ solution. The organic layer was dried over anhydrous MgSO ₄ , and the organic solvent was distilled off under reduced pressure, followed by column chromatography (dichloromethane: methanol = 20: 1) to obtain 260 mg (yield 83%) of the title compound.

¹ H NMR (CDCl ₃ ) δ 0.85-0.98 (m, 12H), 1.47 (d, 6H), 1.71 (m, 1H), 1.83 (m, 1H), 2.10-2.12 (m, 2H), 2.60 (m , 1H), 2.72-3.15 (m, 10H), 3.95-4.10 (m, 2H), 4.95 (d, 1H), 5.93 (d, 1H), 6.95 (d, 1H), 7.10-7.35 (m, 10H ), 7.72 (bs, 1 H)

FAB MS: 628 (M + 1)

Example 2

Except for using methoxyacetic acid instead of isovaleric acid in Example 1 and was carried out in the same manner as in Example 1 to obtain a compound 1 listed in Table 1 below.

Example 3

Except for using isopropyloxyacetic acid instead of isovaleric acid in Example 1 to give a compound 2 listed in Table 1 in the same manner as in Example 1.

Example 4

Except for using thiophenic acid instead of isovaleric acid in Example 1 and was carried out in the same manner as in Example 1 to obtain a compound 4 listed in Table 1 below.

Example 5

Except for using furan acetic acid instead of isovaleric acid in Example 1 to give the compound 5 listed in Table 1 in the same manner as in Example 1.

[Inhibitory Effect Analysis of HIV Protease]

The inhibitory efficacy of the compounds of the present invention against HIV protease was confirmed using the following method.

First, the HIV protease enzyme is mixed with the compound of the present invention in the absence of the reactive material (pre-culture solution), and then a part of the reaction solution is taken and added to the excess reactive solution (analytical solution) to reduce the enzyme activity. It was measured as the degree of remaining activity. Pre-culture solution is 50mM sodium acetate, pH 5.5, 1mM dithiotreitol (DTT), 1mM ethylenediaminetetraacetate (EDTA), 0.75M ammonium sulfate, 0.1% NP 40 (Noniget 40, Sigma Chemical Co.) .) And the compound of the present invention in various concentrations. The inhibitory response was initiated by adding 2.6 nM HIV-1 protease. 10 μl was taken at regular intervals, and the remaining enzyme activity was assayed by adding 80 μl of the solution containing 100 μM of the reactive material to the same buffer. At this time, the oligopeptide consisting of 11 amino acids of H-His-Lys-Ala-Arg-Val-Leu- (p-nitro) -Phe-Glu-Ala-Ile-Ser-NH was used. The substrate breaks the amide bond between (p-nitro) -Phe and Leu by HIV protease. The reaction rate is determined by using a high absorbance at 280 nm of (p-nitro) -Phe to separate the substrate before the reaction and the product after the reaction by high performance liquid chromatography (HPLC) to determine the relative amount of the product. Determined by. The amount of decrease in enzyme activity over time was determined and k was calculated from the slope of the straight line graph of the decrease in natural log value (ln) versus time. Inhibition constant was calculated | required by the following formula.

Where k _obs is the rate constant indicating the extent to which enzyme activity decreases with time in the presence of a constant concentration of inhibitor, and k _ina is the covalent bond formed from the Michaelis-Mente conjugate between the inhibitor and the enzyme. The rate constant at which the chemical reaction occurs, K _I is the Michaelis-Mentene Inhibition Constant between the inhibitor and the enzyme, and [I] is the concentration of the inhibitor. The formula is applicable when the inhibitor concentration is significantly higher than the enzyme concentration (steady state conditions). However, if the inhibitor concentration is high activity and the experiment should be conducted under similar conditions of the inhibitor concentration and the enzyme concentration, E + I than the above formula. EI Using the reaction equation of EI '(E: free enzyme, I: free inhibitor, EI: Michaelis-menten conjugate, EI': covalent combination of enzyme and inhibitor) Inhibition constants k _i and k _ina and secondary by calculating the concentration [E] / ([E] + [EI] + [EI ']) by entering the KINSIM / FITSIM program (Willams and Morrison; Zimmerle and Frieden) A second order rate constant, k _ina / K _I, was obtained and shown in Table 2 below. In the case of the compound of the present invention, since the value of the second inhibitory constant k _ina / K _I is sufficiently large, it can be seen that an irreversible reaction occurred between the enzyme and the inhibitor.

[Anti HIV-1 Virus Activity and Cytotoxicity Measurement]

The antiviral effect of the compounds of the present invention was evaluated by determining the compound concentration (50% Inhibitory Concentration, IC ₅₀ ) that inhibits the amount of HIV-1 virus by 50%.

HIV-1 virus to be used in the experiment was obtained by culturing as follows. First, COS cells (renal cells of African green monkeys transformed with Simian virus 40, source: American Type Culture Collection (ATCC) Cat # CRL-1651) were added to DME medium (Dulbecco's modified). After cultivation in Eagle's medium, 1 ml (10 ⁷ cells) of this COS cell culture solution was placed in an electroporation vessel, which contained 20 µg of HIV-1 gene (pNL 4-3, obtained from: Add and mix US AIDS Research and Reference Reagent Program Cat. # 114, and then in a cell-porator in RPMI 1640 medium (Gibco-RRL) containing 20% Fetal Bovine Serum (FBS) -Porator, BRL) was electroporated at 800μF, 250V. The electroporated cell solution was then left in ice for 10 minutes and then placed in a T75 flask containing 15 ml of DME medium (containing 10% fetal bovine serum) and incubated in a CO ₂ incubator. After 24 hours, the cells were replaced with 10 ml of the same fresh medium, and on the second day, the culture solution was collected and centrifuged, and the supernatant was passed through a 0.22 mm filter to obtain HIV-1 virus, which was distributed and stored at -70 ° C. The resulting HIV-1 virus was then infected with MT-2 cell line (obtained from US AIDS Research and Reference Reagent Program Cat. # 237) to amplify the amount of virus. That is, MT-2 cell lines of 4 × 10 ⁶ cells were mixed with HIV-1 virus at 1500 TCID ₅₀ (50% cell culture infectious dose) and then cultured. Infected MT-2 cells were fused to form a balloon-like synthium (Syncytium) around 7 days again 5 times uninfected MT-2 cells were incubated. After two days, virus cultures were taken daily for three days, centrifuged, and the supernatants were passed through a 0.22 mm filter to obtain amplified HIV-1 virus, which was distributed and stored at -70 ° C. As a result, an HIV-1 virus having 131 ng / ml, a virus amount of 4 × 10 ⁴ TCID ₅₀ / ml, and a reverse transcriptase activity of 3.8 × 10 ⁵ cpm / ml was obtained. .

In order to examine the effects of inhibiting the proliferation of HIV-1 of the compound of the present invention, the following experiment was conducted. Peripheral Blood Mononuclear Cells (PBMC) were isolated from Fibro-Hypaque (Pharmacia) using blood obtained from Daejeon blood sources, followed by 5 μg / ml PHA-P (phytohemagglutinin-P). , Plant erythrocyte aggregate-P, Sigma) was added and cultured. The culture consisted of 20% fetal bovine serum, interleukin-2 (IL-2, 20 Units / ml), penicillin (100 Units / ml), streptomycin (100 μg / ml) and amphotericin B (0.25 Μg / ml) was used using RPMI 1640 medium. After 3 days, the cells were washed with RPMI 1640 medium, and 1.2 × 10 ⁷ cells were infected by mixing 12,000 TCID ₅₀ HIV-1 virus at 37 ° C. for 2 hours. Infected cells obtained after centrifugation were washed twice with RPMI 1640 medium and the cell number was adjusted to 2 × 10 ⁶ cells / ml. The HIV-1 inhibitory compound to be measured was diluted with dimethylsulfoxide (DMSO) to prepare 200,63.2, 20, 6.32, 3.56, 2, 0.632, 0.2, 0.0632 and 0 μM solutions (200 μl each). . 495 μl of RPMI 1640 medium was added to a 24-well culture plate, to which 5 μl of HIV-1 inhibitory compound was added, followed by incubation with 500 μl of HIV-1 virus infected cells (1 * 10 ⁶ x cells). The final concentrations of the compounds are therefore 1000, 316, 100, 31.6, 17.78, 10, 3.16, 1, 0.316 and 0 nM. After 4 days, 750 µl of medium was discarded and 1 ml of the same medium containing the compound was added and cultured. After 7 days, the amount of protein p24 or reverse transcriptase activity in the culture was measured to determine the IC ₅₀ concentration of the compound that inhibits the amount of HIV-1 virus by 50%.

Protein p24 amount was determined by Du Pont's HIV-1 p24 nuclear profile ELISA (Enzyme Linked Immunosorbent Assay) (NEN Cat # NEK-060) or Cellular Products Inc. Measurements were made using Retro-tekTM HIV-1 p24 House Eliza (CPI Cat # 0801111).

Reverse transcriptase activity was measured as follows. First, 800 μl of the culture solution was mixed with 400 μl polyethylene glycol (PEG) solution (30% PEG 8000, 0.4 M NaCl) and then stored at 4 ° C. The following day centrifuged at 15,000 rpm for 20 minutes in an Eppendorf centrifuge to obtain HIV-1 virus, 40 μl of Solution 1 (25 mM Tris-HCl, pH 7.8, 0.25 mM EDTA, 0.025% Triton X-100, 50% Glycerol, 10 mM DTT and 100 mM KCl) and 20 µl of Solution 2 (0.9% Triton X-100, 43.7 mM KCl), followed by 10 µl of 90 µl reverse transcriptase activity assay (46.5 mM Tris-HCl, pH). 7.8, 8.89 mM DTT, 11.1 mM MgCl 2, 3.75mM NaCl, 2.5μCi 3 H-TTP (methyl- 3 H-thymidine-5-triphosphate) (NEN-Du Pont, 10-25 Ci / mmol), 0.556μ / Ml poly (rA): oligo (dT) (Pharmacia Co., Ltd.) and reacted for 1 hour at 37 ° C. After the reaction, 9 µl of 0.25M EDTA was added to stop the reaction. Was transferred to a DEAE (diethylaminoethyl, diethylaminoethyl) filter (DE 81, Whatmann Cat # 3658N323), dried and washed with 2 × SSC buffer (0.3M NaCl, 0.03M Na ₃ Citrate.2H ₂ O pH 7.0). Gun filter It was added a liquid scintillation solution of the following 5㎖ and was quantified using a liquid scintillation counter.

To test for cytotoxicity to determine the maximum tolerated anti-Aid agent, 1 × 10 ⁶ PBMC cells stimulated with PHA-P were added to a 24-well culture plate and the compounds of the invention in the range 0.1-100 μM were added followed by RPMI cultured in 1640 medium with 37 ℃ and 4 days changing the medium at intervals while the proliferation of the cells trypan blue die IX crew former method (trypan blue dye exclusion technique) as observed two weeks in blood calculation plate cytotoxic CT ₅₀ ( 50% of cells die). The results of the measured antiviral activity and cytotoxicity are shown in Table 2 below. As shown in Table 2, it can be seen that the compounds of the present invention are all low in cytotoxicity while having a high HIV protease inhibitory effect.

As described above, the compound of general formula (I) of the present invention is an irreversible inhibitor of HIV protease and has high inhibitory effect and low cytotoxicity, and thus is useful as a pharmaceutical composition for treating or preventing HIV or HIV infection. Can be used.

Claims (6)

A compound having a cis-epoxide structure of formula (I) or a pharmaceutically acceptable salt, hydrate or solvate thereof: Wherein R ¹ is a straight, branched or cyclic alkyl radical having 1 to 8 carbon atoms; Straight, branched or cyclic alkoxy of 1 to 8 carbon atoms; Lower alkoxy substituted with aryl; Or heterocycle, and R ² and R ³ are independently of each other lower alkyl, lower alkyl substituted by aromatic radicals, cyclic alkyl having 3 to 8 carbon atoms, lower alkyl substituted by cyclic alkyl radicals or aromatic radicals. The compound of claim 1, wherein R ₁ is 3-thiophene, R ₂ is isopropyl, and R ₃ is benzyl. The compound of claim 1, wherein [(5S)-[[(N-methoxyacetyl) -β-methanesulfonyl-L-valinyl] amino]-(4R, 3S) -epoxy-6-phenyl-1-hexa Noyl]-[(2S)-[1-phenyl-3-methyl] butyl] amide; [(5S)-[[(N-isopropyloxyacetyl) -β-methanesulfonyl-L-valinyl] amino]-(4R, 3S) -epoxy-6-phenyl-1-hexanoyl]-[( 2S)-[1-phenyl-3-methyl] butyl] amide; [(5S)-[[(N-isovaleroyl) -β-methanesulfonyl-L-valinyl] amino]-(4R, 3S) -epoxy-6-phenyl-1-hexanoyl]-[( 2S)-[1-phenyl-3-methyl] butyl] amide; [(5S)-[[(N-3-thiophenacetyl) -β-methanesulfonyl-L-valinyl] amino]-(4R, 3S) -epoxy-6-phenyl-1-hexanoyl]-[ (2S)-[1-phenyl-3-methyl] butyl] amide; [(5S)-[[(N-3-furanacetyl) -β-methanesulfonyl-L-valinyl] amino]-(4R, 3S) -epoxy-6-phenyl-1-hexanoyl]-[( 2S) -1-phenyl-3-methyl] butyl] amide and pharmaceutically acceptable salts, hydrates and solvates thereof. Coupling the compound of formula (II) with the compound of formula (III) and then oxidizing to obtain a compound of formula (IV); Deprotecting the compound of formula (IV) followed by coupling and oxidizing the compound of formula (V) to obtain a compound of formula (VI); A method for preparing a compound of formula (I), characterized by coupling R ¹ -CH ₂ COOH after deprotecting the compound of formula (VI). In the above, R ¹ , R ² and R ³ are as defined in claim 1, Is an amino group protecting group. The method of claim 4, wherein the compound of general formula (VII) is subjected to a vitig reaction to obtain a compound of general formula (VIII), which is then heated to the reflux temperature of the solvent in t-butanol in the presence of an acid catalyst. Further comprising preparing a compound of formula (II). In the above, X is a halogen atom, Is an amino group protecting group. A composition useful for the prevention or treatment of acquired immunodeficiency or human immunodeficiency virus (HIV) infection, comprising the compound of any one of claims 1 to 3 or a pharmaceutically acceptable salt, hydrate or solvate thereof as an active ingredient. .