KR0161144B1 - 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, the compound having a cis-epoxide structure of the following general formula (I) and a pharmaceutically acceptable salt and hydrate thereof 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.

In the above formula, A, B and D 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 a composition comprising the same, and more particularly, a novel cis-epoxy compound and a method for preparing the same, which irreversibly inhibit HIV protease, and The present invention relates to a composition for treating and preventing 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 the host's enzyme or the appeal of the virus. The most important of these enzymes is a 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 made, and then the polyprotein is broken down into enzymes necessary for the formation of viruses 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., I 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 (transition state analogue, TSA) that is similar to the enzyme's transition stat to increase the affinity of the enzyme, thereby enhancing its binding to the enzyme. HIV protease inhibitors developed in this manner are disclosed in several documents (Roberts, et al., Science, 248, 358 (1990); European Patent Publication No. 0337714; 0346847; No. 356223; 352000; 357332; 362002; and 361341; Bone et al., IACS, 113, 9382 (1991). However, all of these known HIV protease inhibitors are reversible inhibitors that increase the affinity of the enzyme, which is insufficient to use the 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 that mimics the transition state as described above (1993). Korean Patent Application No. 93-10811 dated June 14, 2012), according to the present invention, both ends of the cis-epoxide structure are N-terminus to increase the stability in vivo and the sulfonamide group on the right N-terminus. The introduction has resulted in the development of novel (4R, 3S) -cis epoxide compounds that increase in vivo stability and at the same time have a stronger inhibitory effect on the irreversible HIV virus growth.

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): a pharmaceutically acceptable salt, hydrate or solvate thereof;

Wherein A is a functionalized acyl of the general formula (II)

R ¹ is a lower alkoxy, an aromatic radical containing a lower alkoxy or nitrogen atom substituted with an aromatic radical,

R ² is lower alkyl or lower alkyl substituted with amide, n is 0, 1 or 2),

B is hydrogen, lower alkyl or lower alkyl substituted by aromatic radical,

D is lower alkyl, lower alkoxy, lower alkyl substituted with an aromatic radical or a group of the general formula (III)

(Wherein R ³ and R ⁴ are independently of each other hydrogen, lower alkyl, OH or NH ₂ ).

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, an aromatic radical containing a nitrogen atom means a mono or dicyclic aromatic radical in which one or two nitrogen atoms are contained in an aromatic ring, and examples thereof include quinoline.

Abbreviations for amino acids as used herein are in accordance with the 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 in the form of isomers.

Preferred compounds of the invention

R_One Is 2-quinoline,

R ₂ is an asparagine residue,

n is 1,

B is hydrogen,

D is p-methylphenyl or p-aminophenyl

It is a compound of general formula (I).

Specific examples of representative compounds of the present invention are as follows:

Compound 1: [(4S)-[(N ¹ -Toluenesulfonyl) amino]-[N ^4- [N '-(2-quinolinecarbonyl) -L-asparaginyl] amino] -5-phenyl- ( 2S, 3R) -epoxy pentane

Compound 2: [([(4S)-[(N ¹ -paraaminobenzenesulfonyl) amino]-[N ^4- [N '-(2-quinolinecarbonyl) -L-asparaginyl] amino] -5 -Phenyl- (2S, 3R) -epoxy pentane

Compound 3: [(4S)-[(N ¹ -Toluenesulfonyl-N ¹ -isobutyl) amino]-[N ^4- [N '-(2-quinolinecarbonyl) -L-asparaginyl] amino] -5-phenyl- (2S, 3R) -epoxy pentane

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

Wherein A, B and D are as defined above and X is a halogen atom, for example Br or I.

According to Scheme 1, compound (a) is obtained from N- (2-bromoethyl) phthalimide through a bitig reaction and a reductive amination reaction, and compound (a) is reacted with compound (IV) to give a compound ( V) was obtained and reacted with compound (VI) to obtain compound (VII), followed by oxidation with mCPBA (metachloroperoxybenzoic acid) to obtain an epoxy compound (VII), from which the deprotection reaction and compound (VII) were obtained. The desired compound of general formula (I) is obtained through a coupling reaction with.

The coupling reaction includes, for example, dicyclo hexylcarbodiimide (DCC), 3-ethyl-3 '-(dimethylamino) -propylcarbodiimide (EDC), bis- (2-oxo-) as a coupling reagent. 3-oxazolidinyl) -phosphinic acid chloride (BOP-C1), diphenylphosphoryl azide (DPPA) and the like can be used, but are not limited thereto. The carbolic acid used in the process may be converted to an acid halide or other active ester derivative and then subjected to a coupling reaction. The acid halide derivatives include acid chlorides, and active ester derivatives activate carboxylic acid groups to form amide bonds by coupling reactions with amines or to form ester bonds by culling 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 water bath pressure in the presence of a Pd / C catalyst.

Since the compound of general formula (I) of the present invention has HIV protease inhibitory activity, it can be used as an agent for treating or preventing AIDS 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 can be converted according to the time of administration, the method of administration, the rate of excretion, 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 oral dosage form, the active compounds of the present invention may comprise at least one inert diluent such as sucrose, lactose or starch, and at least one glidant such as magnesium stearate. When prepared in injectable preparations can be prepared according to known techniques, for example, in combination with sterile injectable aqueous or oily suspensions and with suitable dispersing, wetting or suspending agents. Reckle, 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, and any agent other than the above-mentioned formulations can be used as long as it is a useful agent for the treatment and prevention of HIV infection. 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.

Example 1

[(4S)-[(N ¹ -Toluenesulfonyl) amino]-[N ^4- [N '-(2-quinolinecarbonyl) -L-asparaginyl] amino] -5-phenyl- (2S, 3R Preparation of Epoxy Pentane

(1-1) Preparation of N-[(2-triphenylphosphine) ethyl] phthalimide bromide

2.54 g (10 mmol) of N- (2-bromoethyl) phthalimide and 2 equivalents of triphenylphosphine were stirred for 16 hours at 150 ° C without solvent, and then recrystallized with ether with ethanol, 4.5 g of the title compound. (Yield 88%) was obtained.

¹ H NMR (CDCl ₃ ) δ 4.32 (m, 2H), 4.54 (m, 2H), 7.51-7.71 (m, 15H), 7.81-7.93 (m, 4H)

(2-2) Preparation of 4S-[(N ¹ -phthaloyl) amino]-[(N ⁴ -benzyloxycarbonyl) amino] -5-phenyl- (2,3) -pentene

0.568 g (1.1 mmol) of the product of (1-1) was dissolved in 30 mL of anhydrous tetrahydrofuran and 2.2 mL (1.1 mmol) of 0.5 M potassium hexamethyl disilazane solution was slowly added at -78 ° C, followed by 1 Stir for hours.

To this was added 0.283 g (1 mmol) of N-benzyloxycarbonyl-L-phenylalanal in 10 ml of anhydrous tetrahydrofuran and added, followed by stirring at -78 ° C for 1 hour, then at room temperature for 1 hour, and water. Was added to terminate the reaction.

The reaction solution was removed, dissolved in 50 ml of 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 = 70: 30) to obtain 0.374 g (yield 84%) of the title compound.

¹ H NMR (CDCl ₃ ) δ 2.78-2.99 (m, 2H), 4.26 (m, 2H), 4.59 (bs, 1H), 4.85 (m, 1H), 5.10 (s, 2H), 5.44-5.58 (m , 2H), 7.15-7.32 (m, 10H), 7.72-7.95 (m, 4H)

(1-3) Preparation of 4S-[(N ⁴ -benzyloxycarbonyl) amino] -1-amino-5-phenyl- (2,3) -pentene

0.44 g (1 mmol) of the product of (1-2) and 3 equivalents of hydrazine were diluted in 20 ml of ethanol and refluxed for 3 hours. The resulting solid was filtered off to remove the organic solvent, 50 ml of ethyl acetate was added, washed with 0.2N NaOH solution and dried over anhydrous MgSO ₄ to give 0.298 g (96% yield) of the title compound.

¹ H NMR (CDCl ₃ ) δ 2.65-3.23 (m, 4H), 4.64 (m, 1H), 5.11 (s, 2H), 5.15-5.54 (m, 2H), 7.09-7.43 (m, 10H)

(1-4) Preparation of 4S-[(N ¹ -toluenesulfonyl) amino]-[N ⁴ -benzyloxycarbonyl) amino] -5-phenyl- (2,3) -pentane

0.31 g (1 mmol) of the product of (1-3) and 1.1 equivalents of toluenesulfonylchloride and triethylamine, respectively, were dissolved in 20 ml of dimethylformamide and stirred for 16 hours. The reaction solvent was distilled off under reduced pressure, dissolved in 50 ml of ethyl acetate, and then washed with NaHCO ₃ solution. After drying over anhydrous MgSO ₄ and removing the organic solvent, column chromatography (ethyl acetate: hexane = 1: 3) was carried out to obtain 0.36 g (yield 77%) of the title compound having the cis olefin at the 2,3 position.

¹ H NMR (CDCl ₃ ) δ 2.43 (s, 3H), 2.62 (m, 1H), 2.85 (m, 1H), 3.20-3.41 (m, 2H), 4.40 (m, 1H), 4.78 (bs, 1H ), 5.01 (bs, 1H), 5.10 (s, 2H), 5.31 (m, 1H), 5.52 (m, 1H), 7.01-7.75 (m, 14H)

(1-5) Preparation of 4S-[(N ¹ -toluenesulfonyl) amino]-[N ⁴ -benzyloxycarbonyl) amino] -5-phenyl- (2S, 3R) -epoxy pentane

0.464 g (1 mmol) of the product of (1-4) were dissolved in 20 ml of dichloromethane, 3 equivalents of metachloroperoxybenzoic acid was added, followed by stirring at room temperature for 24 hours. 30 ml of 10% Na ₂ S ₂ O ₃ solution was added, 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 (ethyl acetate: hexane = 2: 3) was performed to obtain 0.39 g (yield 81%) of the title compound.

¹ H NMR (CDCl ₃ ) δ 2.39 (m, 1H), 2.42 (s, 3H), 2.74 (m, 2H), 2.97 (m, 2H), 3.14 (m, 1H), 3.68 (m, 1H), 3.89 (m, 1H), 4.95 (bs, 1H), 5.11 (s, 2H), 7.15-7.76 (m, 14H)

(1-6) 4S)-[(N ¹ -toluenesulfonyl) amino]-[N ^4- [N '-(2-quinolinecarbonyl) -L-asparaginyl] amino] -5-phenyl- ( Preparation of 2S, 3R) -Epoxy Pentane

241 mg (0.5 mmol) of (1-5) was dissolved in 20 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. 144 mg (0.5 mmol) of N- (2-quinolinecarbonyl) -asparagine and 1.5 equivalents of EDC, HOBT and triethylamine, respectively, were dissolved in 10 ml dimethylformromamide. Stirred for 3 h. 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 , distilled under reduced pressure to remove the solvent, and then subjected to column chromatography (dichloromethane: methanol = 10: 1) to give 206 mg (yield 67%) of the title compound.

¹ H NMR (CDCl ₃ ) δ 2.41 (s, 3H), 2.46 (m, 2H), 2.65-3.13 (m, 8H), 3.89 (m, 1H), 5.02 (m, 1H), 5.78 (d, 1H ), 6.19 (d, 1H), 6.70 (d, 1H), 7.07-8.29 (m, 16H), 9.37 (d, 1H)

MS: (FAB, m / e): 616 (M + 1)

Example 2

Except for using paraaminoben gensulfonyl chloride instead of toluenesulfonyl chloride in Example 1 was carried out in the same manner as in Example 1 to obtain the following compound 2.

Example 3

In Example 1, the product of step (1-3) using isobutyl bromide was carried out in the same manner as in step (1-4), and then step (1-4) was repeated to obtain compound 3 below.

[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. The pre-culture solution was prepared with 50 mM sodium acetate, pH 5.5, 1 mM dithiothracene (DTT), 1 mM ethylene diaminetetraacetate (EDTA), 0.75 M ammonium sulfate, 0.1% NP 40 (Noniget 40, Sigma Chemical Co.), Compounds of the invention are included in various concentrations. Inhibition was initiated by adding 2.6 nM of HIV-1 protease. 10 μl each was taken at regular intervals, and the remaining enzyme activity was assayed by adding 80 μl of the solution containing 100 μM of reactive mass to the same complete solution. 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 down the amide bond between (p-nitro) -Phe and Leu by HIV protease. The reaction rate was determined by using a strong absorbance at 280 nm of (p-nitro) -Phe to separate the substrate before the reaction and the product after the reaction by HPLC to measure the relative amounts of the product. 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 (1n) versus time. The inhibition constant was calculated 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 agent, and k _ina is the rate constant at which a chemical reaction occurs in which a covalent bond is formed from the Michaelis-Menten conjugate between the inhibitor and the enzyme. ego,

K ₁ is the Inhibition Constant of the Michaelis-Menten conjugate between the inhibitor and the enzyme,

[I] is the concentration of the inhibitor.

The above formula is applicable when the inhibitor concentration is much higher than the enzyme concentration (steady state conditions). However, when the concentration of the inhibitor is high activity and the experiment should be conducted under similar conditions of the inhibitor concentration and the enzyme concentration, (E: free enzyme, I; free inhibitor concentration, EI: Michaelis-Menten conjugate concentration, EI ': covalent conjugate concentration of enzyme and inhibitor). Inhibition constants k _i and k _ina and second order rate constants are calculated by entering values of concentrations E / (E + EI + EI ') into the KINSIM / FITSIM program (Willams and Morrison; Zimmerle and Frieden). To obtain k _ina / K _I is 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]

Antiviral effects were assessed by determining the concentration (IC ₅₀ ) that inhibited viral replication by 50% by the study of C. chitin formation and reverse transcriptase assay.

HIV-1 virus was cultured as follows. First, COS cells (renal cells of African green monkeys transformed with Simian virus 40) were cultured in DME medium (Dulbecco's modified Eagle's medium), and then 1 ml (10 ⁷ cells) of the culture solution was placed in an electroporation vessel. Mix with 20 μl of plasmid containing HIV-1 gene (pNL 4-3), then use 800 μF, 250 V Cell-Porator (BRL) in RPMI medium containing 20% FBS (fetal bovine serum). Electroporation was performed. Subsequently, it was left in ice for 10 minutes and then transferred to a T75 flask containing 15 ml of DME medium (containing 10% FBS) 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, centrifuged, passed through a 0.22 mm filter, distributed, and stored at -70 ° C. The MT-2 cell line HIV-1 was then infected to amplify the amount of virus. In other words, the MT-2 cell line of 4 × 10 ⁶ cells was infected with 1500 TCID ₅₀ (50% tissue culture infectious dose) of NL43 virus, and then, 5 days after infection, a large amount of C. chitin was formed. Unloaded MT-2 cells were taken and virus cultures were taken daily for three days from two days later. The culture was centrifuged, passed through a 0.22 mm filter and then dispensed and stored at -70 ° C. As a result, a virus having an amount of p24 of 131 ng / ml, a TCID ₅₀ value of 4 x 10 ⁴ TCID ₅₀ / ml, and a reverse transcriptase activity of 3.8 x 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 Ficoll-Hypaque (Pharmacia) and then cultured with 5 μl / ml PHA-P (Sigma). The culture was RPMI 1640 medium containing 20% FBS, IL-2 (20 Units / ml), penicillin (100 Units / ml), streptomycin (100 μl / ml) and amphotericin B (0.25 μl / ml). It was performed using. After 3 days, the cells were washed with RPMI medium, and then infected with 1.2 × 10 ⁷ cells mixed with 12,000 TCID ₅₀ virus for 2 hours at 37 ° C. The cells obtained after centrifugation were washed twice with RPMI medium and the cell number was adjusted to 2 × 10 ⁶ cells / ml. The HIV-1 inhibitory compounds to be measured were diluted with 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 used above was added to a 24-well culture plate, to which 5 μl of HIV-1 inhibitory compound was added, followed by 500 μl of HIV-1 virus infected cells (1 × 10 ⁶ 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 after 7 days, the amount of virus was reduced to 50% and IC was determined by measuring the amount of p24 or reverse transcriptase. ₅₀ was measured.

The p24 amount was determined by Du Pont's HIV-1 p24 Core Profile ELISA (NEN Cat # NEK-060) or Cellular Products Inc. Retro-tekTM HIV-1 p24 antigen ELISA (CPI Cat #). 0801111). Reverse transcriptase activity was measured as follows. Cells were first infected with HIV-1 and after 7 days 800 μl of culture was mixed with 400 μl of PEG (polyethylene glycol) solution (30% PEG 8000, 0.4 M NaC1) and then stored at 4 ° C. The next day, the virus obtained by centrifugation at 15,000 rpm for 20 minutes in an Eppendorf centrifuge was collected in 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 μl KC1) and 20 μl of solution 2 (0.9% Triton X-100, 43.7 mM KC1), then take 10 μl of 90 μl reverse transcriptase activity assay [46.5 mM Tris-HC1, pH 7.8, 8.89 mM DTT, 11.1 mM MgC1 ₂ , 3.75 mM NaC1, 2.5 μCi 3H-TTP (NEN-Du Pont, 10-25 Ci / mmol), 0.556 μ / ml poly (rA): oligo (dT) (Pharmacia) )] And reacted at 37 ° C. for 1 hour. After the reaction was stopped by adding 9 μl of 0.25 M EDTA, the solution was transferred to a DEAE filter ((DE 81, Whatmann Cat # 3658N323), dried and washed with 2 × SSC buffer. 5 ml of liquid scintillation solution was added and quantified using a liquid scintillation counter.

To test the cytotoxicity to determine the maximum tolerated anti-Aid agent, 1 x 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 of 0.1-100 μM were added RPMI cultured in 1640 medium with 37 ℃ and 4 days while replacing the medium at intervals proliferation of trypan blue die IX crew before the cell method (Trypanblue 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 mixture of the formula (I) of the present invention is useful as a pharmaceutical composition for the treatment or prevention of HIV or HIV infection because of its high inhibitory effect and low cytotoxicity as an irreversible inhibitor of HIV protease. Can be used.

Claims (5)

Compounds having a cis-epoxide structure of formula (I) or a pharmaceutically acceptable salt, hydrate or solvate thereof: Wherein A is a functionalized acyl of the general formula (II) Wherein R ¹ is quinoline, R ² is an asparagine residue, n is 0, 1 or 2, B is hydrogen or C _1-4 alkyl, D is C _1-4 alkyl, or III). (Wherein R ³ and R ⁴ are independently of each other hydrogen, C _1-4 alkyl or NH ₂ ). The compound of claim 1 wherein n is 1, B is hydrogen and D is p-methylphenyl or p-aminophenyl. The compound of claim 1, wherein 4S-[(N ¹ -toluenesulfonyl) amino]-[N ^4- [N ′-(2-quinolinecarbonyl) -L-asparaginyl] amino] -5-phenyl- ( 2S, 3R) -epoxy pentane; 4S-[(N ¹ -paraaminobenzenesulfonyl) amino]-[N ^4- [N '-(2-quinolinecarbonyl) -L-asparaginyl] amino] -5-phenyl- (2S, 3R) Epoxy pentane; 4S-[(N ¹ -Toluenesulfonyl-N ¹ -isobutyl) amino]-[N ^4- [N '-(2-quinolinecarbonyl) -L-asparaginyl] amino] -5-phenyl- ( 2S, 3R) -epoxy pentane and pharmaceutically acceptable salts, hydrates and solvates thereof. Compound (a) is obtained from N- (2-bromoethyl) phthalimide via a Bitig reaction and a reductive amination reaction; Reacting compound (a) with compound (IV) to obtain compound (V); Reacting compound (V) with compound (VI) to obtain compound (VII), and then oxidizing to obtain compound (VII); A method for producing a compound of formula (I), wherein the compound (VII) is deprotected and then coupled with the compound (VII). In the above, A, B and D are as defined in claim 1. Prevention of acquired immunodeficiency or human immunodeficiency virus (HIV) infection, comprising as an active ingredient a compound having a cis-epoxide structure of the general formula (I) or a pharmaceutically acceptable salt, hydrate or solvate thereof Useful compositions for treatment. Wherein A is a functionalized acyl of the general formula (II) Wherein R ¹ is quinoline, R ² is an asparagine residue, n is 0, 1 or 2, B is hydrogen or C _1-4 alkyl, D is C _1-14 alkyl, or Ⅲ) is a group of: (Wherein R ³ and R ⁴ are independently of each other hydrogen, C _1-4 alkyl or NH ₂ ).