JPH08165274A - Hiv protease inhibitor - Google Patents

Abstract

PURPOSE: To provide an HIV protease inhibitor which is useful in prophylaxis and treatment for HIV virus infections such as AIDS because it has the HIV protease inhibitory action, suppress the HIV infection and shows high absorptivity, when orally given. CONSTITUTION: This compound is represented by formula I [R1 is a (substituted) aryl, a (substituted) hetero ring, a (substituted) heteroarylalkyl; R2 is a fluorine- substituted lower alkyl, a fluorine-substituted alkylthio; R3 and R4 are independently H, a lower alkyl; X is S, SO, CH2 9147/28} or a salt thereof, for example, the compound of formula II. The compound of formula I is obtained, for example, by treating a compound of formula III with an alkali at 7-9 of pH.

Description

Detailed Description of the Invention

The present invention relates to a novel norstatin derivative having an inhibitory activity against HIV protease and effective in the prevention and treatment of HIV infection.

[0002]

2. Description of the Related Art Conventionally, it has been proposed to use an immunodeficiency virus (HIV) RNA reverse transcriptase inhibitor or a protease inhibitor for the treatment of acquired immunodeficiency syndrome (AIDS). It has been. Of these,
Various peptide derivatives have been disclosed as active ingredients of protease inhibitors (for example, JP-A No. 2-1176).
No. 15, JP-A-2-202898, JP-A-2
-202899, Japanese Patent Application Laid-Open No. 2-204475, Japanese Patent Application Laid-Open No. 5-78311, Japanese Patent Application Laid-Open No. 5-1707.
No. 22, JP-A-5-178824, JP-A-6
-100533 publication, and WO93 / 3066 etc.). In particular, a derivative having a β-amino-α-hydroxycarboxylic acid group as a hydroxyamino acid isostere in a peptide has been attracting attention (EP0490667A).
2, JP-A-5-170722 and JP-A-5-178
No. 824, and WO93 / 3066). However, there are problems in terms of absorption rate upon oral administration, metabolic stability, transferability to lymph nodes, affinity with HIV protease, etc., especially in terms of absorption rate upon oral administration.
In order to properly treat or prevent HIV infections such as IDS, it is necessary to develop more compounds and examine their clinical applicability.

[0003]

DISCLOSURE OF THE INVENTION The inventors of the present invention have conducted extensive studies for the purpose of providing a novel compound having HIV protease inhibitory activity which can solve the above-mentioned problems, and as a result, Formula I:

[Chemical 4] (In the formula, R ₁ is an optionally substituted aryl, an optionally substituted heterocyclic group, or an optionally substituted heteroarylalkyl; R ₂ is a lower alkyl substituted with fluorine or a fluorine substituted Alkylthio, R ₃
And R ₄ are each independently hydrogen or lower alkyl, X
Represents a compound represented by S, SO or CH ₂ ) or a salt thereof is suitable for achieving this object, and has completed the present invention.

In the present specification, a lower alkyl substituted with fluorine means a straight or branched carbon number in which one or more hydrogen atoms are substituted with one or more fluorine atoms. Means 1-8 alkyl. For the purpose of the present invention, a fluorine-substituted linear or branched carbon number 1 to 6
Is preferred, and specific examples thereof include trifluoromethyl and trifluoroethyl.

The alkylthio substituted with fluorine means that a carbon atom in a hydrocarbon chain is substituted with a sulfur atom, and one or more hydrogen atoms are substituted with one or more fluorine atoms. It means a group, and examples thereof include trifluoromethylthio and trifluoroethylthio. The "lower alkyl" means a straight or branched alkyl having 1 to 8 carbon atoms. For example, methyl, ethyl, propyl, isopropyl and the like are exemplified. “Aryl” means an aromatic ring group having 6 to 12 carbon atoms, and examples thereof include phenyl and naphthyl. Examples of the substituent in the "optionally substituted aryl" include lower alkyl, halogen, nitro, amino, hydroxyl group, alkoxy, aralkyloxy, trifluoromethyl, phenyl and the like. Here, lower alkyl and halogen are as defined above, with methyl, fluorine and chlorine being particularly preferred. For example, tolyl, xylyl,
Biphenyl etc. are illustrated. The “heterocyclic group” means a ring optionally containing one or more oxygen atom, sulfur atom or nitrogen atom in the ring and optionally condensed with a carbocycle. Mention may be made of pyridyl, triazolyl (eg 1,2,3-triazolyl or 1,2,4-triazolyl), thiadiazolyl (eg 1,2,3-thiadiazolyl or 1,2,4-thiadiazolyl), tetrazolyl, quinolyl and isoquinolyl. Quinolyl and xoxoquinolyl are preferred. Examples of the substituent in the "optionally substituted heterocycle" include lower alkyl such as methyl and ethyl, and trifluoromethyl. Examples of the "heteroarylalkyl" include quinolylmethyl, quinolylethyl, isoquinolylmethyl, isoquinolylethyl and the like. The substituent is the same as the substituent in the “optionally substituted heterocycle”.

Salts of compounds of formula I are pharmaceutically acceptable salts of hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid,
Salts derived from inorganic acids such as hydroiodic acid, nitrous acid and phosphorous acid, and aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxy-alkanemonoic acids and alkanedioic acids, aromatic acids and aliphatics And salts derived from non-toxic organic acids such as aromatic sulfonic acids. Such pharmaceutically acceptable acid addition salts include sulfates, pyrosulfates,
Bisulfate, sulfite, bisulfite, nitrate, phosphate,
Dibasic phosphate, primary phosphate, metaphosphate, pyrophosphate, hydrochloride, hydrobromide, hydroiodide, hydrofluoride, acetate, propionate, Decanoate, caprylate, acrylate, formate, isobutyrate, caprate, heptanoate, propiolate, oxalate, malonate, succinate, suberate, sebacate , Fumarate, maleate, mandelate, butyne-1,
4-diacid salt, hexyne-1,6-diacid salt, benzoate,
Chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, phthalate, terephthalate, benzenesulfonate, toluenesulfonate,
Chlorobenzene sulfonate, xylene sulfonate,
Phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, β-hydroxybutyrate, glycolate, malate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1 Includes sulfonates, naphthalene-2-sulfonates and other salts. Preferred for the present invention are salts of inorganic acids, with hydrochlorides and nitrates being especially preferred.

Since the compound of the present invention contains a fluorine atom in the R ₂ group, its hydrophobicity is increased and its affinity with HIV protease is increased as compared with the conventional corresponding compound, and in vivo Therefore, it is expected that it will easily migrate to lymph nodes and that the absorption rate will be high especially when administered orally. Compound I of the present invention has HIV protease inhibitory activity as well as HIV (HTLV-IIIB), as shown in Experimental Examples described later.
Strain), has a high absorption rate upon oral administration and is expected to be useful for the prevention and treatment of HIV infection. All of the compounds I as defined above are useful for the purposes of the invention above, but especially those compounds wherein R ₁ is 5-isoquinolyl, R ₂ is trifluoromethyl and X is S are directed against HIV protease. It has a strong inhibitory activity and is preferable.

The compound of the present invention can be produced by a method known to those skilled in the art. As a general synthetic method, condensation is carried out stepwise by a liquid phase method. For example, in the presence of HOBt / DCC or HOBt / EDC as a condensing agent, CH ₂ Cl ₂ , DMF, THF, AcOE as a solvent.
Any one of t is used. Reaction temperature is -10 ° C to 30
C., preferably 0.degree. C. to 25.degree. C., and the reaction time is 1 to 16 hours, preferably 3 to 5 hours. Deprotection of intermediates is 4
It is carried out using N-HCl / dioxane or 6NaqHCl / dioxane at a reaction temperature of 0 to 25 ° C and a reaction time of 1 to 3 hours. The deprotected amine component may be used in the next reaction by neutralizing hydrochloric acid and taking out in a free state, or after distilling off the solvent under reduced pressure and adding an equivalent amount of a base (for example, TEA or NMM). . The compound I of the present invention can be produced, for example, according to the following reaction formula.

[Chemical 21] 1) Preparation of compound (a): Condensation and deprotection of commercial Boc amino acid with t-butylamine 2) Preparation of compound (b): M.P. T. Reeze et al., THL 29
3259 (1988) 3) Preparation of compound (e): T.I. Tushima et al., Tetrahedron
44 (17) 5375 (1988) 4) Preparation of compound (f): R ₁ —OH and BrCH ₂ CO ₂ R ₄
More induction

Further, the compounds of the present invention have the formula II:

Embedded image (Wherein R ₁ , R ₂ , R ₃ , R ₄ and X are as defined above) at pH 7-9 at 2
It can be produced by treating at 0 to 25 ° C. The conversion of Compound II to Compound I having HIV protease inhibitory activity can be carried out at a normal physiological pH, as will be apparent from the above reaction conditions and the description of Example 3 below.
And over the temperature range. This indicates that compound II, which does not have anti-protease activity by itself, can be readily converted to a compound of formula I that is active under in vivo conditions when administered to a subject such as a human. ing. That is, the compound II is also suitable for the purpose of the present invention and can function as a precursor or an intramolecular prodrug of the compound I of the present invention. Therefore, the compound II is included in the scope of the present invention, and like the compound I, any pharmaceutically acceptable salt thereof is also included in the scope of the present invention.

This ester type compound II is water-soluble and is converted into the compound of formula I in the intestine when it is orally administered and absorbed, and when it is intravenously administered, it is immediately converted to the compound of formula I in plasma. It is useful for both oral and parenteral administration because it is converted into the compound to exert a pharmacological effect.

In order to use Compound I or Compound II of the present invention or a salt thereof for the prevention or treatment of HIV infection, it is administered in the form of a formulation suitable for oral or parenteral administration. In the case of oral administration, the compound of the present invention is either a usual preparation, for example, a solid preparation such as tablets, powders, granules and capsules; water preparation; oily suspension; or liquid preparation such as syrup or elixir. It can also be used as a dosage form. When administered parenterally, the compound of the present invention can be used as an aqueous or oily suspension injection. In the preparation, any of conventional excipients, binders, lubricants, aqueous solvents, oily solvents, emulsifiers, suspending agents and the like can be used, and other additives such as preservatives, It may contain a stabilizer or the like. The dose of the compound of the present invention or a salt thereof varies depending on the administration method, age, weight, condition of the patient and type of disease, but usually, orally, 3 mg to 2 g per day, preferably 10 mg to 1 g. It may be administered in 1 to 5 divided doses.

[0012]

The present invention will be described in detail below with reference to examples, but the present invention is not limited thereto. The abbreviations used in the examples are explained below. TEA: triethylamine (Boc) ₂ O: di-t-butyl carbonate H ₂ N +: t-butylamine EDC (HC1): 1-ethyl-3- (3-dimethylaminopropylcarbodiimide hydrochloride HOBt (H ₂ O) : N-hydroxybezosotriazole
Hydrate FmocOSU: 9-fluorenyl-N-succinimidyl carbonate DIC: diisopropylcarbodiimide

Production Example 1

[Chemical 6] Compound 1 (5.00 g, 37.55 mmol) was added to CH ₂ Cl
Suspend in ₂ (50ml) and TEA (5.8ml, 41.38mmo
l) was added, and (Boc) ₂ O (9.80 g, 4
(4.9 mmol) is added and reacted for 3 hours. Aqueous Na in the reaction solution
HCO ₃ is added to redissolve the acidic part, and then citric acid is added to the basic solution to bring it to pH 3 and extracted with ethyl acetate (× 3). The extracts were combined and washed with saturated NaCl (× 2),
After drying over MgSO ₄ , the solvent was distilled off under reduced pressure to give compound 2 (7.
2 g, yield 82.2%) was obtained. Compound 2 (7.20 g,
3.09 mmol), t-butylamine (3.89 ml, 3.70 m)
mol) and HOBt (4.84 g, 3.58 mmol) in CH ₂ C
Suspend in l ₂ (40 ml) and stir in an N ₂ stream under ice-cooling and ED
After adding C (7.1 g, 3.6 mmol) and reacting for 1 hour,
React overnight at room temperature. The solvent was distilled off under reduced pressure, and 6N was added to the residue.
-HCl (50 ml) -dioxane (20 ml) was added and 5
React on time. The reaction solution was washed with _{CH 2 Cl 2 (× 3)} , made alkaline with _{NaHCO 3, CH 2 Cl 2 (} × 3)
Compound 4 (5.80 g, yield: quantitative) was obtained by extraction with.

Production Example 2

[Chemical 7]Compound11(Tetrahedron Letters, Vol.29, No.2
7,3295-3298 (1988)) (4.77g, 16.1)
5 mmol), compoundFour(2.53 g, 13.44 mmol) and H
OBt (2.18 g, 16.13 mmol) in CH₂Cl₂(30m
l) -dissolved in MeCN (10 ml), N₂In the air flow, ice cooling
EDC (3.18 g, 16.14 mmol) is added with stirring.
After 1 hour, the mixture is allowed to react at room temperature for 16 hours. The reaction solution is aqueous
NaHCO₃(X2), water, 10% citric acid (x2) and
After washing sequentially with water, MgSO_FourAnd dry the solvent under reduced pressure.
The crude (2S, 3S) (Nt-butoxycarbonyl-
3-amino-2-hydroxy-4-phenyl-butyri
) -L-thiazolidine-4-carboxylic acid t-butyl ester
The amide (Compound 23) (7.0 g) was obtained. Crude compound23
To (7.00 g), add 50 ml of 4N-HCl / dioxane.
Then, the mixture is reacted for 2 hours at room temperature with stirring. Evaporate the solvent under reduced pressure
And CH in the residue₂Cl₂Add (15 ml) and water (15 ml)
Eh, under stirring, aqueous NaHCO₃At pH8 and precipitated
A compound showing an mp of 213 to 215 ° C., which was obtained by filtering crystals.24
(4.60 g, yield 93.7%) was obtained.

Production Example 3 (2S, 3S) -N- (t-butoxycarbonyl trifluoromethyl-L-alanyl)-
3-amino-2-hydroxy-4-phenyl --t-butyryl -L- thiazolidine-4-carboxylic acid butylamide (Compound 25 a)

Embedded image Compound 24 (2.50 g, 6.84 mol), Compound 18 a
(Prepared according to Tetrahedron Vol. 44, No. 17, 5375-5387 (1988)) (2.00 g, 7.78 mmol) and HOBt
(1.05 g, 7.77 mmol) was suspended in CH ₂ Cl ₂ (30 ml) and MeCN (10 ml), and EDC (1.53 g, 7.77 mmol) was added under ice-cooling stirring in a N ₂ stream, React for 1 hour (clear in about 30 minutes), then 2 hours at room temperature. The precipitated crystals were collected by filtration, washed with CH ₂ Cl ₂ and m
Crystals showing p215-217 ° C (3.627 g, yield: 8
7.7%). After washing the mother liquor with aqueous NaHCO ₃ ,
The crystalline residue obtained by drying over SO ₄ and evaporating the solvent under reduced pressure was triturated with CH ₂ Cl ₂ and mp 215-217 ° C.
To give a crystal (0.357 g, yield: 8.6%). The products were totaled to obtain 3.994 g (yield: 96.6%). Using compound <br/> compound 18 b and 18 c as starting materials, the corresponding compounds were treated in the same manner as above 25 a (90% yield) and 2
5 was obtained b (88% yield).

[Chemical 9]

Production Example 4

[Chemical 10] Compound 25 a (3.99g, 6.60mmol) in 4N-HCl
40 ml of dioxane was added, and the mixture was reacted at room temperature for 2 hours with stirring. The solvent is distilled off under reduced pressure, and water is added to the residue to dissolve it. After washing with CH ₂ Cl ₂ (10 ml × 3), NaHClO ₃
Made alkaline with 5% MeOH / CH ₂ Cl (25 ml
Extract with × 3). The CH ₂ Cl ₂ solution was dried over MgSO ₄ and then evaporated under reduced pressure to give a residue of 3.00 g (yield: 90.1%).
I got Compound 25 b (0.304 g) and Compound 25
c (0.260 g) was treated in the same manner as above to give the corresponding compound 26 b (0.245 g, yield 96.1%) and compound 26 c (0.200 g, yield 91.3%). Obtained.

Production Example 5

[Chemical 11] Amine 18 a (about 0.700 g) aqueous Na ₂ CO ₃ (8.9
ml; Na ₂ CO ₃ 5.30 g / water 100 ml) and MeCN (6
ml) and stirred under ice-cooling with FmocOSU (1.522).
g, 4.68 mmol) in MeCN (12 ml) is added.
Then, the mixture is reacted at room temperature for 14 hours. 20 ml of water in the reaction solution
Was added and the precipitated insoluble matter was filtered off, and then N-HCl was added.
The pH is adjusted to pH3 by adding and extracted with ethyl acetate (30 ml × 3). The ethyl acetate solution was combined and washed with a saturated NaCl solution (20 ml × 3), dried over MgSO ₄ , and the solvent was distilled off under reduced pressure to obtain a crystalline residue (1.62 g). CH ₂ Cl ₂ :
Recrystallized from hexane (1: 5) mixture to give mp159-1
Compound 27 a showing 60 ° C. (1.44 g, yield 85.2%)
I got Compound 18 b of the amine shown in Production Example 3 (0.
754 g) and FmocOSU (1.502 g)
No crystal form compound showing the p127-128 ℃ 27 b (1.4
2 g, yield 80%) was obtained. Similarly, amine compounds 18 c shown in Production Example 3 (0.576 g) and FmocOS
Amorphous compound 27 c (0.96 g) using U (1.04 g)
0 g, yield 75.5%) was obtained.

Production Example 6

[Chemical 12] Compound 27 a (0.293g, 0.77mmol) and HOBt
(0.115 g, 0.85 mmol) was dissolved in CH ₂ Cl ₂ (3 ml) and DMF (1 ml), and DI was stirred in an N ₂ stream under ice-cooling.
C (0.14 ml, 0.644 mmol) is added. 20 minutes later,
Compound 23 (0.800 g, 0.644 mmol) / CH ₂ Cl ₂
(2 ml) solution was added, and after 30 minutes, the temperature was raised to room temperature and the reaction was continued for 5 hours. The solvent was distilled off under reduced pressure, the residue was dissolved in ethyl acetate, washed with 7% NaHCO ₃ (× 2) and saturated NaCl (× 2), and dried with MgSO ₄ . The solvent was distilled off under reduced pressure,
A crude product (0.75 g) was obtained, and silica gel (230-40
0 mesh, 120 g) and CH ₂ Cl ₂ containing 1% MeOH
Compound 28a (amorphous form) (0.5
08 g, yield 95.3%) was obtained. Compound as starting material
27 b (0.180g), HOBt ( 0.065g), DIC
(0.076 ml) and Compound 23 (0.70 g) were used,
Was treated in the same manner as the above compound 28 b (amorphous) (0.23
8 g, yield 77.5%) was obtained. Similarly, compound 27 c
(0.236g), HOBt (0.083g), DIC (0.0
96 ml) and compound 23 (0.200 g)
28 c (amorphous form) (0.278 g, yield: 76.8%) was obtained.

Production Example 7

[Chemical 13] Compound 28 a (0.500g, 0.61mmol) in morpholine (2 ml) was added, under stirring, to the reaction for 2.5 hours. The morpholine was distilled off under reduced pressure, and the residue was converted to silica gel (230-400).
Mesh, 100g) and concentrated NH ₄ OH water / MeOH / C
Medium pressure chromatography was carried out using a mixed solution of H ₂ Cl ₂ (0.4: 4: 95.6) to obtain the target compound 29 a (0.288 g, yield 78.9).
%) Was obtained. Compound 28 b (0.238 g) and was treated similarly, using morpholine (1 ml) Compound 29 b (0.
130 g, yield 74.3%) were obtained. Similarly, compound 28
From c (0.190 g) and morpholine (1 ml), the compound
29 c was obtained (0.118 g, yield: 77.0%).

Production Example 8

Embedded image Compound 29 a (0.276 g, 0.46 mmol), compound 22
(0.112 g, 0.55 mmol) and HOBt (0.074)
g, 0.55 mmol) in CH ₂ Cl ₂ (2 ml), MeCN (0.5
ml) and EDC (0.118 g, 0.55 mol),
Reacted in the same manner as in Production Example 3, compound 30 a (0.280 g,
Yield 78%) was obtained. Compound as a starting material was 29 b (0.
268 g, 0.43 mmol), compound 22 (0.105 g, 0.14 g).
52mmol), HOBt (0.070g, 0.52mmol ) and EDC (0.102 g, with 0.52 mmol), in the same manner as described above reacts with compound 30 b (0.245 g, yield: 71.4
%) Was obtained. Similarly, compound 29c (0.260 g, 0.
4 mmol), compound 22 (0.100 g, 0.5 mmol), HO
Bt (0.068g, 0.5mmol) and EDC (0.097)
g, using 0.5 mmol), compound 30 c (0.25g, 77.4% yield, amorphous) was obtained.

Example 1 (2S) (3S)-[N- (5-isoquinoliniuoxyacetyl) -trifluoromethyl-L-
Alanyl] -3-amino-2-hydroxy-4phenyl-
Butyryl-L-thiazolidine-4-carboxylic acid t-butylamide (Compound Ia)

[Chemical 15] Compound 22 (1.400 g, 6.89 mmol), Compound 26 a
(3.30 g, 6.54 mmol) and HOBt (0.466)
g, 3.45 mmol) in CH ₂ Cl ₂ (50 ml), MeCN (1
0 mL) and stirred in an N ₂ stream under ice-cooling with EDC (1.
36 g, 6.9 ml) was added and the reaction was carried out for 1 hour and then at room temperature for 2 hours. The reaction solution was washed with aqueous NaHCO ₃ , water, 10% citric acid, water, aqueous NaHCO ₃ and water, and then flash chromatographed using silica gel (15 g) and CH ₂ Cl ₂ / MeOH (95: 5) to flow out. The product was collected and crystallized from ethyl acetate to give crystals (3 g) showing mp 142-144 ° C. Furthermore, the mother liquor is silica gel (80 g) and 2% MeO.
Chromatography was carried out using a mixed solvent of H / CH ₂ Cl ₂ (containing 0.2% concentrated aqueous ammonia) to obtain crystals (0.20 g) having an mp of 143 to 144 ° C. Since this crystal has 1/2 of ethyl acetate as a crystal solvent, it was dissolved in CH ₂ Cl ₂ and evaporated under reduced pressure (× 3), ethyl ether was added to the residue, and the mixture was stirred.
After treated for 0.5 hours, filtered and dried, mp143-1
Compound Ia showing 45 ° C. (3.30 g, yield: 73.2%)
I got In the same manner as in Example 1 above, the following compounds Ib to Ih were synthesized from the corresponding starting materials. The structures, NMR, IR, [α] _D, and elemental analysis results of these compounds Ia to Ih are summarized below.

[0022]

Embedded image

[Chemical 17]

[0023]

[Table 1] c: calculated value; f: measured value

[0024] Example 2 Compound 30 was prepared in Preparation Example 8 a (0.254g, 0.3
4N-HCl / dioxane solution (2 ml) was added to 2 mmol), and the mixture was reacted for 2 hours at room temperature with stirring. The reaction mixture was evaporated to dryness under reduced pressure, and the crystalline residue was triturated with Et ₂ O-CH ₂ Cl ₂ to give compound IIa showing mp167-170 ° C.
(0.245 g, yield 97.6%) was obtained. Example 2 above
From the corresponding starting materials in the same manner as
Ib and IIc were synthesized. These compounds IIa to II
The structure, melting point, and elemental analysis result of c are summarized below.

[0025]

Embedded image

[0026]

[Table 2]

Example 3

[Chemical 19] Compound IIa prepared in Example 2 (0.150 g, 0.19)
mmol) with 7% aqueous NaHCO ₃ (4 ml) at room temperature.
Stir for 0 minutes. The reaction solution is extracted with CH ₂ Cl ₂ (5 ml × 3). The extracts were combined, washed with water and dried over MgSO ₄ , and the solvent was distilled off to obtain a crystalline residue (0.133 g).
This was crystallized with ethyl ether and mp143-145 ° C.
(0.126 g, yield 94.7%) was obtained.
The product was confirmed to be the compound (Ia) because it did not show melting point drop due to melting with the compound Ia synthesized in Example 1 and the NMR, IR [α] and TLCRf values were the same. It was Compound IIb or IIc was treated in the same manner as above to give the corresponding compound Ib (yield 95.5, respectively).
%) And compound Ic (96.5%). The HIV protease inhibitory action and HIV infection inhibitory action of the compounds obtained in Examples were examined by the following methods.

Experimental Example 1 Measurement of HIV-1 Protease Inhibitory Action of Compound The experiment was carried out according to the following general procedure. Material A sample solution (DMSO) of 5-fold dilution series is prepared in advance, and 5 μl is taken from the sample solution and placed in an Eppendorf microtube. To this, the reaction solution that had been ice-cooled (95 μl)
Add. The final concentrations of the components after mixing were set as follows. Mes-NaOH buffer solution (pH 6.0) 50 mM NaCl 200 mM EDTA.2Na 5 mM DGGpE ¹⁾ 5 μM HIV-1 protease ²⁾ 0.27 mU / ml DMSO 10% Note: 1) Fluorescent substrate 4- (4-dimethylaminophenenyl) (Razo)
Benzoyl (DABCYL) -γ-aminobutyryl (GA
BA) -Ser-Gln-Asn-Tyr-Pro-Ile-Val-
Gln-5-[(2-aminoethyl) amino] naphthalene-1
-Sulfonic acid (EDANS) 2) HIV-1 protease was added at 1 µM for 1 minute at 37 ° C.
The activity of degrading DGGpE of is defined as 1 unit.

Method The reaction solution was reacted at 37 ° C. for 2 hours, and the reaction was stopped by adding 2% trifluoroacetic acid (TFA) (100 μl). The decomposition product in the reaction solution was analyzed using a TSK-gel ODS-80TM column, 0.1% TFA-17% acetonitrile,
The mixture was separated by HPLC under the condition of 0.8 ml / min, excited at 365 nm, and the fluorescence intensity at 490 nm was measured. The percentage inhibition of protease by the compound was calculated according to the following formula.

## EQU1 ## Inhibition rate (%) = [1-{(peak area when sample is added) / (peak area when sample is not added)] × 100 50% inhibitory concentration (IC ₅₀ ng / ml) Was calculated from the semilog plot of the inhibition rate% at each concentration of the compound.

Experimental Example 2 Inhibitory Action of Compounds The anti-HIV activity and cytotoxicity of the compounds prepared in Examples were tested by the following methods. Antiviral activity (1) HIV (HTLV-IIIB strain) persistently infected human T cell line MOLT-4clone8 with 10% fetal bovine serum added RP
Culture in MI-1640 medium and filter the supernatant to determine virus titer and store at -80 ° C. On the other hand, the test compound is diluted with the above culture medium to a predetermined concentration,
Dispense 100 μl each into a 96-well microplate. Next, 50 μl of MT-4 cell suspension (2.5 × 10 ⁴ cells)
50 μl (600 pfu (plaque formi) of the above HIV-containing supernatant diluted with the above culture medium
ng unit)) each.

(2) After culturing at 37 ° C. for 5 days in a carbon dioxide incubator, MTT (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide) was added to all wells. 5 mg / ml, PBS) is added in an amount of 30 μl each, and the mixture is further cultured for 1 hour. At this time, the surviving cells reduce MTT to precipitate formazan. 150 μl of culture supernatant was removed from all wells, and instead 150 μl of lysate (10% Triton X-100 and 0.
Add 4% (v / v) HCl in isopropanol) and shake with a plate mixer to elute formazan.
Formazan is measured at OD540 nm and the results are compared to controls. The ED ₅₀ is the concentration of the compound that suppresses the cell damage caused by the virus by 50%. The test results are shown in Table 3.

Cytotoxicity In the above (1), 50 μl of culture medium was added to each well instead of the HIV-containing supernatant (virus solution), and (2)
The same procedure as above is used to examine the cytotoxicity of the compound. The CD ₅₀ is defined as the compound concentration at which the compound has 50% cytotoxicity.
The above test results are summarized in Table 3 below.

Experimental Example 3 Measurement by Oral Administration to Rats Rats fasted overnight (male Jcl-SD, 8-9 weeks old, 2
40-300g) to HIV protease inhibitor (20mg
/ 4 ml / kg of 0.01 M citric acid aqueous solution or suspension) was orally administered, and then blood was collected from the jugular vein over time, and changes in plasma concentration were followed under non-anesthesia.

[0034] AUC (area under the concentration-time curve in plasma) measured deproteinized blood samples, plasma (200μl) MeCN (750μ
l) was added, the mixture was stirred with a mixer, centrifuged with a cooling centrifuge, and the resulting supernatant (850 μl) was evaporated to dryness, and the eluent (0.1% aqueous trifluoroacetic acid solution-MeCN solvent) ( 1
After dissolving in 50 μl), 100 μl was injected into HPLC and quantified under the following conditions. Quantification Quantification of HIV protease inhibitors is done by SPD-M6A
LC-6A HPLC equipped with a UV detector (Shimadzu Corporation; Kyoto) was used (column: Nucleosil 5).
C ₁₈ ; eluent: 0.1% trifluoroacetic acid aqueous solution-MeC
N-based solvent). The results are shown in Table 3 below and FIG.

[0035]

[Table 3] Note 1) MTT assay 2) HIV protease inhibition assay

Embedded image

[0036]

As is clear from the results shown in Table 3,
The compound of the present invention or a salt thereof has an HIV protease inhibitory activity, exhibits an HIV infection inhibitory action, and has a high absorption rate upon oral administration. Therefore, the compound of the present invention can be used to prevent or treat HIV virus infections such as AIDS.

[Brief description of drawings]

FIG. 1 is a graph showing changes in plasma concentration after oral administration of the compound of the present invention to rats.

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Claims (6)

[Claims] 1. Formula I: (In the formula, R ₁ is an optionally substituted aryl, an optionally substituted heterocyclic group, or an optionally substituted heteroarylalkyl; R ₂ is a lower alkyl substituted with fluorine or a fluorine substituted Alkylthio, R ₃
And R ₄ are each independently hydrogen or lower alkyl, X
Represents S, SO or CH ₂ ) or a salt thereof. 2. Formula II: Preparation of a compound of formula I according to claim 1 which comprises treating a compound of formula (wherein R ₁ , R ₂ , R ₃ , R ₄ and X are as defined above) at pH 7-9. Method. 3. Formula II: embedded image (Wherein R ₁ , R ₂ , R ₃ , R ₄ and X are as defined above) or a salt thereof. 4. The compound according to claim 1 or 3, wherein R ₁ is a heterocyclic group, R ₂ is trifluoromethyl and X is S. 5. The compound according to claim 4, wherein R ₁ is isoquinolyl. 6. An antiviral agent containing an effective amount of the compound according to claim 1.