JPH11116480A - Phaemaceutical composition containing acridone derivative - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject composition, capable of sufficiently inhibiting the transcription from a DNA to an RNA in a cell sustainedly infected with a retrovirus, especially a human immunodeficiency virus(HIV) and having antiretroviral activity (anti HIV activity) by including an acridone derivative having a specific structure as an active ingredient therein. SOLUTION: This pharmaceutical composition contains an acridone derivative represented by formula I (R is an alkyl; R<1> to R<4> are each H, OH, a halogen or an alkoxy), e.g. 1-methoxy-10-methylacridone as an active ingredient. Furthermore, the derivative is preferably obtained by cooling a solution of, e.g. lithium N-isopropylcyclohexylamide in an organic solvent to -80 to -60 deg.C, then adding an anthranilic acid derivative represented by formula II thereto, stirring the resultant mixture, heating up the mixture to -20 to 0 deg.C, adding a benzene derivative represented by formula III thereto and reacting both the compounds while stirring the mixture at 0-40 deg.C.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a pharmaceutical composition useful as an antiretroviral agent, particularly as an anti-HIV (human immunodeficiency virus) agent.

[0002]

2. Description of the Related Art In recent years, various compounds having anti-HIV activity have been found, and some of them have already been widely used clinically as pharmaceuticals. Most of these conventional pharmaceuticals are intended to improve the symptoms of patients in the acute phase, and the mechanism of action is to inhibit reverse transcriptase or protease.

[0003]

However, in the case of conventional pharmaceuticals, after the infection with HIV, the replication of HIV DNA integrated into the genome of immune cells is not performed very much. However, there has been a strong demand for the development of a drug that does not sufficiently prevent the transition from the incubation period to the acute stage, which does not sufficiently prevent the transition to the acute stage.

Accordingly, an object of the present invention is to provide an excellent anti-HIV which is capable of sufficiently inhibiting the replication of the above viral DNA integrated into the genome of immune cells to RNA after infection with a retrovirus, particularly HIV. It is to provide a pharmaceutical composition having activity.

[0005]

Means for Solving the Problems The present inventors have made intensive studies to achieve the above object, and as a result, have found that an acridone derivative having a specific structure can be converted from DNA in a cell persistently infected with a retrovirus, particularly HIV, to RNA. Replication, and has excellent antiretroviral activity (anti-HIV
Activity), thereby completing the present invention.

That is, the present invention provides the following formula (I):

[0007]

Embedded image (In the formula (I), R represents an alkyl group, R ^{1 to} R ⁴ may be the same or different, and each represents a hydrogen atom,
Shows any one selected from the group consisting of a hydroxyl group, a halogen atom and an alkoxy group. A pharmaceutical composition comprising the acridone derivative represented by the formula (1) as an active ingredient.

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION The active ingredient of the pharmaceutical composition of the present invention has the following formula (I):

[0009]

Embedded image Is an acridone derivative represented by

R in the above formula (I) is an alkyl group,
A lower alkyl group having 1 to 5 carbon atoms such as methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, sec-butyl and tert-butyl is preferred, and a methyl group is particularly preferred.

Further, R ^{1 to} R ⁴ in the above formula (I) may be the same or different and are each selected from the group consisting of a hydrogen atom, a hydroxyl group, a halogen atom and an alkoxy group. Is shown. Examples of such a halogen atom include fluorine, iodine, iodine, and chlorine atoms. The alkoxy group is preferably a lower alkoxy group having 1 to 5 carbon atoms such as methoxy, ethoxy, n-propoxy, iso-propoxy, n-butoxy, iso-butoxy, sec-butoxy, tert-butoxy, and the like. Groups are particularly preferred.

More specifically, R ¹ is preferably a hydroxyl group, a hydrogen atom or a lower alkoxy group having 1 to 5 carbon atoms, since the anti-HIV activity of the acridone derivative tends to be improved. Further, it is preferable that R ² is a hydrogen atom or a hydroxyl group, since the anti-HIV activity of the acridone derivative tends to be improved. In particular, when R ² is a hydrogen atom, an acridone derivative having particularly excellent anti-HIV activity tends to be obtained. It is particularly preferred because of its existence. Further, it is preferable that R ³ is a hydroxyl group, a hydrogen atom or a lower alkoxy group having 1 to 5 carbon atoms because the anti-HIV activity of the acridone derivative tends to be improved. Further, R ⁴ is preferably a hydrogen atom, a hydroxyl group or a halogen atom, since the anti-HIV activity of the acridone derivative tends to be improved.

Accordingly, among the acridone derivatives represented by the above formula (I), an acridone derivative in which R is a methyl group, more preferably an acridone derivative in which R is a methyl group and R ² is a hydrogen atom, particularly preferably R Is a methyl group, R
¹ is a hydroxyl group, it is preferable that R ² to R ⁴ is a 1-hydroxy-10-methyl acridone, the active ingredient is a hydrogen atom.

The above acridone derivative according to the present invention may be any pharmaceutically acceptable salt (eg,
Acid addition salts such as hydrochloride and tartrate), hydrates and solvates.

Such an acridone derivative is a known compound or can be prepared according to a known method such as the following reaction scheme (for example, Chem. Pharm. B
ull., 32 (3), 1264-1267 (1984), J. Org.Chem., 1988,
53, 880-882, J. Chem. Soc., Perkin Trans. 1 (199
5), (18), 2333-2337, etc.).

[0016]

Embedded image In the above formulas (II), (III) and (I) [(I-1) and (I-2)], R represents an alkyl group, and R ^{1 to} R ⁴ are the same, ^May be different from each other and represents any one selected from the group consisting of a hydrogen atom, a hydroxyl group, a halogen atom and an alkoxy group, and R ⁵ represents a hydrogen atom or a halogen atom.

That is, a solution of lithium N-isopropylcyclohexylamide in an organic solvent is cooled to -80 to -60 ° C, an anthranilic acid derivative (compound of the formula (II)) is added, and the mixture is stirred. After raising the temperature, a benzene derivative (compound of the formula (III)) is added, and the mixture is reacted at 0 to 40 ° C. with stirring to obtain a compound of the formula (I-1)
An acridone derivative represented by the following formula is obtained. Next, the acridone derivative represented by the formula (I-2) is obtained by reacting the acridone derivative of the formula (I-1) with tribromoboron at 0 to 40 ° C. with stirring in an organic solvent.
The acridone derivative thus obtained can be isolated and purified by a chromatography method using silica gel or the like.

The pharmaceutical composition of the present invention may contain the above-mentioned acridone derivative as an active ingredient, and other ingredients are not particularly limited. For example, the pharmaceutical composition of the present invention is used as a composition containing excipients and other additives usually used in medicine. Examples of such other components include, as solid components, lactose, kaolin, sucrose, crystalline cellulose, corn starch, talc, agar, pectin, stearic acid, magnesium stearate, lecithin, sodium chloride, and the like. ,
Examples of the liquid form include glycerin, peanut oil, polyvinylpyrrolidone, olive oil, ethanol, benzyl alcohol, propylene glycol, and water.

The pharmaceutical composition of the present invention can take any form, for example, solid preparations such as tablets, powders, granules, capsules, suppositories, troches and the like.
Liquid preparations such as syrup, emulsion, soft gelatin capsule, cream, gel, paste, spray, injection and the like can be mentioned.

The content of the above acridone derivative in the pharmaceutical composition of the present invention is not particularly limited and is appropriately selected depending on the dosage form and the like.
It is preferably about 99% by weight.

The method of administering the pharmaceutical composition of the present invention may be oral,
Any administration route such as enteral, parenteral, topical administration and the like may be used, and the dose is appropriately determined according to the patient's age, disease state, body weight, etc., but is usually 0.001 to 1 per day.
The dose is selected from the range of 000 mg / kg body weight, and is administered once or in multiple doses.

As is clear from the test examples described below, the pharmaceutical composition of the present invention can be used for HIV persistently infected cells.
Excellent inhibition of DNA to RNA replication and excellent anti-HI
Since it has V activity, it is useful as an antiretroviral agent, particularly as an anti-HIV agent. Therefore, the pharmaceutical composition of the present invention can be effectively used for the treatment and prevention of retroviral infection, particularly for the treatment of AIDS.

[0023]

Next, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples.

Examples 1 to 13 ( Synthesis Examples ) Preparation of Acridone Derivatives According to the synthesis methods shown in the following (1) and (2), various acridone derivatives shown in the following reaction schemes were synthesized.

[0025]

Embedded image

(1) n-butyllithium (8.25 equivalents) and N-isopropylcyclohexylamine (8.2
50 ml of a tetrahydrofuran solution (LICA / THF) containing 8.25 equivalents of lithium N-isopropylcyclohexylamide generated from the above (5 equivalents) was cooled to −78 ° C. using dry ice / acetone. A solution of N-alkylanthranilic acid methyl ester (compound of formula (II), 2.5 equivalents) in THF (5 m
l) was added slowly using a syringe. After stirring the obtained mixture at -78 ° C overnight, the temperature was raised to -10 ° C,
T of halobenzene (compound of formula (III), 5.0 equivalents)
An HF solution (5 ml) was added. After stirring the obtained mixture at room temperature for 12 hours, the reaction was stopped by adding saturated saline, and the solvent was distilled off under reduced pressure. The product was extracted from the obtained residue using dichloromethane, the dichloromethane layer was dried, and the solvent was distilled off to obtain an acridone derivative represented by the formula (I-1). These acridone derivatives were purified by silica gel column chromatography (eluent: dichloromethane) to obtain the acridone derivatives of Examples 1 to 7.

(2) Next, a chloroform solution (10 ml) of the acridone derivative (1.0 equivalent) represented by the formula (I-1) is prepared.
Was cooled to 0 ° C. using an ice bath, a chloroform solution of tribromoboron (2 equivalents) was added, and the resulting mixture was stirred at room temperature for 12 hours to react. After completion of the reaction, a 5% sodium hydrogencarbonate solution was added to the mixture, a chloroform layer was extracted, dried, and the solvent was distilled off under reduced pressure to obtain an acridone derivative represented by the formula (I-2). . These acridone derivatives were purified by silica gel column chromatography (eluent: dichloromethane).
13 acridone derivatives were obtained.

The yield in each of the examples, the structural formula, molecular weight, melting point, NMR measurement value and I
The R measurements are listed below. In addition, NMR measurement values are manufactured by Varian, trade names: Gemini200 and 300NM.
R spectrometer and IR measurement values were measured using a JASCO IR810 spectrometer (trade name, manufactured by JASCO Corporation).

Example 1 The following acridone derivative 3a was obtained from the compound 1a of the formula (II) and the compound 2a of the formula (III) according to the method of (1), and the yield was 50%.
Met.

[0030]

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Example 2 The following acridone derivative 3b was obtained from the compound 1a of the formula (II) and the compound 2b of the formula (III) according to the method of (1), and the yield was 50%.
Met.

[0032]

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Example 3 The acridone derivative 3c shown below was obtained from the compound 1a of the formula (II) and the compound 2c of the formula (III) according to the method of (1). The yield was 68%.
Met.

[0034]

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Example 4 The following acridone derivative 3d was obtained from the compound 1a of the formula (II) and the compound 2d of the formula (III) according to the method of (1), and the yield was 59%.
Met.

[0036]

Embedded image

Example 5 The following acridone derivative 3e was obtained from the compound 1a of the formula (II) and the compound 2e of the formula (III) according to the method of (1), and the yield was 13%.
Met.

[0038]

Embedded image

Example 6 The following acridone derivative 3f was obtained from the compound 1a of the formula (II) and the compound 2f of the formula (III) according to the method of (1), and the yield was 67%.
Met. This 10-methylacridone is commercially available as Wako Pure Chemicals 550-24431, Aldrich 19250-3.

[0040]

Embedded image

Example 7 The acridone derivative 3g shown below was obtained from the compound 1b of the formula (II) and the compound 2a of the formula (III) according to the method of (1), and the yield was 57%.
Met.

[0042]

Embedded image

Example 8 Acridone derivatives shown below according to the method of compounds (3a) to (2) of formula (I-1)
When 4a was obtained, the yield was 80%.

[0044]

Embedded image

Example 9 An acridone derivative shown below from the compound 3b of the formula (I-1) according to the method of (2)
When 4b was obtained, the yield was 75%.

[0046]

Embedded image

Example 10 Acridone derivatives shown below from compounds 3c of formula (I-1) according to the method of (2)
When 4c was obtained, the yield was 80%.

[0048]

Embedded image

Example 11 An acridone derivative shown below according to the method of (2) from compound 3d of formula (I-1)
When 4d was obtained, the yield was 75%.

[0050]

Embedded image

Example 12 An acridone derivative shown below is prepared from the compound 3e of the formula (I-1) according to the method of (2).
When 4e was obtained, the yield was 80%.

[0052]

Embedded image

Example 13 Acridone derivative shown below from compound 3g of formula (I-1) according to the method of (2)
When 4f was obtained, the yield was 80%.

[0054]

Embedded image

( Test Example 1 ) Using HIV persistently infected cells
Anti-HIV activity test Each of the acridone derivatives (drugs) obtained in Examples 1, 3, 6, 8 to 13 was used in a culture solution (trade name: RPMI164, manufactured by Nissui Pharmaceutical Co., Ltd.) using each well of a 96-well microplate.
0, 100 μl) to prepare a solution having a concentration of 0.16 to 100 μg / ml for each acridone derivative.
Then, OM10.1 cell culture (1 × 10 ⁴
Cells / 100 μl) and left at 37 ° C. for 30 minutes. Thereafter, TNF (tumor necrosis factor, manufactured by Genzyme, trade name: TNF-α, 1 ng / ml) was added to each solution and cultured for 2 days. Two days later, 50 μl of the supernatant was collected, and the p24 antigen was measured using an HIV-1 p24 antigengen ELISA kit (manufactured by Celluler). Compared to control without drug, the increase in p24 antigen was
% Inhibitory drug concentration (50% effective concentration (EC ₅₀ ), μg
/ Ml). The results obtained are shown in Table 1 below.

[0056]

[Table 1]

As is clear from the results shown in Table 1, the acridone derivatives of the present invention obtained in Examples 1, 3, 6, 8 to 13 sufficiently suppressed the increase of the p24 antigen in cells persistently infected with HIV. From the results, it was confirmed that the compound has an excellent anti-HIV activity that sufficiently prevents the replication of HIV DNA integrated into the genome of immune cells into RNA.

( Test Example 2 ) Cytotoxicity test The culture of each of the above solutions was continued for two more days.
The cytotoxicity of the acridone derivative (drug) obtained in 3,6,8,12 was measured by MTT (3- (4,5-dimethylthiazol-2-yl) 2,5-
diphenyltetrazolium bromide) method. That is, 20 μl of an MTT solution (manufactured by Sigma, trade name: MTT, 7.5 mg / ml) was added to each of the cultured solutions, the mixture was allowed to stand at 37 ° C. for 3 hours, and the eluate (10% (v / v)) was added.
Triton X-100-containing isopropanol hydrochloride solution (hydrochloric acid: isopropanol = 1: 250)) was further added to solubilize formazan, and the color tone change of each solution was measured at 540 nm and 690 nm using a micro plate reader manufactured by Bio-Rad. Measured in wavelength. The concentration of the drug that inhibits the growth of normal cells by 50% (50% inhibitory concentration (CC ₅₀ ), μg / ml) was determined as compared to a control to which no drug was added.
The results obtained are shown in Table 1 above.

As is evident from the results shown in Table 1, the acridone derivatives of the present invention obtained in Examples 1, 3, 6, 8, and 12 showed a drug concentration (CC ₅₀ ) was sufficiently higher than the 50% effective concentration (EC ₅₀ ), indicating that the compound was effective as an anti-HIV agent and had relatively little effect on normal cells. In particular, the acridone derivative of the present invention obtained in Examples 3, 6, 8, and 12 was confirmed to be promising as a pharmaceutical because the value of CC ₅₀ / EC ₅₀ was 2 or more.

( Formulation Example 1 ) Tablet acridone derivative 30.0 mg Fine powdered cellulose 25.0 mg Lactose 39.5 mg Starch 40.0 mg Talc 5.0 mg Magnesium stearate 5 mg tablets having the above composition were prepared by a conventional method.
An anti-HIV tablet could be obtained without inhibiting the anti-HIV activity of the acridone derivative of the present invention.

Formulation Example 2 Capsule Acridone Derivative 30.0 mg Lactose 40.0 mg Starch 15.0 mg Talc 5.0 mg When a capsule having the above composition is prepared by a conventional method, the anti-HIV activity of the acridone derivative of the present invention is obtained. Was obtained, and an anti-HIV capsule was obtained.

Formulation Example 3 Injectable Acridone Derivative 30.0 mg Glucose 100.0 mg The above components were dissolved in purified water for injection to prepare an injectable having the above composition. The anti-HIV activity of the acridone derivative of the present invention was obtained. Was obtained, and an anti-HIV injection was obtained.

[0063]

Industrial Applicability According to the present invention, an excellent anti-virus capable of sufficiently inhibiting the replication of the viral DNA integrated into the genome of an immune cell to RNA after infection with a retrovirus, particularly HIV, is provided. It is possible to obtain a pharmaceutical composition having HIV activity.

 ──────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor Shiro Shigeta 147-28 Omori Kubouchi, Fukushima City, Fukushima Prefecture (72) Inventor Tomoyuki Yokota 20-1 Kitashimozato, Iizaka-cho Hirano, Fukushima City, Fukushima Prefecture

Claims (7)

[Claims] 1. A pharmaceutical composition containing an acridone derivative represented by the following formula (I) as an active ingredient. Embedded image (In the formula (I), R represents an alkyl group, R ^{1 to} R ⁴ may be the same or different, and each represents a hydrogen atom,
Shows any one selected from the group consisting of a hydroxyl group, a halogen atom and an alkoxy group. ) 2. The pharmaceutical composition according to claim 1, which is an antiretroviral agent. 3. The pharmaceutical composition according to claim 1, which is an anti-HIV agent. 4. In the above formula (I), R is a lower alkyl group having 1 to 5 carbon atoms, R ¹ is a hydroxyl group, a hydrogen atom or a carbon atom having 1 to 5 carbon atoms.
5, a lower alkoxy group, R ² is a hydrogen atom or a hydroxyl group, R ³
Is a hydroxyl group, a hydrogen atom or a lower alkoxy group having 1 to 5 carbon atoms, R ⁴ is a hydrogen atom, a hydroxyl group or a halogen atom,
The pharmaceutical composition according to claim 1. 5. In the formula (I), R is a methyl group.
The pharmaceutical composition according to claim 1. 6. The pharmaceutical composition according to claim 1, wherein in the formula (I), R is a methyl group and R ² is a hydrogen atom. 7. The pharmaceutical composition according to claim 1, wherein in the formula (I), R is a methyl group, R ¹ is a hydroxyl group, and R ^{2 to} R ⁴ are hydrogen atoms.