JPS63107924A - Antiretroviral agent - Google Patents

Abstract

PURPOSE:To obtain an antiretroviral agent, containing 2',3'-didehydropyrimidine nucleoside as an active ingredient having excellent antiretroviral action as well as high safety. CONSTITUTION:An antiretroviral agent containing a compound expressed by formula I (X is NH2 or OH: Y is H when X is NH2 and CH3 when X is OH), a e.g. 2',3'-dideoxy-2',3'-didehydrocytidine as an active ingredient. The dose of the compound expressed by formula I is normally 0.1-10g, preferably 0.2-5g per day for an adult and administered once or in several divided portions. The compound expressed by formula I is obtained by treating a 3',5'- anhydropyrimidine nucleoside expressed by formula II with a strong base, e.g. potassium tert-butoxide, etc., in dimethyl sulfoxide.

Description

[Detailed description of the invention] [Industrial application field] hair The present invention relates to antiretroviral agents.

[Conventional technology]

The causes of immunodeficiency syndrome (AIDS) and adult T-cell leukemia (ATL), which are caused by retrovirus infection, have only recently been discovered. In other words, in the case of AIDS, the human immunodeficiency virus (HIV), which is the cause of AIDS, preferentially infects T cells of human lymphocytes, causing cell degeneration that selectively kills T cells, resulting in immunodeficiency. (Igaku no Ayumi, Vol. 137, No. 12, p. 973 (1986))
.

Although an effective treatment for AIDS has not yet been established, various countermeasures have been studied, and there are currently 3 treatments available.
(Monthly Yakuji, Vol. 28, 3)
Monthly issue, pp. 61-65 (1986)).

A. Treatment with antiviral drugs for HIV Antiviral drugs for HIV include suramin, ribavirin, H
Current amounts of PA-23, azidothymidine, and interferon are also being studied. However, many of them do not have sufficient medicinal efficacy or have problems in use due to side effects.

B. Treatment with an immune enhancer This treatment method is a method of normalizing the immune function, which has been reduced due to a virus, using an immune enhancer. Interleukin ■
, isoprinosine, etc. have been studied, but since this method is a symptomatic treatment, it requires combination with the above-mentioned antiviral agents.

C. Vaccines In the past, many viral diseases have been eradicated or significantly reduced by vaccines. However, HIV mutates frequently, making it extremely difficult to develop a vaccine for AIDS.

[Problem that the invention seeks to solve]

As mentioned above, several types of compounds have been reported to have anti-HIV effects, but it is still difficult to find an antiviral agent that has excellent antiretroviral effects, has less toxicity and side effects, and can be used for a long period of time. At present, development is strongly desired.

Therefore, an object of the present invention is to provide a drug that has excellent antiretroviral effects and is highly safe.

[Means for solving problems]

As a result of repeated research to develop pyrimidine nucleoside derivatives useful as novel antiretroviral agents, the present inventors have discovered that 2',3'-didehydropyrimidine nucleosides exhibit excellent properties as antiretroviral agents. They discovered this and completed the present invention.

That is, the present invention provides a 2' compound represented by the general formula (13 [wherein, X represents an amino group or a hydroxyl group, and Y represents a hydrogen atom when X is an amino group, and represents a methyl group when X is a hydroxyl group]. , 3'-didehydropyrimidine nucleoside as an active ingredient.

Specifically, the 2',3'-didehydropyrimidine nucleoside represented by the general formula (1), which is the active ingredient of the drug of the present invention, is 2',3'-dideoxy-2',3'-
Didehydrocytidine (hereinafter abbreviated as rdd-Cy d J) or 3'-deoxy-2',3'-didehydrothymidine (hereinafter abbreviated as rdd-ThdJ)
It also includes the use of any pharmaceutically acceptable salt thereof such as hydrochloride or sulfate. These compounds are known compounds, for example, the general formula [■] [wherein, X and Y have the same meanings as above. ] 3'
, 5'-anhydropyrimidine nucleoside can be synthesized by treatment of potassium t-butoxide in dimethyl sulfoxide (J, Org, Che
m,, Vol, 31, p205 (1966)), J.
Org, Chem, Vol. 32, p817 (1
967)) - This reaction uses aprotic polar solvents such as dimethylacetamide, dimethylformamide, and hexamethylphosphoric acid triamide in addition to dimethyl sulfoxide as a reaction solvent, and sodium methoxide and sodium isopropoxide in addition to potassium t-butoxide as reaction reagents. This can be carried out by applying a strong base such as di, diazabicycloundecene, or n-butyllithium. The reaction conditions are 0-100°C for several hours.

The reaction product may be isolated by conventional methods, such as extraction,
Recrystallization, adsorption chromatography, ion exchange chromatography, etc. can be appropriately selected and employed.

The drug of the present invention is used in the treatment of retrovirus infections.

The dosage of 2',3'-dehydropyrimidine nucleoside, which is the active ingredient of the drug of the present invention, varies depending on the patient's severity of illness, drug tolerance, etc., and should ultimately be determined by the physician's judgment. However, the amount is usually 0.1 to Log, preferably 0.2 to 5 g per day for adults, and this is administered once or in divided doses. The method of administration can take any form appropriate to the route of administration.

The medicament of the present invention can be prepared for administration by any conventional method of formulation. Therefore, the present invention includes a pharmaceutical composition containing a 2',3'-dehydropyrimidine nucleoside represented by general formula (I) suitable for use as a human medicine.

Such compositions are subjected to administration in a conventional manner with any required pharmaceutical carriers or adjuvants.

For example, in the case of a composition preparation for oral administration, it is provided in a form suitable for absorption from the gastrointestinal tract, such as solid preparations such as tablets, capsules, powders, sugar-coated tablets, and granules, syrups, suspensions, etc. It may be prepared as a liquid preparation such as an elixir.

For solid agents, binders such as syrup, gum arabic, gelatin, sorbitol, tragacanth, polyvinylpyrrolidone.

Excipients such as lactose, sugar, corn starch, calcium phosphate, sorbitol, glycine, lubricants such as magnesium stearate, talc, polyethylene glycol, silica, disintegrants such as potato starch, wetting agents, stabilizers, and flavoring agents. The formulation can be prepared by appropriately selecting and using adjuvants such as the following, depending on pharmaceutical considerations. In the case of liquid preparations, as adjuvants, suspending agents such as sorbitol syrup, methyl cellulose, glucose/sugar syrup, gelatin, hydroxyethyl cellulose, carboxymethyl cellulose, aluminum stearate gel, hydrogenated edible fat, emulsifying agents, p -methyl hydroxybenzoate, p
- Preservatives such as propyl hydroxybenzoate, sorbic acid, etc. can be used.

In addition, when preparing a composition formulation for injection administration, 2',3'-didehydropyrimidine nucleoside, which is the active ingredient of the present invention, may be added with a pH adjuster, a buffer, a stabilizer, a preservative, a soluble agent, etc. as necessary. It can be administered as a subcutaneous, intramuscular, or intravenous injection using conventional methods.

〔Effect of the invention〕

Below, test results regarding the anti-HIV effect of 2',3'-didehydropyrimidine nucleoside, which is the active ingredient of the drug of the present invention, are shown.

Test Example 1 In this test of the anti-HIV properties of W based on the proliferation rate of cells, the established human T cell line MT-4 cells (Gann
monogr,, Vol, 28, pP2i, 9-22
8 (1982)) and HTL- which is a type of HIV (7).
Type m virus was used.

HTL-III-infected MT-4 cells were cultured in RPM 11640 medium containing 10% decomplemented fetal calf serum, 100 IU/Q of penicillin and 1100 tt/mQ of streptomycin supplemented with various concentrations of drugs for 30 min. The cells were seeded at 10,000/- and cultured at 37°C in a carbon dioxide gas incubator. After 3 days, half of the culture was separated, and the number of viable cells (X 10'/m) and viability (%) of each were determined by trypan blue staining. An equal volume of a medium containing the same concentration of drug was added to each remaining culture, culture was continued for an additional 3 days, and the number of viable cells was counted in the same manner.

The respective measured values are shown in Table 1. The numerical values in the table indicate the number of viable cells (X 10'/m12) and the survival rate (%) in parentheses.

As is clear from Table 1, the control (drug concentration 0 fish/-)
In , almost no cell proliferation was observed after 3 days due to virus proliferation, and most of the cells died after 6 days; The presence of .25t1g/mQ or more can almost completely prevent cell death caused by HTL-m type virus.

Test Example 2 Effect of drugs on proliferation of T cells
The effects of drugs on the proliferation of -4 cells were observed. The results are shown in Table 2. The numbers in Table 1 are the number of living cells (XIO'/
mQ) and the survival rate is shown in parentheses.

As is clear from Table 2, dd-Thd is 10IJg
However, dd-cyct has no effect on the proliferation of MT-4 cells at concentrations below 2 ttg/mQ.

Test example 3 HI Vi by firefly method.

Each living cell measured in Test Example 1 was dried on a slide glass, fixed with cold methanol for 3 minutes, and treated with human anti-HTLV-III positive serum diluted to 1/1000 at 37°C for 30 minutes. . This was followed by a 15-minute wash with phosphate-buffered saline (PBS) and 3 minutes with fluorescein-isothiocyanate-conjugated anti-human IgG.
It was treated at 7°C for 30 minutes. After washing with PBS again, the number of cells emitting fluorescence was measured using a fluorescence microscope.

The results are shown in Table 3. The numbers in the table are the ratio (%) of the number of fluorescent cells to the total number of cells.

As is clear from Table 3, on day 6 of infection, almost 100% of the cells in the drug-free control were positive for viral antigen, whereas in the drug-treated group, dd-Thd, dd-Cy.
In both cases, less than 1% of antigen-positive cells were observed at concentrations of 0.5 fish/- or more, and the expression of viral antigens was significantly suppressed.

Test Example 4 Virus Production Same as Test Example 1 for anti-HIV activity by side method 4. : HTLV-m infected MT-4 cells were cultured for 4 days in the presence of various concentrations of the drug, and the amount of virus released into the culture medium was measured by plaque assay as follows.

A 50x/mfi poly-L-lysine solution 1- was dropped into a 35+m plastic Petri dish and treated at room temperature for 1 hour to prepare a poly-L-lysine coated Petri dish. After washing each petri dish three times with PBS.

150 x 10'/IIIQ MT-4 cells 1.5
mQ was added and treated at room temperature for 1 hour. This on PBS 2
After washing gently twice, 100ml of culture supernatant of HTLV-m-infected cells in the presence of the above-mentioned drug was gently added, and the mixture was left at room temperature for 1 hour to adsorb the virus. Agarose overlay medium (RPM11640 medium supplemented with 10% tallow serum, antibiotics, and 0.6% agarose) 1- was added to each petri dish, and incubated at 37°C in a carbon dioxide gas incubator.
After culturing for 3 days, 1 ml of agarose overlay medium containing Neutral Red (1) was added, and the cells were further cultured at 37°C for 3 days, and the amount of virus was measured.

The above test was repeated three times, and the average amount of virus was calculated.

The results are shown in Table 4. The values in Table 0 are the average viral load (PFU/mQ+s, D, n=3).

Table 4 As is clear from Table 4, the virus amount in the drug-free control was 25.7 x 10'PFU/-, whereas
The virus load was significantly reduced in the drug-treated group.

Test Example 5 HIV by Plaque Method A poly-L-lysine coating treated in the same manner as in Test Example 4 was added to the MT-4 cell culture medium in a petri dish at 2000 PFU/
- 1 ounce of the virus solution was gently added and left at room temperature for 1 hour to adsorb the virus. Agarose overlay medium 1- was added to each petri dish, and after culturing in a carbon dioxide gas incubator at 37°C for 3 days, agarose overlay medium 1-1 containing neutral red was overlaid. Each petri dish was further cultured at 37°C for 3 days, and the number of plaques was measured.

The above test was repeated three times and the average value was calculated.

The results are shown in Table 5. The numbers in the table are the average number of plaques (/dish + S, D, , n = 3)
It is.

Table 5 As is clear from Table 5, the drug-free control was 192
In contrast, in the dd-Thd and dd-C7d treatment groups, only 21.3 and 91 plaques were formed at drug concentrations of o and 05IIg/-, respectively; No plaque formation was observed in either drug-treated group.

As is clear from the above test results, the 2',3'-didehydropyrimidine nucleoside, which is the active ingredient of the present invention, has anti-HIV activity at low concentrations that do not affect host cells. Therefore, the drug of the present invention is useful as a low-toxicity antiretroviral agent.

〔Example〕

Examples of the present invention will be shown below for more specific explanation. However, the present invention is not limited to these Examples.

Example 1 Tablet dd-Thd 10g Cornstarch 65g Carboxycellulose
20g polyvinylpyrrolidone
3g calcium stearate 2g total
Amount 100g 1 by conventional method
Prepare 100 square tablets. dd-Th in one tablet
Contains 10 mg of d.

Example 2 Powder, capsule dd-Cyd 20g crystalline cellulose 80g total amount
Mix 100g of both powders to make a powder. Further, 100 μm of the powder was filled into No. 5 hard capsules to prepare capsules.

Synthesis Example 1 Synthesis of dd-Thd 3.6 g of potassium t-butoxide was added to 100-dimethyl sulfoxide, and further 3'-deoxy-3', 5
3.6 g of '-anhydrothymidine was added and stirred at room temperature for 2 hours. After neutralization with an acetic acid-ethanol solution, the mixture was concentrated to dryness under reduced pressure at about 50°C. This was suspended in acetone, insoluble salts were filtered off, and the filtrate was concentrated to dryness. Dilute the residue with 50% ethanol
The solution was dissolved in mQ, 250 mΩ of benzene was added, and the solution was concentrated to about 50 mQ to obtain 3.2 g of crystals.

This was recrystallized from ethanol-benzene to give dd-Th.
d2.9g (yield: 80%) was obtained.

Melting point: 165-166°C Ultraviolet absorption: λ”0 266nm (g9910)a
x Elemental analysis: C engineering. Calculated value as H1□N, H4 C,
53,56H, 5,40N, 12.50 Actual value C, 5
3,39H, 5,43N, 12.38 Example 2dd-
C: Synthesis of yd 4.8 g of 2',3'-dideoxy-3'-5'-anhydrocytidine was added and dissolved in 150 mQ of dimethyl sulfoxide solution containing 5.3 g of potassium t-butoxide, and 2' was dissolved at room temperature. Stir for hours. Approximately 50% of this reaction solution
Concentrate to dryness under reduced pressure at °C, dissolve the residue in approximately 20°C of water,
Weakly acidic cation exchange resin Amberlite IRC-50 (
It was neutralized by passing it through a column of H0 type. The permeate was concentrated to dryness, and the remaining anion exchange resin DOWEX-1 (OH
- type) column (2 x 45 am) and eluted with methanol-water. Both fractions containing the target compound were collected, concentrated to dryness, and crystallized from ethanol to give dd-Cyd2.
．． 9 g (yield 61%) was obtained.

Melting point: 160-162℃ Ultraviolet absorption = F'0 271nm (ε8710)a
x

Claims (1)

[Claims] General formula [I] ▲ Numerical formulas, chemical formulas, tables, etc.▼ [I] [In the formula, X represents an amino group or a hydroxyl group, and Y represents a hydrogen atom when X is an amino group; When it is a hydroxyl group, it indicates a methyl group. An antiretroviral agent comprising a 2',3'-didehydropyrimidine nucleoside represented by the following as an active ingredient.