JPH03209321A - Anti-retrovirus agent - Google Patents

Abstract

PURPOSE:To obtain an anti-retrovirus agent, containing delphinidin or a derivative thereof as an active ingredient and effectively used as an inhibitor against infection with retroviruses and proliferation thereof. CONSTITUTION:An anti-retrovirus agent containing a compound expressed by the formula (R1 and R2 are H, hydrocarbon group or glucoside-forming residue of a six-membered ring pyranose type hexose or five-membered ring furanose type pentose; R3 and R4 are H or hydrocarbon group; X<-> is a residue of an acid), e.g. delphinidin chloride, delphinidin chloride-3-glucoside or malvinidin chloride as an active ingredient. The aforementioned compound has inhibitory action on reverse transcriptases derived from retroviruses and is capable of inhibiting cell degeneration with the retroviruses in cells infected therewith and effective against pathogenic viruses of acquired immunodeficiency syndromes, pathogenic viruses of adult T cell leukemia, animal retroviruses, etc.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Field of Application) The present invention relates to an antiretroviral drug effective in treating and preventing various viral diseases caused by retroviruses.

HIV (Huma
n I++aunodeficiency Viru
s) and HTL V-1 (Human
T-cell Lymphotropic Vir
us Type I) is feared as a human retrovirus. The present invention relates to antiretroviral drugs that are also effective against these human retroviruses.

[Prior art] Retroviruses contain RNA-dependent DNA within their virus particles.
It is a unique virus that contains reverse transcriptase, a synthetic enzyme. This retrovirus-specific reverse transcriptase produces DNA from viral RNA within cells, and this DNA is integrated into the chromosome of the host cell. incorporated dna
mRNA is synthesized from the virus, various viral proteins are produced, and the proteins are further modified and then budded as a virus.

One reason for the increased interest in retroviruses and antiretroviral drugs in recent years is the discovery and worldwide spread of AIDS. Since the first AIDS patient was discovered in the United States in 1981, research has begun to elucidate the causative virus and find ways to treat it. It has been revealed that the virus that causes AIDS is HIV, and it has become possible to detect carriers of HIV infection, but an effective treatment method has not yet been established. There are already more than 50,000 AIDS patients in the United States alone, and more than half have already died.

It is thought that there are 10 million people around the world who are infected with HIV, and if things continue at this rate, by the end of this century
It is predicted that the number will exceed 100 million. until now,
People infected with HIV go through the carrier state and develop pre-AIDS A.
It is thought that the disease will develop into RC and eventually AIDS and death will occur within 15 years of infection. It is a terrifying infectious disease that humanity has never experienced before.

In addition to AIDS, there is Seihachi T-cell leukemia (ATL) as a human disease caused by a retrovirus. Research is also being conducted into the infection route and prevention methods for HTLV-1, the virus that causes ATL.

In Japan alone, it is thought that there are 1 million carriers of HTLV-I infection, and it is said that ATL develops several decades after infection, and the incidence rate is said to be about 1/1000. ~This is also a very scary infectious disease, as you will die within a few years. It has been found that carrier mothers can infect their newborns through breast milk, and in places such as Nagasaki Prefecture, prevention of mother-to-child transmission is being implemented by stopping carrier mothers from breastfeeding. An antiviral drug effective against HTLV-I has not yet been found.

Diseases caused by various retroviruses are also known in animals. Animal retroviruses include the avian myeloblastosis virus (AMV; avianm7elobl).
ostosis virus), chicken leukemia virus, chicken sarcoma virus, chicken reticulosarcomatosis virus, mouse breast cancer virus, mouse leukemia virus, mouse sarcoma virus, feline leukemia virus, cat sarcoma virus,
Many retroviruses are known, including feline AIDS virus, ovine leukemia virus, bovine leukemia virus, equine infectious anemia virus, simian leukemia virus, monkey AIDS virus, baboon type C virus, and others. Antiretroviral drugs effective for treating or preventing diseases caused by retroviruses in these animals have not yet been found.

Nucleic acid-based azidothymidine (A
ZT) has been approved and is said to have a life-prolonging effect, but it is still not considered to be sufficient because it causes bone marrow suppression and other serious side effects.

The research on anti-AIDS drugs is too numerous to mention, but for example, recent related review papers include R.
．． Yashoan, Hiroaki Mitsuya, S. Science journal article by Broder et al. [Science, 18 (12). 96
~108 (1988)), Chemistry and Industry journal article by Koro Hoshino [Chemistry and Industry, 42 (8), 1356~
1358 (1989) ), E. de Clera
q's TIPS journal paper (TIPS, 8, 339~
345 (1.987):l, Hiroaki Mitsuya, S. A paper in Nature by Broder et al. (Nature. 32
5773-778 (1987)).

Research is being carried out on unique drugs for each process from HIV infection to multiplication. That is, adsorption of HIV to the cell surface, entry into the cell, synthesis of DNA from RNA by reverse transcriptase in the cell, integration of DNA into the host gene, synthesis of RNA and virus protein from the integrated DNA, modification, assembly, budding, cell fusion and cell destruction by HIV-infected cells, etc.
Drug research is being conducted for each process. Among them, reverse transcriptase in particular is an enzyme specific to the proliferation of retroviruses that is not involved in the proliferation of host cells, and if we can find a drug that is selectively effective in this reverse transcription process, it will not have a negative effect on host cells. It is expected to be able to inhibit the target retrovirus.

[Problems to be Solved by the Invention] An object of the present invention is to provide an antiretroviral drug that is effective against retroviruses.

In particular, it is an object of the present invention to provide a new antiretroviral drug that has an inhibitory effect on reverse transcriptase, which is a retrovirus-specific enzyme.

[Means for Solving the Problems] The present inventors thought that there may be compounds that can be used as antiretroviral drugs among natural products that widely exist in nature, and after conducting extensive studies, they arrived at the present invention. . In particular, initial screening was performed using a retrovirus-specific reverse transcriptase inhibition test. Furthermore, the effect of inhibiting cell degeneration caused by HIV infection in lymphocytes infected with HIV was also investigated.

As a result, the general formula (I): [wherein R1 and R2 are hydrogen, a hydrocarbon group, or a glycoside-forming residue of a six-membered ring bilanose-type hexose or a five-membered ring furanose-type pentose; , R3, R4
is hydrogen or a hydrocarbon group, and X- is an acid residue. It's arrived.

Delphinidin or a derivative thereof represented by the general formula (I) of the present invention can be obtained by extraction and purification from natural plant materials, or can be synthesized by a synthetic reaction.

In the general formula (I), R1 and R2 are each independently hydrogen, a linear, branched, or cyclic saturated or unsaturated hydrocarbon group, a six-membered biranose-type hexose, or a five-membered furanose. It is a glycoside-forming residue of a type of pentose, preferably hydrogen, a lower alkyl group such as a methyl group, or a glycoside-forming residue of glucose or fructose, and particularly preferably at least R1 is a group of glycol or fructose. It is a glycoforming residue.

In general formula (I), Residues of organic acids are preferred, and 01-, which is a residue of hydrochloric acid, is particularly preferred.

Specifically, examples of the compound of general formula (I) include delphinidin chloride, malvidin chloride, delphinidin chloride-3-glucoside, malvidin chloride-3-glucoside, and delphinidin chloride-3.
,5-diglucoside,malvidinuclide-3,5-
Examples include diglucoside and delphinidin chloride-3-galactoside. However, it is not necessarily limited to these compounds.

It is known that delphinidin chloride-3-glucoside is contained in adzuki bean seeds, delphinidin chloride-3,5-diglucoside is contained in flowers of dayflower, and malvidin chloride-3,5-diglucoside is contained in flowers of malva. It is also known that the aglycones delphinidin chloride and malvidin chloride can be obtained by acid hydrolysis of these glycosides. Delphinidin and its derivatives obtained by extraction, purification, or hydrolysis from natural plants are commercially available and can be easily obtained.

It is also possible to obtain the compound (I) of the present invention by a synthetic reaction, in which 2,4,6-trihydroxybenzaldehyde or its derivative and 3,4,5,ω-tetrahydroxyacetophenone or its Synthesis can be carried out by a condensation cyclization reaction with a derivative, or by performing hydrolysis or other modification reactions after the condensation cyclization reaction (J.Ch.
et Soc. +1934. 1235-1243 (
1934) ).

Myricetin, a flavonol, is combined with Mg, MCI, and Li.
When reduced with AIH4, Na-amalgam, etc.,
It is also known that delphinidin can be obtained.

Regarding the antiviral effect of the compound (I) of the present invention, it has been reported that it has an antiviral effect on influenza virus (Azerb. Med. Zh.. 84 (
4), 33 ~ 3B (19g?) ). That is, the Soviet Union (7) Z, A. Lazymoya et al. found that an anthocyanin mixture consisting of cyanidin, belargonidin, and delphinidin derivatives isolated from tulip flowers had antiviral activity against influenza A virus in a chicken fetal fibroblast culture system.

However, the virus replication process is different between influenza viruses and retroviruses, and it goes without saying that even if it has an antiviral effect on influenza viruses, it is not known whether it has an inhibitory effect on reverse transcriptase. .

The present inventors conducted tests to find compounds derived from various natural products that have a reverse transcriptase inhibitory effect, and as a result, they found that the compound (I) of the present invention has a reverse transcriptase inhibitory effect, and arrived at the present invention. It is.

Reactions using reverse transcriptase are m in gene manipulation reactions.
It is used to synthesize cDNA from RNA. The present inventors used AMV-derived reverse transcriptase to improve the results of Akira Ishihama's paper [Protein, Nucleic Acids, Enzymes, Utsubo (10), 1127
An enzyme inhibition test was conducted according to the method described in (1985).

Furthermore, a cytopathic inhibition test was conducted on lymphocytes infected with HIV. As lymphocytes, human leukemia T-cell cell line Molt-4 clone 8 (R.
Kikukawa et al., J. V1rol. , 57. 1
159-1162 (1986)] was used. HIV
The Molt-4/HTLV-m cell line [S. Ha
rada, V1rology, 146. 272-
281 (1985)) HTLV obtained from the culture supernatant of
-III was used.

Compound (I) of the present invention has an inhibitory effect on retrovirus-derived reverse transcriptase and inhibits retrovirus-induced cell degeneration in retrovirus-infected cells, thus inhibiting retrovirus infection and proliferation. It was shown to be effective as a drug.

The antiretroviral drug of the present invention is administered to humans or animals before retrovirus infection, when the patient is in a carrier state after retrovirus infection, or at the onset of retrovirus-induced pathologies and symptoms.

The therapeutic and prophylactic pharmaceutical compositions containing the antiretroviral drug of the present invention, i.e. compound (I), can be formulated into powders, suspensions, solutions, syrups, emulsions, ointments or creams. . Parenteral administration (intravenous, intramuscular, intraperitoneal, etc.) or oral administration can also be carried out. Topical application, for example intranasally, rectally or vaginally, is also possible.

The concentration of compound (I) in the antiretroviral agents of the invention can range widely from 0.1% to 100% and depends on the mode of administration.

Furthermore, the amount of compound (i) is 0.1 per kg of body weight.
It can be set from ■ to 100■.

[Implementation example] The present invention will be specifically described below with reference to Examples.

Example 1 The results of a reverse transcriptase (hereinafter abbreviated as RT) inhibition test of delphinidin chloride are shown.

AMV-derived RT was used for RT. The reaction is performed using p(dT)12 using poly(rA) as a template.
-18 is used to measure RT activity based on the amount of uptake of the substrate 3H-dTTP (tritium-labeled thymidine triphosphate).

Eppendorf polyethylene microtube (1
．． 5 ml) was used as the reaction vessel, and the composition of the reaction solution was as follows.

(RT inhibition reaction solution composition) 1M Tris-HCI (pH 7.8) 2.5μl LM Mg (OAc)2 0.5ttl
LM NaCl 2.5μ10.
5M DTT (dithiothreitol) 0.5μl 5mM 3H-dTTP 1. Ottl
5U/mlp oly (r A) o (d
T) 12-181.0 μl AMV-RT 1. O μl drug solution 2.0 μl The drug solution used was a 10% DMSO solution of the drug at a concentration 25 times the target drug concentration during the reaction.

Therefore, the DMSO concentration during the reaction was 0.4%.

The reaction was carried out at 37°C for 30 minutes, and after the reaction, 10% TCA
Add 50 ul of (}lichloroacetic acid) solution, cool with ice water for 30 minutes, and then filter with a glass microfiber filter (Wba
Suction r at tman GF/C diameter 2.1an)
filtered, further washed with 5% TCA and ethyl alcohol, dried, placed in toluene scintillator solution, and placed in a liquid scintillation counter (Pa-ckard Instr.
P by TRI-CARB 4000)
The amount of 3H on the paper was measured. control, i.e. RT
The RT activity when no inhibitory drug was added was taken as 100%, and the RT activity when the drug was added was determined by relative comparison.

The results of the test using delphinidin chloride are the first
As shown in the figure. Delphinidine chloride is AMV-derived RT
It is found that it has inhibitory activity.

The 50% inhibitory concentration is approximately 50 μg/ml.

Example 2 Using delphinidin chloride-3-glucoside,
An inhibition test of AMV-derived RT was conducted in the same manner as in Example 1.

Figure 2 shows the results. Delphinidine chloride-3-
It can be seen that glucoside has AMV-derived RT inhibitory activity. The 50% inhibitory concentration is approximately 10 μg/ml.

Example 3 An inhibition test of AMV-derived RT was conducted in the same manner as in Example 1 using delphinidin chloride-3,5-diglucoside.

The results are shown in Figure 3. delphinidin chloride}'-3
．． It can be seen that 5-diglucoside has AMV-derived RT inhibitory activity.

Example 4 The anti-HIV effect of delphinidin chloride was tested.

Human leukemia T-cell cell line Molt-4 (ku0-:/
8) Cells [R. Kikukawax et al., J4irol.
57. 1159-1162 (1986)),
2×105 cells/ml were prepared in a 24-well microplate, infected with HTLV-I [I, a type of HIV, at a multiplicity of infection (MOI) of 0.002, and incubated at 37°C with CO2.
The cells were cultured in two incubators. The medium contained 10% fetal calf serum (FCS), 1001 U/ml.
RPMI-1640 medium supplemented with penicillin G and 100 μg/ml streptomycin was used. Also used were those obtained from culture supernatants of HTLV-m and MOlt-4/HTLv-■ cell lines [S,}lxradl (1985)]. During the HIV infection operation,
At the same time, a test drug, in this case delphinidin chloride dissolved in DMSO water, was added to each well of the microplate, and then cultured for 3 days at 37°C and further in a CO2 incubator. Drug administration was continued on the first day and on the second day.
Additional additions were made in the same manner on the second and third days.

After 3 days, half of each well was removed to reduce the number of cells by half, and the same amount of new culture medium as the removed amount was added, followed by the addition of drugs in the same manner as on the first day, and culture was continued for another 2 days. The drug was further added on the second day after replacing half of the medium. After culturing for a total of 5 days, the cells were extracted and stained with trypan blue, and the number of living cells that were not stained was counted under a microscope.

Only dead cells are stained and identified. HTL for comparison
A similar test was also conducted without infection with V-III.

Figure 4 shows the test results for delphinidin chloride.

At a drug concentration of 12 μg/nut, cell growth proceeded in the same manner as when uninfected even when infected with HTLV, and no cell degeneration due to HTLV-III infection was observed. That is, anti-HIV effect was confirmed.

Example 5 Using delphinidin chloride-3-glucoside,
A test similar to Example 4 was conducted.

The results are shown in Figure 5. It can be seen that cell degeneration caused by HIV infection is significantly inhibited at a drug concentration of 0 μg/ml. This means that anti-HIV action was confirmed.

Example 6 A test similar to Example 5 was conducted using malvidin chloride-3,5-diglucoside. However, the experiment was carried out under slightly different conditions. That is, the cells were cultured for 1 hour after being infected with HIV, and then the drug was administered. After culturing for 3 days, half of the medium was replaced, and after drug administration, the cells were cultured for 1 day, and the number of cells was counted after a total of 4 days of culture.

The results are shown in Figure 6. Cell degeneration caused by HIV could be completely inhibited at 60 μg/ml. Therefore, it was confirmed that malvidinuclide-3.5-diglucoside has an anti-HIV effect.

[Effects of the Invention] The antiretroviral drug of the present invention has an inhibitory effect on retrovirus-specific reverse transcriptase and has an action to prevent cell degeneration in retrovirus-infected cells. It is effective in treating and preventing various viral diseases caused by the virus.

[Brief explanation of drawings]

Figure 1 shows the results of the reverse transcriptase inhibition test using delphinidin chloride, and Figure 2 shows the results of the reverse transcriptase inhibition test using delphinidin chloride.
Figure 3 shows the results of a reverse transcriptase inhibition test using 3-glucoside, and Figure 3 shows the results of a reverse transcriptase inhibition test using malvidin 3,5-diglucoside. Figure 4 shows the anti-HIV effect of delphinidin chloride.
Figure 5 shows the anti-HIV effect of delphinigincylide-3 monoglucoside, and Figure 6 shows the anti-HIV effect of delphinigincylide-3,
Each figure shows the anti-HIV effect of 5-diglucoside.

Claims (10)

[Claims] (1) General formula (I): ▲Mathematical formula, chemical formula, table, etc.▼(I) It is a glycoside-forming residue of furanose-type pentose, and R_3
, R_4 is hydrogen or a hydrocarbon group, and X^- is an acid residue.] An antiretroviral drug containing delphinidin or a derivative thereof as an active ingredient. (2) R_1, R_2, R_3, R_ of general formula (I)
The antiretroviral drug according to claim (1), which contains delphinidin chloride in which both 4 are hydrogen and X^- is Cl^-. (3) Delphinidin chloride-3-glucoside in which R_1 in general formula (I) is a glycoside-forming residue of D-glucose, R_2, R_3, and R_4 are all hydrogen, and X^- is Cl^-. The antiretroviral drug according to claim (1), comprising: (4) Delphinidin chloride-3, in which R_1 and R_2 of general formula (I) are both glycoside-forming residues of D-glucose, R_3 and R_4 are both hydrogen, and X^- is Cl^-; The antiretroviral drug according to claim (1), which contains 5-diglucoside. (5) In general formula (I), R_1 and R_2 are both hydrogen, R_3 and R_4 are both methyl groups, and X^-
The antiretroviral drug according to claim (1), which contains malvidin chloride in which is Cl^-. (6) Malvidin chloride-3 in which R_1 and R_2 of general formula (I) are both glycoside-forming residues of D-glucose, R_3 and R_4 are both methyl groups, and X^- is Cl-. , 5-diglucoside (
1) The antiretroviral drug described above. (7) The antiretroviral drug according to claim (1), wherein the retrovirus is a human retrovirus. (8) The antiretroviral drug according to claim (7), wherein the retrovirus is a pathogenic virus of human immunodeficiency syndrome or a pathogenic virus of adult T-cell leukemia. (9) The antiretroviral drug according to claim (1), wherein the retrovirus is an animal retrovirus. (10) A claim in which the retrovirus is a myeloblastosis virus or a pathogenic virus of feline immunodeficiency syndrome (
9) The antiretroviral drug described above.