JPH0687737A - Anti-aids agent - Google Patents

Abstract

PURPOSE:To obtain an anti-AIDS agent having activity capable of suppressing proliferation of HIV in infected cell. CONSTITUTION:The anti-AIDS agent contains 1-methoxy-2-formyl-3- hydroxyanthraquinone expressed by the formula as an active ingredient. This compound is obtained by extracting Morinda citrifolia of a tropical plant, preferably with a non-polar solvent, especially a hydrocarbon solvent or a halogenated hydrocarbon solvent and concentrating the resultant extract once and then purifying the extract by chromatography. This anti-AIDS agent is mainly orally administered and its dose is 50-500mg/1kg body daily.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to an anti-AIDS agent containing 1-methoxy-2-formyl-3-hydroxyanthraquinone.

[0002]

BACKGROUND OF THE INVENTION Human Immunodeficiency Virus
ficiency virus (HIV) destroys helper T cells that play an important role in the human immune system, and also infects cells of the monocyte / macrophage system to infect B cells, NK cells,
It is a type of retrovirus that causes abnormalities in the entire immune system, including red blood cells and IL-2.

[0003] Therefore, when infected with HIV, it becomes extremely susceptible to infection due to a decrease in immune function, and it is accompanied by opportunistic infections such as Carini pneumonia and malignant tumors such as Kaposi's sarcoma, and eventually leads to death. Therefore, the disease is acquired immunodeficiency syndrome (AIDS, Acquired Immune Deficien
cy Syndrome).

[0004] In AIDS, HIV is extremely susceptible to mutation, and the target of HIV is T cells themselves, which are the main players of immunity, so induction of neutralizing antibody is not sufficient.
Vaccine development presents many major challenges due to lack of suitable animal models, etc., resulting in high mortality and global social problems.

Currently, AIDS therapeutic agents used worldwide are AZT (3'-azido-2 ', 3'-dideoxythymidine), DDI (2', 3'-dideoxyinosine),
DDC (2 ', 3'-dideoxycytidine). These are all nucleic acid compounds and reverse transcriptase inhibitors. However, AIDS drugs need to be administered to patients over a long period of time, and these drugs are not
There is a problem that it also inhibits the synthesis and causes serious side effects such as myelosuppression and carcinogenesis. In addition, there is a new problem of the emergence of resistant viruses, and the development of therapeutic agents by another mechanism is awaited.

[0006]

Since a therapeutic drug for AIDS is premised on long-term administration to patients, it is necessary to provide a drug which suppresses HIV proliferation and has few side effects.

As a result of searching for plant-derived anti-AIDS substances, the present inventors have found that the extract of tropical plant Morinda citrifolia has an activity of suppressing the growth of HIV virus, and completed the present invention. Let

An object of the present invention is to provide an anti-AIDS agent containing 1-methoxy-2-formyl-3-hydroxyanthraquinone as an active ingredient.

Another object of the present invention is 1-methoxy-2-
It is to provide a method for isolating formyl-3-hydroxyanthraquinone.

[0010]

The present invention provides an anti-AIDS agent containing 1-methoxy-2-formyl-3-hydroxyanthraquinone as an active ingredient and optionally a pharmaceutically acceptable carrier or the like.

1-Methoxy-2-formyl-3-hydroxyanthraquinone is an anthraquinone derivative having the following structure.

[0012]

[Chemical 1]

When the above compound is administered to HIV-infected cells, the proliferation of HIV in the cells is suppressed.

HI was used in a medium containing the compound at various concentrations.
When V-infected MT-4 cells were cultured for 6 days, CPE (cytopathic effect) was suppressed without substantially inhibiting cell growth when the concentration of the present compound in the medium was 63 to 2 μg / ml.

The pharmaceutically acceptable carrier used in the anti-AIDS agent of the present invention includes, for example, natural or synthetic aluminum silicate, microcrystalline cellulose, talc, dextrin, starch, magnesium aluminometasilicate, lactose and the like. Excipients and diluents such as vegetable oils, propylene glycol and the like can be mentioned. In addition to this, pharmaceutically acceptable stabilizers, disintegrating agents, coating agents, suspending agents, emulsifying agents, solubilizing agents, preservatives, buffers, sweeteners and the like may be present as other optional ingredients. Known additives may be appropriately combined and used as these additives. The dosage form may be any form such as powder, granules, tablets, capsules and injections.

The anti-AIDS agent of the present invention is mainly orally administered, but the present invention is not limited by the administration method. The daily dose for an adult varies depending on the administration method, medical condition and the like. In the case of oral administration, which is the main method of administration, weigh 1
The usual dose is 50 to 500 mg / kg / day. However, the present invention is not limited by dose.

1-Methoxy-2-formyl-3-hydroxyanthraquinone can be isolated from Morinda citrifolia by solvent extraction followed by purification.

Morinda ci
trifolia L., Rubiaceae, Japanese name Yaeyama Aoki)
A small tree with white flowers that grows naturally in Southeast Asia or is partly cultivated. As the raw material plant, dried plants are preferably used for reasons such as storage and transportation. In addition, crushed ones are preferable for extraction.

The solvent used for the extraction is preferably a non-polar solvent, particularly a hydrocarbon solvent or a halogenated hydrocarbon solvent. Examples of the hydrocarbon solvent include pentane, hexane, heptane, octane, benzene, toluene, xylene and the like, and hexane is particularly preferable. Further, as the halogenated hydrocarbon solvent, for example,
Examples thereof include chloroform, methylene chloride, carbon tetrachloride, etc., and chloroform is particularly preferable. The most preferred solvent is chloroform. Of course, the present invention is not limited to these solvents, and any solvent capable of stably extracting the present active ingredient is included in the scope of the method of the present invention.

The amount of the solvent is not particularly limited as long as it can extract the present active ingredient, and generally 200 to 1,000 per 100 g of Morinda citrifolia dried and ground.
ml. The extraction temperature is also not particularly limited, but may be any temperature at which the present compound can be stably extracted without decomposition, and is preferably room temperature to 60 ° C. The extraction time varies depending on the temperature, but is, for example, 2 to 20 hours when extracting at room temperature. The extraction may be repeated twice or more depending on the extraction rate.

The extract obtained is purified. Purification can be performed by, for example, chromatography after once concentrating the extract.

Chromatography may preferably include column chromatography, gel filtration chromatography, high performance liquid chromatography, liquid-liquid countercurrent partition chromatography, thin layer chromatography or a combination thereof. Appropriate carrier and solvent systems, respectively, can be used for column chromatography such as MerckSil.
icagel 60 (Merck) and hexane: ethyl acetate = 1
It is 0: 1 (v / v). However, the types of chromatography, carrier and solvent system are not limited to those mentioned above.

In addition, recrystallization may be applied. As a solvent for this purpose, for example, a mixed solvent of dichloromethane and hexane is suitable.

1-Methoxy-2-formyl-3-hydroxyanthraquinone itself has been described by Gopalakirhsnan et al., J. In.
dian Chem . Soc. , 67 (5), 390-393 (1990) and could be obtained by chemical synthesis.

[0025]

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

In the present specification, "%" indicates% by weight unless otherwise specified.

1. Morinda Citrifo with organic solvent
Extraction of the active substance from the rear 50 g of Morinda citrifolia L. roots dried in the sun or in an oven at 60 ° C are crushed and then placed in a 1 liter Erlenmeyer flask and 200 ml of chloroform
Was added and the mixture was stirred at room temperature for 7 hours. The solvent was renewed, the same operation was repeated, and extraction was performed three times in total. The three extracts were combined and the chloroform solvent was distilled off under reduced pressure, and the resulting extract was freeze-dried to give 0.7 g of a yellowish brown powder.
Got This powder was used as a raw material for the next purification.

The CPE inhibitory activity was observed in the extract with chloroform or hexane, but the same activity was not observed in the extract with methanol or water.

2. Purification of the active substance from the extract 200 mg of the extract were dissolved in a little chloroform and deposited on a little silica gel. After removing the solvent, the column was packed.
Place on 20 g of silica gel, hexane: ethyl acetate =
The mixture was sequentially eluted with 11: 1, 10: 1 and 5: 1 (v / v) mixtures of hexane and ethyl acetate, and fractionated by about 10 ml each. The portion eluted with the 10: 1 eluate in which activity was observed was collected and dried, and the weight was 51.8 mg. This was mixed with methanol to obtain 35.4 mg of the active substance as an insoluble fraction.

Further, for crystallization, this was dissolved in a small amount of dichloromethane, a few drops of hexane were added, and the mixture was left standing to obtain 12.6 mg of crystals.

3. The physicochemical properties of the purified product and the physicochemical properties of the structural purified product were analyzed. The results are shown below.

[0032]

[Table 1]

From these physicochemical properties, NMR spectrum and X-ray crystallographic analysis, it was found that the purified product was an anthraquinone derivative having the structure of Chemical formula 1.

4. HIV Growth Inhibitory Action and Cytotoxicity The compound is RPMI1640 containing 2% dimethylsulfoxide (DMSO) and 10% fetal calf serum (FSC).
It was dissolved in a medium (manufactured by Nissui) and a dilution series was prepared on a round-bottom 96-well microplate (manufactured by Nunc) so that the final concentration was 500 to 8 μg / ml. On the other hand, MT-4 cells in the logarithmic growth phase (CD4 antigen-positive, HTLV-positive cells)
[Science, 229 , 563-566 (1985)] was collected by centrifugation and suspended in a very small amount of the above medium to give a final concentration of 3.0 × 10 ⁵ cells / ml. HIV stock was added to the cell suspension so that the moi was 0.001 and the cells were adsorbed at 37 ° C for 1 hour. This HIV stock is a persistent infection Molt-4 / HI
The culture supernatant of V-1 cells [J. Virology, 57 (3), 1159-1162] was filtered with a 22 μm Millipore filter and stored at −80 ° C. The price has been decided. After the adsorption, the cells were suspended in the medium, 100 μl / well was added to the sample diluent, and the cells were cultured at 37 ° C. in the presence of 5% CO ₂ . At this time,
The cell concentration was adjusted to about 300,000 cells / ml. On day 6 after HIV infection, cytotoxicity and H
The presence or absence of the expression of cytopathic effect (CPE) due to IV infection was observed. As a control, H in the absence of this compound was used.
IV uninfected MT-4 cells and HIV infected MT-4 cells were used.

The results are shown in Table 2.

[0036]

[Table 2]

As can be seen from the results shown in Table 2, when the concentration of this compound was 63 to 2 μg / ml, CPE was not observed and the cells were proliferated. These results indicate that the compound has anti-HIV activity.

[0038]

INDUSTRIAL APPLICABILITY 1-Methoxy-2-formyl-3-hydroxyanthraquinone can be effectively used as an anti-AIDS agent that suppresses the proliferation of HIV.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Kaoru Asano Kaoru Asano 1-3-1, Nishitsurugaoka, Oi-cho, Iruma-gun, Saitama Prefecture Tonen Corporation Research Institute (72) Masao Yoshizawa, Nishitsurugaoka, Nishii-gun, Saitama-ken 3-3-1 Tonen Co., Ltd. Research Institute (72) Inventor Kazuo Umezawa 3-1-2-505 Hiroo, Shibuya-ku, Tokyo

Claims (1)

[Claims] 1. An anti-AIDS agent containing 1-methoxy-2-formyl-3-hydroxyanthraquinone as an active ingredient.