JP2001253823A - Hiv gene expression inhibitor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a medicine exhibiting an HIV promoter activity-inhibiting activity more excellent than those of conventional medicines. SOLUTION: This HIV promoter activity inhibitor contains a compound having an isoflavone skeleton, a 3-phenylcoumarin skeleton or a chalcone skeleton, or their glycosides and especially contains glycylisoflavone, irridine, glycycoumarin, lycopyranocoumarin, lycochalcone A, lycochalcone B or tetrahydroxymethoxychalcone.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a human immunodeficiency virus (hereinafter referred to as H) containing a compound having an isoflavone skeleton, 3-phenylcoumarin skeleton or chalcone skeleton.
(Referred to as IV) gene expression inhibitor, particularly an HIV promoter activity inhibitor.

[0002]

2. Description of the Related Art HIV, which is a virus that causes acquired immunodeficiency syndrome (AIDS), is one of retroviruses. There are many ways in which a drug may exhibit antiretroviral activity. Viral reverse transcriptase inhibitors represented by AZT, 3TC, and the like suppress proviral conversion by inhibiting the reverse transcription reaction of a retroviral RNA genome.
On the other hand, a viral protease inhibitor, which was approved by the US FDA in 1996, appeared to inhibit the reaction of cleaving a precursor protein, which is a transcriptional translation product of a provirus, into each mature viral protein, thereby increasing the growth of the virus. Suppress. Furthermore, integrase inhibitors that are being developed recently include viral DNA generated by reverse transcription reaction.
Inhibits the process of incorporation into the chromosome of the cell, thereby suppressing proviral transformation.

[0003] Suppressing viral growth by inhibiting gene expression from HIV proviral DNA, particularly transcription, is also an attractive strategy for preventing (or delaying) the development of AIDS in HIV-infected individuals. It is. So far, for example, it has been known that tannic acid and several kinds of low-molecular-weight polyphenols have an inhibitory action on HIV promoter activity, but the effect is not sufficient, and a search for a stronger inhibitor of HIV promoter activity has been conducted. This is an important issue.

[0004]

SUMMARY OF THE INVENTION Accordingly, an object of the present invention is to provide a substance having a higher inhibitory activity on HIV promoter activity than tannic acid or the like.
It is to provide a new means of prevention and treatment of S.

[0005]

Means for Solving the Problems The expression of HIV gene is induced by activation of kinase by binding of ligands such as TNF and IL-1 to a receptor on the cell side.
IκB of the NFκB / IκB complex is phosphorylated and released, and the activated NFκB translocates to the nucleus and binds to a specific cis element in the HIV promoter (LTR) to initiate transcription of viral RNA. It is described as being caused by things. NFκB is also activated by TPA stimulation. That is, upon TPA stimulation, cellular C-kinase is activated, and its action causes
κB is phosphorylated and released, and NFκB is activated. Therefore, an assay system for transfecting a cultured cell with a plasmid having a reporter gene downstream of the HIV promoter to induce NFκB activation by TPA stimulation and examining the transient expression of the reporter gene is known as an HIV inhibitor activity inhibitor. This is a very effective tool as a screening system for drugs.

The present inventors have investigated the inhibitory activity of various compounds extracted and isolated from medicinal plants on HIV promoter activity using this assay system. The present inventors have found that a compound having a phenylcoumarin skeleton or a chalcone skeleton exhibits a stronger inhibitory action on HIV promoter activity than tannic acid, thereby completing the present invention.

That is, the present invention is as follows. 1. The following general formula (I)

[0008]

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[0009] (wherein, R ¹ is hydroxy or C _1-4 alkoxy, R ² is hydrogen, hydroxy, C _{1 -4} alkoxy or C _2-5 alkenyl, R ³ is hydrogen, hydroxy or C
_1-4 alkoxy, R ⁴ is hydrogen, hydroxy or C _1-4 alkoxy, R ⁵ is hydrogen, hydroxy or C _1-4 alkoxy, and R ⁶ is hydrogen, hydroxy or C _2-5 alkenyl) It contains at least one compound selected from the group consisting of isoflavone compounds and glycosides thereof, preferably at least one isoflavone compound selected from the group consisting of glycyl isoflavone and iridine, more preferably iridine. Human immunodeficiency virus promoter activity inhibitor. 2. The following general formula (II)

[0010]

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Wherein R ¹ is hydroxy or C _1-4 alkoxy; R ² is hydrogen or C _2-5 alkenyl;
¹ and R ² may together form a chroman ring;
³ is hydroxy or C _1-4 alkoxy, R ⁴ is hydrogen, hydroxy or C _1-4 alkoxy, R ⁵ is hydrogen, hydroxy, C _1-4 alkoxy or C _2-5 alkenyl, and R
⁶ is hydrogen, hydroxy or C _1-4 alkoxy), and at least one compound selected from the group consisting of 3-phenylcoumarin compounds and glycosides thereof;
A human immunodeficiency virus promoter activity inhibitor, preferably containing at least one 3-phenylcoumarin-based compound selected from the group consisting of glycicoumarin and lycopyranocoumarin. 3. The following general formula (III)

[0012]

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(Wherein R ¹ is hydroxy or C _1-4 alkoxy, R ² is hydrogen, hydroxy or C _1-4 alkoxy, R ³ is hydroxy or C _1-4 alkoxy, R ⁴ is hydrogen, hydroxy or C _1-4 alkoxy, R ⁵ is hydroxy or C _1-4 alkoxy, R ⁶ is hydrogen, hydroxy or C _2-5 alkenyl), and at least one selected from the group consisting of chalcone compounds and glycosides thereof Human immunodeficiency virus promoter activity inhibitor containing one compound, preferably at least one chalcone-based compound selected from the group consisting of lycochalcone A, lycochalcone B and tetrahydroxymethoxychalcone, more preferably tetrahydroxymethoxychalcone .

[0014]

BEST MODE FOR CARRYING OUT THE INVENTION The isoflavone-based compound which is an active ingredient of the HIV promoter activity inhibitor of the present invention is a compound having an isoflavone skeleton represented by the above general formula (I) or a glycoside thereof, which is described below. In the case of using the assay system described in detail in Examples, there is no particular limitation as long as the compound can inhibit HIV promoter activity in the HIV promoter-containing plasmid introduced into cells by about 80% or more at a concentration of 100 μg / ml or less. Absent. Here, “C _1-4 alkoxy” may be any alkoxy group having 1 to 4 carbon atoms, and is preferably methoxy. Also, "C
" _2-5 alkenyl" may be any alkenyl group having 2 to 5 carbon atoms, but is preferably 2-methyl-2-buten-4-yl. Specific examples of the isoflavone-based compound of the present invention include glycyl isoflavone (Chem. 7) and iridine (Chem. 8), and more preferably irisine.

[0015]

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[0016]

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These isoflavone compounds may be obtained by any method.
It can be obtained as a free compound or glycoside from a plant belonging to the legume family, the iris family, the rose family, etc. by using a conventional extraction and purification method.

For example, glycyl isoflavone is obtained by homogenizing commercially available northwest licorice with 70% aqueous acetone, removing the insoluble matter by centrifugation, partitioning the supernatant concentrate with ether and water, and removing the ether-soluble portion. The aqueous layer removed was further extracted with ethyl acetate, and the concentrated solution was subjected to silica gel column chromatography (eluted with chloroform-water 98: 2) and column chromatography using MCI-gel CHP-20P as an adsorbent (methanol-water 6). : Eluted at 4: 4) and then separated and purified to obtain colorless crystals.

For example, iris can be obtained by extracting a dried rhizome of German iris ( Iris germanica ) with methanol at room temperature, extracting a concentrated solution with ether, ethyl acetate and butanol in that order, and converting the obtained ethyl acetate extract into a silica gel column. By adsorbing and sequentially eluting with chloroform and a mixed solution of chloroform and methanol (the methanol content is increased by 10% from 10% to 40%), yellow crystals can be obtained.

The isoflavone compounds of the present invention not found in nature can be prepared, for example, by modifying isoflavone compounds which can be isolated from nature using a conventional method.

A 3-phenylcoumarin compound, which is another active ingredient of the HIV promoter activity inhibitor of the present invention, comprises:
A compound having a 3-phenylcoumarin skeleton represented by the above general formula (II) or a glycoside thereof, wherein an HIV promoter-containing plasmid introduced into a cell when the assay system described in Examples below is used. HIV promoter activity at a concentration of 100 μg / ml or less
There is no particular limitation as long as the compound can inhibit the above. Here, “C _1-4 alkoxy” may be any alkoxy group having 1 to 4 carbon atoms, and is preferably methoxy.
“C _2-5 alkenyl” may be any alkenyl group having 2 to 5 carbon atoms, but is preferably 2-methyl-alkenyl.
2-buten-4-yl. Specific examples of the 3-phenylcoumarin-based compound of the present invention preferably include glycicoumarin (Chem. 9) and lycopyranoccoumarin (Chem. 10).

[0022]

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[0023]

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These 3-phenylcoumarin compounds may be obtained by any method,
For example, it can be obtained as a free compound or a glycoside from a plant belonging to the Asteraceae, Leguminosae, Umbelliferae, Gramineae, Rutaceae, etc. using a conventional extraction and purification method.

For example, in the same manner as the above-mentioned glycyl isoflavone, glycicoumarin is obtained by homogenizing northwest licorice with 70% aqueous acetone, removing insolubles by centrifugation, and dispersing the concentrate of the supernatant with ether and water. The aqueous layer from which the solvent had been removed was further extracted with ethyl acetate, and the concentrated solution was subjected to silica gel column chromatography (chloroform-water 9).
8: 2) and column chromatography using MCI-gel CHP-20P as adsorbent (methanol-
(Elution with water 6: 4) and separation and purification to obtain yellow crystals. On the other hand, lycopyranocoumarin was obtained from ethyl acetate extract by droplet countercurrent distribution chromatography (chloroform-methanol-water 7: 13: 8; descending method), MCI-gel CHP-20.
Column chromatography using P as an adsorbent (eluted with methanol-water 7: 3) and preparative chromatography on a thin layer of silica gel (chloroform-acetone-formic acid 8)
0: 17: 3) to obtain yellow crystals.

Also, the present invention, which has not been found in nature.
The -phenylcoumarin-based compound can be prepared, for example, by modifying a 3-phenylcoumarin-based compound that can be isolated from nature using a conventional method.

The chalcone compound, which is another active ingredient of the HIV promoter activity inhibitor of the present invention, is a compound having a chalcone skeleton represented by the above general formula (III) or a glycoside thereof, as described in Examples below. In the case of using the assay system described in detail in item (1), the HIV promoter activity in the HIV promoter-containing plasmid introduced into the cells was 1
There is no particular limitation as long as it is a compound that can inhibit about 80% or more at a concentration of 00 μg / ml or less. Here, “C _1-4 alkoxy” may be any alkoxy group having 1 to 4 carbon atoms, and is preferably methoxy. “C _2-5 alkenyl” may be any alkenyl group having 2 to 5 carbon atoms, but is preferably 3-methyl-1-butene-3-
Ill. Specific examples of the chalcone-based compound of the present invention preferably include lycochalcone A (chemical formula 11), lycochalcone B (chemical formula 12), tetrahydroxymethoxychalcone (chemical formula 13), and more preferably tetrahydroxymethoxychalcone.

[0028]

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[0029]

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[0030]

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These chalcone compounds may be obtained by any method. For example, they can be obtained as free compounds or glycosides from asteraceous plants and the like using a conventional extraction and purification method.

For example, Ricochalcones A and B are prepared by homogenizing commercially available Xinjiang licorice in 70% aqueous acetone, removing insolubles by centrifugation, and dispersing the supernatant concentrate with ether and water. Centrifugal countercurrent partition chromatography (chloroform-methanol-water 7: 1) for the extract
3: 8; descending method) and silica gel column chromatography (eluted with chloroform-methanol 98: 2) to separate and obtain yellow crystals.

For the tetrahydroxymethoxychalcone, for example, Tohoku licorice is extracted with aqueous acetone in the same manner as described above, and the ether extract is subjected to droplet countercurrent distribution chromatography (chloroform-methanol-propanol-water 45: 60: 10: 40). Descending method), MCI-Gel CHP-20P as an adsorbent, column chromatography (eluted with aqueous methanol), and preparative chromatography on a thin layer of silica gel to separate and separate them together with lycochalcone B and glycicumarin. It can be obtained as crystals.

The chalcone compound of the present invention not found in nature can be prepared, for example, by modifying a chalcone compound which can be isolated from nature by a conventional method.

The HIV promoter activity inhibitor of the present invention comprises one or more of the above isoflavone compounds,
Alternatively, two or more of the above-mentioned 3-phenylcoumarin-based compounds or one or more of the above-mentioned chalcone-based compounds are contained as active ingredients. Further, two or more compounds having these three different basic skeletons can be contained.

The inhibitor of the HIV promoter activity of the present invention is very useful as a research reagent in basic or applied research on HIV gene expression.
It is particularly useful as an inhibitor of HIV gene expression in cells containing HIV provirus of S patients or HIV-infected individuals.

H in cells containing the HIV provirus
When used as an IV gene expression inhibitor, the H
The IV promoter activity inhibitor comprises one or more of the above isoflavone-based compounds and one or more of the above-mentioned 3- or 3-
The phenylcoumarin-based compound or one or more chalcone-based compounds described above are appropriately mixed with necessary components such as pharmaceutically acceptable carriers (eg, excipients, diluents, etc.) to prepare liquid preparations, powders, and granules. , Tablets, capsules, syrups, injections, aerosols and the like, and can be administered orally or parenterally.

[0038] Pharmaceutically acceptable carriers include, for example,
Excipients such as sucrose, starch, mannitol, sorbitol, lactose, glucose, cellulose, talc, calcium phosphate, calcium carbonate, cellulose, methylcellulose, hydroxypropylcellulose, polypropylpyrrolidone, gelatin, gum arabic, polyethylene glycol, sucrose , A binder such as starch, starch, carboxymethylcellulose, hydroxypropyl starch,
Disintegrants such as sodium-glycol-starch, sodium bicarbonate, calcium phosphate, calcium citrate, magnesium stearate, aerosil, talc,
Lubricants such as sodium lauryl sulfate, citric acid, menthol, glycyrrhizin ammonium salt, fragrance such as glycine, orange powder, preservatives such as sodium benzoate, sodium bisulfite, methylparaban, propylparaban, citric acid, sodium citrate , Acetic acid and other stabilizers,
Methylcellulose, polyvinylpyrrolidone, suspending agents such as aluminum stearate, dispersants such as surfactants,
Examples include, but are not limited to, water, physiological saline, diluents such as orange juice, cocoa butter, polyethylene glycol, and base wax such as white kerosene.

Preferably, the preparation of the present invention is an oral or injection preparation. Formulations suitable for oral administration include a solution prepared by dissolving an effective amount of an inhibitor of HIV promoter activity in a diluent such as water, saline, orange juice, and a capsule containing an effective amount of the compound as a solid or a granule. Agent, sachet or tablet, a suspension in which an effective amount of the compound is suspended in a suitable dispersion medium, an emulsion in which a solution in which an effective amount of the compound is dissolved is dispersed in a suitable dispersion medium and emulsified And so on.

Suitable formulations for parenteral administration include aqueous and non-aqueous isotonic sterile injection solutions, which include antioxidants, buffers, bacteriostats, isotonic agents and the like. May be included. Aqueous and non-aqueous aseptic suspensions may also be mentioned, which may contain suspending agents, solubilizers, thickeners, stabilizers, preservatives and the like. The preparation of the present invention
A unit dose or a plurality of doses can be enclosed in a container like an ampoule or a vial. Alternatively, the HIV promoter activity inhibitor of the present invention and a pharmaceutically acceptable carrier may be freeze-dried (freeze-dried) and stored in a state that may be dissolved or suspended in an appropriate sterile vehicle immediately before use.

The dosage of the preparation of the present invention varies depending on the effective amount of the active ingredient, ie, the inhibitor of the HIV promoter activity, the cytotoxicity of the compound, the degree of disease progression, the drug receptivity of the administration subject, body weight, age and the like. , Usually 3 per adult per day
0 μg to 30 mg / kg body weight, preferably 300 μg to
3 mg / kg body weight, and this amount can be administered once or in several divided doses.

[0042]

The present invention will be described in more detail with reference to the following examples, which are merely illustrative and do not limit the scope of the present invention in any way.

Reference Example 1 Construction of HIV promoter activity inhibitor screening system (1) Construction of HIV promoter-reporter gene chimeric plasmid vector Promoter region of HIV LTR (-452 to +8
0; the transcription start point is +1) About 500 bp of the expression vector pHIVLuc inserted upstream of the luciferase gene of a commercially available pGL2 vector (manufactured by Promega; hereinafter, referred to as pLucC vector when used as a control vector). It was constructed. (2) Transfection of Cultured Human Cells 2 × 10 ⁶ Jurkat cells cultured in RPMI1640 medium at 37 ° C. under 5% CO ₂ were treated with TBS buffer (25 mM Tris, 137 mM NaCl, 5 mM).
KCl, 0.5 mM Na ₂ HPO ₄ , 0.5 mM MgC
After washing with 4 ml of l ₂ , 0.68 mM CaCl ₂ )
DEAE-dextran / DNA mix (1 × TB
S, 500 μg / ml DEAE-dextran, plasmid pHIVLuc or pLucC 2.5-5.0
μg) Incubated for 30 minutes at room temperature in 4 ml.
After washing and removing excess DNA with 4 ml of TBS, 10%
10 ml of RPMI1640 medium containing fetal calf serum was added, and the mixture was incubated at 37 ° C. under 5% CO ₂ for 48 hours.

Example 1 Screening of HIV Promoter Activity Inhibitor (1) Treatment with TPA and Test Compound After transfection treatment and incubation for 48 hours as in Reference Example 1, TPA (5 ng / ml) and various natural compounds ( 12.5-100 μg / ml) was added to the medium of the cells, and the cells were further cultured under the same conditions for 15 hours. (2) Preparation of cell extract After completion of the incubation, the culture solution was removed to collect the cells, and 1 ml of phosphate buffered saline (PBS) was added.
The cells were washed by centrifugation at 3000 rpm at 4 ° C. for 5 minutes (twice). 1 × lysis buffer (25 mM Tris-
Phosphoric acid (pH 7.8), 2 mM DTT, 2 mM 1,2
-Diaminocyclohexane-N, N, N ', N'-tetraacetic acid, 10% glycerol, 1% Triton X-1
00) Add 250 μl and mix well, then add 14,000
Flash centrifugation was performed at 0 rpm at 4 ° C for 5 seconds. The supernatant was collected and a part was used for protein quantification. (3) Luciferase assay The assay was performed according to the method described in Mol. Cell. Biol. , 7 , 725-737. The cell extract obtained in the above (2) (protein amount: about 20 μm)
g) was placed in a 1.5 ml microtube, and a luciferase assay reagent (Luciferase Assay S manufactured by Promega) was used.
ystem (product number E1500) (100 μl) and the luminescence intensity (unit: RLU) was measured immediately using a luminometer. The results are shown in FIG.

Glycyl isoflavone, glycicoumarin,
Lycopyranocoumarin, lycochalcone A, lycochalcone B, tetrahydroxymethoxychalcone, and lycocoumarone (Formula 14;
[b] the structure replaced by a furan skeleton) showed remarkably higher HIV promoter inhibitory activity than tannic acid.

[0046]

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[0047]

Industrial Applicability The HIV promoter activity inhibitor of the present invention can inhibit the transcriptional activity of the HIV promoter extremely strongly as compared with conventionally known tannic acid and some low molecular weight polyphenol compounds.
It can suppress the expression of viral genes from HIV-infected T cells in DS patients or HIV-infected individuals, and is useful for suppressing the growth of the virus and preventing the development or progression of AIDS. In addition, it has been found that these natural compounds are structurally similar and roughly classified into three basic skeletons. Therefore, by changing the substituents by chemical synthesis, compounds having more excellent HIV gene expression inhibitory action can be obtained. It is very useful in that it offers the possibility to be obtained.

[Brief description of the drawings]

FIG. 1 shows a physical map of plasmid pHIVLuc.

FIG. 2 shows the inhibitory effects of tannic acid (shaded bars) and various test compounds (solid bars) on HIV promoter activity. For each compound, 12.
5 (only tetrahydroxymethoxychalcone in FIG. 2B), 25, 50 and 100 μg / ml. TPA
(Open bar) shows the case where only TPA stimulation was added without adding an inhibitor.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12N 7/04 C12N 7/04 4C0715 15/09 C07D 307/80 4C086 // C07D 307/80 311/18 4C206 311/18 311/36 311/36 493/04 106C 493/04 106 C07H 17/07 C07H 17/07 (C12N 7/04 (C12N 7/04 C12R 1:93) C12R 1:93)) (C12N 15 / 09 C12N 15/00 A C12R 1:93) C12R 1:93) (72) Inventor Takashi Yoshida 1444-118 Tsudaka, Okayama-shi, Okayama Prefecture (72) Inventor Tsutomu Hatano 2104-6, Sayama, Okayama-shi, Okayama F-term ( Reference) 4B024 AA20 FA02 HA20 4B065 AA97X BD13 BD34 CA48 CA60 4C037 PA07 4C057 BB02 CC01 DD01 KK08 4C062 EE27 EE44 4C071 AA01 BB01 CC12 EE07 FF17 GG03 HH09 JJ01 LL01 4C086 AA01 A01A01A 11 GA17 MA01 MA04 NA14 ZC55 4C206 AA01 AA02 CB19 KA01 KA18 MA01 MA04 MA36 MA42 MA43 MA55 MA57 MA72 MA86 NA14 ZC55

Claims (8)

[Claims] 1. A compound represented by the following general formula (I) Wherein R ¹ is hydroxy or C _1-4 alkoxy, R ²
Is hydrogen, hydroxy, C _{1 -4} alkoxy or C _2-5 alkenyl, R ³ is hydrogen, hydroxy or C _1-4 alkoxy, R ⁴ is hydrogen, hydroxy or C _1-4 alkoxy, R
⁵ is hydrogen, hydroxy or C _1-4 alkoxy, and R
⁶ is hydrogen, hydroxy or C _2-5 alkenyl), and a human immunodeficiency virus promoter activity inhibitor comprising at least one compound selected from the group consisting of isoflavone compounds represented by the formula: 2. The human immunodeficiency virus promoter activity inhibitor according to claim 1, which comprises at least one isoflavone compound selected from the group consisting of glycyl isoflavone and iridine. 3. The human immunodeficiency virus promoter activity inhibitor according to claim 1, which contains iridine. 4. The following general formula (II): Wherein R ¹ is hydroxy or C _1-4 alkoxy, R ²
_{May be} hydrogen or C _2-5 alkenyl, or R ¹ and R ² may together form a chroman ring, R ³ is hydroxy or C _1-4 alkoxy, R ⁴ is hydrogen, hydroxy or C _{1- 4} alkoxy, R ⁵ is hydrogen, hydroxy, C _1-4
Alkoxy or C _2-5 alkenyl, and R ⁶ is hydrogen,
A human immunodeficiency virus promoter activity inhibitor comprising at least one compound selected from the group consisting of 3-phenylcoumarin compounds represented by the formula: hydroxy or C _1-4 alkoxy) and glycosides thereof. 5. The human immunodeficiency virus promoter activity inhibitor according to claim 4, which comprises at least one 3-phenylcoumarin-based compound selected from the group consisting of glycicoumarin and lycopyranocoumarin. 6. A compound represented by the following general formula (III): Wherein R ¹ is hydroxy or C _1-4 alkoxy, R ²
Is hydrogen, hydroxy or C _1-4 alkoxy, R ³ is hydroxy or C _1-4 alkoxy, R ⁴ is hydrogen, hydroxy or C _1-4 alkoxy, R ⁵ is hydroxy or C _1-4
Alkoxy, R ⁶ is hydrogen, hydroxy or C _2-5 alkenyl), and inhibits human immunodeficiency virus promoter activity containing at least one compound selected from the group consisting of chalcone compounds represented by the formula: Agent. 7. The human immunodeficiency virus promoter activity inhibitor according to claim 6, comprising at least one chalcone-based compound selected from the group consisting of lycochalcone A, lycochalcone B and tetrahydroxymethoxychalcone. 8. The inhibitor of human immunodeficiency virus promoter activity according to claim 6, which comprises tetrahydroxymethoxychalcone.