JPH02196725A - Antiretrovioral agent - Google Patents

Abstract

PURPOSE:To obtain an antiretroviral agent containing sulfate of a hydrophilic solvent extract NF-86I, NF-86II, etc., of Areca catechu L. or salt thereof as an active ingredient, having extremely low toxicity and capable of administering in large quantities. CONSTITUTION:A degreased Areca catechu L. is extracted with a hydrophilic solvent and the extract is concentrated to dryness and water is added thereto to give a suspension. The suspension is filtered and a non-hydrophilic organic solvent is added to the filtrate and solvent soluble part is removed and the treated filtrate is fractionated using a membrane filter to afford a fraction (NF-86I) having 1000-10000 molecular weight and a fraction (NF-86II) having >=10000 molecular weight. The above-mentioned fractions are each separated using high speed liquid chromatograph to give NPF-86IA (having 5620 molecular weight), NPF-86IB (having 5000 molecular weight), NPF-86IIA (having 29400 molecular weight) and NPF-86IIB (having 8610 molecular weight). The above- mentioned 6 kinds of compounds are subjected to esterification (sulfur content: 0.1-30%) to provide the antiretroviral substance.

Description

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

[Background of the invention]

AIDS (acquired immune defect)
198
It was first reported in the United States in 1999 [Gotzlieb].
ttlieb), M, S, etc. (N, Engl,
J, Med, 305.1425 (1981)) and Siegal, F, P et al.
N, εng1. J0Med, 305°1439 (
1981)')). Later, in 1983, Montagnier (Barr6-Sinoussi), F. et al. (Science), 220°868
(1983)) ), followed by the American Gi + O (G
al lo) [Popovic,
M, et al. (Science), 224.49
7 (1984)), the virus responsible for AIDS, HIV (human immunodeficiency v.
irus) was discovered.

However, no reliable therapeutic or preventive drugs have yet been developed. In terms of treatment methods, as a result of searching for substances that have the effect of inhibiting HIV infection, the nucleic acid analog azidothymidine (AZT) was developed by Hiroaki Cmya et al., Proc., Natl.
, Acad, Sci, Li5A, 82.709
6 (1985), Hideki Nakajima et al., Antimicrob,
Agents Chemother,, 30.933
(1986) 3 and various dideoxynucleosides [Hiroaki Mitsuya et al., Proc, Natl, Acad.

Sci, USA, 83.1911 (1986), Hamamoto, Y.

etc., ^ntimicrob, ^gents, Che
Mother, 31.907 (1987)] was found. AZT has been reported to have a life-prolonging effect through in vivo research, and has been approved as a therapeutic agent for AIDS.

However, AZT requires long-term administration and many problems remain, including side effects.

Recently, Nakajima and Sansui et al. discovered that anti-HIV effects are produced by sulfating lentinan and other polysaccharides [Hideki Nakashima, Ho et al., G.
Ann, 78.1164 (1987)), we investigated the anti-HIV effect of sulfate esterified polymeric polyphenols having a tertiary structure, such as polysaccharides such as halentinan. As a result, strong anti-H
The present invention was completed by demonstrating IV action.

On the other hand, betel nut is a variety of betel nut grown in various parts of Southeast Asia.
chu L, ) ) (Aracaceae), with the pericarp removed, and is known as an astringent, saliva secretion stimulant, tapeworm repellent, etc., and as a raw material for the miotic drug arecoline hydrobromide.

Polyphenol carcinogens extracted from betel nut NF-86I, NF-86TI, NPF-86I
A.

NPF-86I B, NPF-86n A, NPF-
86If B has already been patented by the applicant (Japanese Patent Application No. 1983-233246). A patent application has also been filed for these sulfuric acid esters as anticancer agents (Japanese Patent Application No. 89744/1989).

[Purpose of the invention]

Until now, almost no antiretroviral agents were known, and the only drug available, AZT, was highly toxic and had side effects as described above.

Therefore, an object of the present invention is to provide an antiretroviral agent made from a natural substance that has extremely low toxicity and can be administered in large quantities.

[Structure of the invention]

The present invention has the following physical and chemical properties, NF-86I,
NF-86n, NPF-86IA.

NPF 861B, NPF 86IA and NPF-
The present invention provides an antiretroviral agent containing as an active ingredient a sulfate ester of 86IIB and a substance selected from the group consisting of the sulfate ester.

(NF-86I) (i) Shape: Light yellowish brown powder.

(ii) Melting point: shows no clear melting point or decomposition point.

(iii) Elemental analysis: Carbon 56.30% Hydrogen 4.
61% Nitrogen 0.2% or less Ash 0.3% or less (iv) Molecular weight: 1.000-10.000 (by membrane filter) (v) Infrared absorption spectrum (see Figure 5): 'ma
, ICm-': 3430.2940.1610.
1520, (vi) (yj) (viii) 1440.1370.1280.111O11060,
800 Ultraviolet absorption spectrum (see Figures 7 and 9): Water 1% λsaw nm (E 1cm) 279 (13
5,7) Salt lit% λmawt+m (E 1cm) 279 (13
4,5) 290 shoulder (291,2), 420 shoulder (96,4), 500 shoulder (60,6) Solubility: Soluble in water and ethanol. Insoluble in hexane, ether, ethyl acetate, and chloroform.

Color reaction: Ferric chloride reaction Positive Ninhydrin reaction Negative p-7°'9>-7 phosphoric acid negative reaction Aniline-diphenylamine Reaction Negative Lagendorf reaction Negative (IX) Stability: Stable in powder state.

(NF-86m) (i) Shape: Light yellowish brown powder.

(11) Melting point: Shows no clear melting point or decomposition point.

(iii) Elemental analysis: Carbon 56.64% Hydrogen 4
．． 59% Nitrogen 0.2% or less Ash 0.3% or less (iv) Molecular weight: 10,000 or more (by membrane filter) (v) Infrared absorption spectrum (see Figure 6): 1450
．． 1370.1290.1110.1060.800
．． 500 Sodium hydroxide 1%λ1Iax
nff1 (Elcm)2
90 shoulder (306,1), 415 shoulder (100,0), 505 shoulder (61,2) (register) Solubility: Soluble in water, methanol, and ethanol. Insoluble in hexane, ether, ethyl acetate, and chloroform.

(vii) Color reaction: Ferric chloride reaction Positive Ninhydrin reaction Negative p-anisidine-phthalic acid Reaction Negative 7° “J′-ゞ7”=tlz7E7 Negative reaction Dragendorff reaction Negative (ix) Stability: Stable in powder state.

(NPF-86IA) (i) Shape: Light yellowish brown powder.

(ii) Melting point: shows no clear melting point or decomposition point.

(ji) Elemental analysis: Carbon 54.82% Hydrogen 4.5
2% Oxygen 37.93% Nitrogen 0.2% or less Ash 0.2% or less (1v) Molecule 1:5.620 (by gel permeation chromatography using polyethylene glycol as standard) (v) Infrared absorption spectrum ( (See Figure 1): 1440
．． 1380.1280.1260.1210.1160
．． 1110.1060.820.800 (vi) Ultraviolet absorption spectrum: Water λ@axnm279 (vii) Solubility: Soluble in water, methanol, and ethanol. Insoluble in hexane, ether, ethyl acetate, and chloroform.

(v Blood) Color reaction: Ferric chloride reaction Positive Ninhydrin reaction Negative p-anisidine-phthalic acid Reaction Negative Aniline-diphenylamine Reaction Negative Dragendorff reaction Negative (ix) Stability: Stable in powder state.

(NPF-86IB) (i) Shape: Light yellowish brown powder.

(ii) Melting point: shows no clear melting point or decomposition point.

(ii) Elemental analysis: Carbon 57.09% Hydrogen 4.4
5% Oxygen 35.03% Nitrogen 0.2% or less Ash 0.2% or less (iv) Molecular weight: 5,000 (by gel permeation chromatography using polyethylene glycol as standard) (v) Infrared absorption spectrum (See Figure 2): Lid-r ν, am Cm″″.

3400.2930.1610.1520.1440.
1360.1280.1250.1200.1160.
111O11060, (vi) Ultraviolet absorption spectrum: Water λ,, Xruy+279 (vj) Solubility: Soluble in water, methanol, ethanol.

hexane, ether, acetic acid Insoluble in chill and chloroform.

(vffi) Color reaction: Ferric chloride reaction Positive Ninhydrin reaction Negative Dragendorff reaction Negative (ix) Stability: Stable in powder state.

(NPF-86nA) (i) Shape: Light yellowish brown powder.

(ii) Melting point: shows no clear melting point or decomposition point.

(iii) Elemental analysis: Carbon 51.44% Hydrogen 4
．． 44% Nitrogen 0.1% or less Ash 0.2% or less (iv) Molecular weight: 29.400 (by gel permeation chromatography using polyethylene glycol as standard) (v) Infrared absorption spectrum (see Figure 3) 2 144
0.1370.1280.1250.1210.116
0.1110.1060.820.800 (vi) Ultraviolet absorption spectrum: Water λ, □nm279 (vii) Solubility: Soluble in water, methanol, and ethanol. Insoluble in hexane, ether, ethyl acetate, and chloroform.

(vn+) Color reaction: Ferric chloride reaction Positive Ninhydrin reaction Negative p-anisidine-phthalic acid Reaction Negative Dragendorff reaction Negative (ix) Stability: Stable in powder state.

(NPF-86I [B) (i) Shape: Light yellowish brown powder.

(ii) Melting point: shows no clear melting point or decomposition point.

(ji) Elemental analysis: Carbon 52.46% Hydrogen 4.42% Nitrogen 0.1% or less Ash 0.2% or less (iv) Molecular weight: 8.610 (by gel permeation chromatography using polyethylene glycol as standard). ) (v) Infrared absorption spectrum (see Figure 4): 144
0.1370.1280.1250.1200.116
0.1110.1060, (vi) Ultraviolet absorption spectrum: Water λ□, Inm 279 (vj) Solubility: Soluble in water, methanol, and ethanol.

hexane, ether, acetic acid Insoluble in chill and chloroform.

(vi) Color reaction: Ferric chloride reaction Positive Ninhydrin reaction Negative &i Anisidine-7-talic acid negative 7°□J:/-97"=A7E:/, U reaction Dragendorff reaction Negative (ix) Stability: Stable in powder state.

The present invention also provides an antiretroviral agent containing a sulfate ester of a hydrophilic solvent extract of areca nut or a salt of the sulfate ester as an active ingredient.

The present invention will be explained in more detail below.

(a) Raw material The above-mentioned betel nut is used as the raw material, but it is preferable to use one that has been subjected to treatments such as drying, coarse crushing, and pulverization so that it can be easily processed and extracted.

It is also convenient to use commercially available crude drugs.

b) Extraction The areca nut extract used in the present invention is a hydrophilic solvent extract such as water or alcohol such as methanol, and when a hydrophobic solvent extract is used, the effect is extremely low.

Moreover, it is preferable to use the areca nut extract after further extraction and purification as described below.

NF-86I, NF-86I [NPF-
86IASNPF-86IB, NPF-86IIA and NPF-86IIB are phenolic substances, 5'-
Since it is characterized by nucleotidase inhibitory activity and anticancer activity, it can be isolated and purified using appropriate purification methods using water, organic solvents, centrifugation, filtration, etc., using these inhibitory activities as an indicator. These methods can be applied singly, in combination in any order, or repeatedly as necessary. Below are NF-861 and NF-8
An example of a method for extracting 6II will be explained.

(a) Degrease the raw material using a degreasing solvent such as hexane or ether at room temperature or by heating.

(b) The defatted raw material is air-dried or vacuum-dried to remove the defatting solvent.

(c) Next, methanol is added to the extraction raw material and extracted according to a conventional method. Although extraction is usually carried out under boiling conditions, active ingredients can also be obtained by extraction in a cold room at 4°C.

(d) After concentrating the obtained extract to dryness, water is added to form a suspension. Filter this using filter paper.

For insoluble matter, add water, stir well, filter, and combine with the previous filtrate.

(e) Add an equal amount of a non-hydrophilic organic solvent such as ethyl acetate or chloroform to this aqueous solution to remove the organic solvent soluble portion.

(f) The aqueous layer from which the non-hydrophilic organic solvent soluble portion has been removed has a molecular weight cutoff of 1. The solution is placed in an OOO membrane filter (Spectra/Pore 6; manufactured by Spectrum Medical Industries), dialyzed against water, and fractionated into an internal solution and an external solution.

(g) The dialysis fluid fractionated with a membrane filter with a molecular weight cut off of 1.000 is further added to the membrane filter with a molecular weight cut off 110.000.
The solution is placed in a membrane filter (Spectra/Pore 6; manufactured by Spectrum Medical Industries), dialyzed against water, and fractionated into an internal solution and an external solution.

(H) When fractionated in this way, the molecular weight is 1.000 to 10.
．． The desired inhibitory activity was observed in the fraction of 0.000, 10.000 or more, and both active substances could be obtained as light brown powders by freeze-drying or other operations.

The present inventor has determined that the molecular weight is 1.000 to 10,000 and 10.
, 000 or more fractionated active substances are each NF-86I.
and NF-86II.

(NPF-861A, NPF 86IB, NPF-8
6IIA and NPF-86IIB) Thus,
NF-86I was fractionated using a membrane filter.
and NF-86II can each be further classified into two fractions. That is, NF-861 has a molecular weight of 5.620
(NPF-86IA) and a fraction with a molecular weight of 5.000 (NPF-86IB).
．． 610 (N, PF-86IIB) (see Fig. 11).

These four substances (NPF 86 IASNPF-8
6IBSNPF-86IIA and NPF-86IIB)
The separation and purification can be carried out by various known methods, but it is preferably carried out using a high performance liquid chromatograph under the following conditions.

(i) Separation Column In this case, a separation column filled with a distribution/adsorption type resin, an ion exchange resin, a gel filtration type separation agent, etc. can be used. It is also preferable to additionally introduce a device for automatic injection and automatic fractionation.

(1i) Eluent In addition to water-methanol, various buffer solutions such as water, acetonitrinopedimethylformamide, acetic acid, butanol, hexane, and others can be used as the eluent, either singly or mixed in any ratio. be able to.

(iii) Indicator The indicator for detecting each of the above substances is 280n
Absorbance at a wavelength of m and 5'-nucleotidase inhibitory activity can be used.

(iv) Processing method As the processing method, an open column, medium pressure or high pressure method can be used.

NPF-861ASNPF-86IBSNPF-86I
[A and NPF-86I[B] each showed a single peak in high performance liquid chromatography, and their 5'-nucleotidase inhibitory activities matched.

In addition, a single peak was observed in gel permeation chromatography performed to measure the molecular weight of this substance.

When examined using the standard molecular weight of various polyethylene glycols, the molecular weight of NPF-861A was 5,620, NPF
-86I B is 5.000, NPF-86IIA is 29
．． 400 and NPF-86IIB was determined to be 8.610.

As mentioned above, NF-861 is NPF-861
A and NPF-86IB mixture, while NF 86I
[was a mixture of NPF 86IIA and NPF-86IIB. Analysis of high-performance liquid chromatograms revealed that N
F-86I is an approximately 1:1 mixture of NPF-861A and NPF-861B, and NF-8611 is an approximately 1:1 mixture of NPF-86IIA and NPF-86IIB.
It was recognized that it was a mixture of

The above extraction operation is optimal as a method for removing the fragrance and color peculiar to the original plant and obtaining a substance for sulfuric acid esterification. In addition, since the effective substance is soluble in methanol and water,
The above extraction method starts from a methanol extract as a raw material, but in order to save on expensive organic solvents, the same operation may be performed after first extracting with a large amount of water or hot water.

Furthermore, as is clear from the ultraviolet absorption spectra above, when made alkaline, all of the above substances turn yellowish-brown to reddish-brown in color, so it is also effective to perform the entire extraction process under weakly acidic conditions using mineral acids or organic acids. It is a method of extraction.

The inhibitory activity is also effective in crude extracts such as methanol extracts. However, the above-mentioned extraction method is optimal as a method for removing the aroma and color peculiar to the original plant and obtaining a substance with stronger usefulness. Furthermore, since the non-hydrophilic organic solvent soluble portion is removed, it is possible to dissolve it uniformly and transparently even as an aqueous solution, which is even more preferable.

(C) Sulfuric acid esterification In the present invention, the hydrophilic solvent extract of areca nut obtained by the above method or NF obtained by further extraction and purification of this is used.
-86l5NF-86II, NPF-86IASNPF
-8618%NPF 86IA and NPF-86II
B (hereinafter, these may be collectively referred to as raw materials) are then converted into sulfuric acid esters.

These raw materials themselves do not have antiretroviral activity, but by converting them into sulfuric acid esters, they become antiretroviral substances, increasing their solubility in water and making the aqueous solution stable. In addition, when an aqueous solution of raw materials is left for a long time, insoluble substances precipitate and turn brown, but this does not occur in the case of a sulfuric acid ester.

In the present invention, the sulfur content of the sulfuric acid ester is 0.
．． It is preferably 1 to 30%.

As the reagent used for sulfuric acid esterification, any known sulfuric acid esterifying reagent may be used, but chlorosulfonic acid is suitable in terms of reactivity and ease of handling.

The reaction is carried out under basic conditions, but the solvent is not particularly limited.

However, pyridine is preferred, and anhydrous pyridine is particularly preferred, since the solvent itself is under basic conditions.

The reaction temperature may be room temperature, but in organic chemistry, it is common to add a sulfuric acid esterification reagent under water cooling, and then react at room temperature or by heating.

The amount of the sulfuric acid esterification reagent, such as chlorosulfonic acid, significantly influences the degree of sulfuric acid esterification obtained. However, the antiretroviral activity, which is the purpose of this derivative,
Stability and solubility of the specimen can be sufficiently achieved even if the degree of sulfuric acid esterification is low. However, industrially, it is impossible to produce, separate, and purify products with various degrees of sulfuric acid esterification, so it is preferable to use a large excess of the sulfuric acid esterifying reagent to carry out sulfuric acid esterification as much as possible.

The sulfur (S) content of the sulfuric acid ester obtained by using a large excess of the sulfuric acid esterifying reagent in this manner is 5 to 20%, and it is possible to easily obtain a sulfuric acid ester with approximately the same content every time.

Although the reaction proceeds almost simultaneously with the addition of the sulfuric acid esterification reagent, it is preferable to stir the mixture for a while at room temperature. Regarding the reaction times, there was almost no change in the sulfur content when the reaction time was 1 hour, 24 hours, or 48 hours.

Various methods can be considered for obtaining the sulfuric acid ester after the reaction, including a method in which the reaction solution is directly desalted to obtain the sulfuric acid ester, a method in which the reaction solution is neutralized, and an alkali salt is obtained.

Although both are effective, it is preferable to obtain the sulfate ester in the form of a salt such as a sodium salt or a potassium salt.

It is also preferable to remove the malodorous pyridine with a non-hydrophilic solvent such as chloroform, ethyl acetate, etc.

Furthermore, the above sulfuric acid esterification method was repeated several times,
It is also possible to obtain a product with a higher degree of sulfation.

(B) Antiretroviral agent The present inventor has developed a hydrophilic solvent extract of areca nut or its further extracted and purified NF-861, NF-86■, NP.
F-86IASNPF-86IB.

We studied various pharmacological effects of sulfate esters of NPF 86IIA and NPF-86JIB and their salts,
We discovered that these substances have antiretroviral effects, and completed the antiretroviral agent of the present invention. In the present invention, the active ingredient of the antiretroviral agent is a hydrophilic solvent extract of areca nut or N.
PF-86IA.

NPF 861B, NPF 86 IIA, NPF
-86IIB, NF-861 and NF-86If sulfate esters, and salts of the sulfate esters, either singly or in any mixture.

i) Administration method The antiretroviral agent of the present invention can be administered either orally or parenterally, and when administered orally, it can be administered in the form of soft or hard capsules, tablets, granules, fine granules, or powders. When administered parenterally, it can be administered in the form of an injection, an infusion, or a solid or suspended viscous liquid in the form of a suppository to maintain sustained mucosal absorption; External administration methods such as intradermal, subcutaneous, intramuscular, intravenous injection, local application, spraying, halving, and intravesical injection can also be used.

ii) Dosage The dosage varies depending on the administration method, the malignancy of the disease, the age of the patient, the medical condition and general condition, the degree of progression of the disease, etc., but for adults, it is usually 0.00% of the active ingredient per day. 5~
5,000mg, usually 0.5-3,000m for dwarfs
It is g.

iii) Formulation method The proportion of the active ingredient in the antiretroviral composition of the present invention may vary depending on the dosage form, but usually it is about 0.3 to 15.0 when administered orally or by mucosal absorption. % by weight is suitable, and when administered parenterally, from 0.01 to 10% by weight.

In addition, when formulating the active ingredient of the present invention, it can be made into an aqueous solution, oil-based preparation, etc., into a subcutaneous or intravenous injection preparation, or into a capsule, tablet, fine granule, etc. It can be formulated into a dosage form and administered orally.

In addition, in order to provide the active ingredient with stability and acid resistance that can withstand long-term storage and to fully maintain its medicinal efficacy, it is also possible to formulate a formulation with a pharmaceutically acceptable coating to achieve excellent stability. It can be an antiretroviral composition.

The surfactants, excipients, lubricants, adjuvants, pharmaceutically acceptable film-forming substances, etc. used in formulating the active ingredient of the present invention are as follows.

In order to improve the disintegration and elution of the composition of the present invention,
One or more surfactants such as alcohols, esters, polyethylene glycol derivatives, fatty acid esters of sorbidan, sulfated fatty alcohols, etc. can be added.

In addition, as excipients, for example, sucrose, lactose, starch, crystalline cellulose, mannitol, light silicic anhydride, magnesium aluminate, magnesium metasilicate aluminate, synthetic aluminum silicate, calcium carbonate, sodium hydrogen carbonate, calcium hydrogen phosphate, Carboxymethyl cellulose calcium and the like can be added alone or in combination of two or more.

As lubricants, for example, one or more types of magnesium stearate, talc, hydrogenated oil, etc. can be added, and as flavoring and flavoring agents, salt, saccharin, sugar, mannitol, orange oil, licorice, etc. can be added. Extracts, sweeteners such as citric acid, glucose, menthol, eucalyptus oil, and malic acid, fragrances, coloring agents, preservatives, and the like may be included.

Examples of adjuvants such as suspending agents and wetting agents include coconut oil, olive oil, sesame oil, peanut oil, calcium lactate,
Safflower oil, soybean phospholipids, etc. can be contained.

Film-forming substances include cellulose acetate phthalate (CAP), which is a carbohydrate derivative such as cellulose and sugars.
Examples of polyvinyl derivatives such as acrylic acid copolymers and dibasic acid monoesters include methyl acrylate/methacrylic acid copolymers and methyl methacrylate/methacrylic acid copolymers.

In addition, when coating the above-mentioned film-forming substances, in addition to commonly used coating aids such as plasticizers, the properties of the film-forming agent can be improved by adding various additives to prevent the chemicals from adhering to each other during coating operations. The coating process can be improved and the coating operation made easier.

iv) Assay method for antiretroviral activity, we will describe the test method that confirmed the antiretroviral activity of the active ingredient.

Test Example I Inhibitory effect of drugs on cell damage caused by HIV infection This test was conducted using the established human T cell line MT of adult T cells.
-4 cells (Gann monogr., Vol. 2
L pp219-228 (1982)) and HTLV-nIB, a strain of HIV, were used.

HTLV-I [[B-infected MT-4 cells were treated with various concentrations of N
Sodium salt of sulfate ester of F-86II (hereinafter referred to as NF-86II-3 sodium salt of sulfate ester)
RPM containing 10% complement-depleted fetal bovine serum, 1001 U/mt' of penicillin and 1100 p/rnll of streptomycin supplemented with -8, as in
300,000 pieces/mj in 1164Q medium! The cells were seeded and cultured in a carbon dioxide gas incubator at 37°C. After 3 days, half of the culture was collected and the number of viable cells (X 10' /
ml) and viability (%) were determined by trypan blue staining. An equal volume of RPM11640 medium containing the same concentration of drug was added to each of the remaining cultured cells, culture was continued for an additional 3 days, and the number of viable cells and survival rate were measured in the same manner.

The results are shown in Table 1.

As is clear from this table, the control (drug concentration 0 μg/
ml), most of them die after 6 days due to virus multiplication, whereas with the addition of drugs, HTLV
-I [Cell death caused by type 1 virus: Keibo was completely prevented.

Test Example 2 Inhibitory effect of drugs on HIV antigen expression The cells measured in Test Example 1 were dried on a slide glass, fixed for 3 minutes with cold methanol, and then treated with human anti-HT diluted to 1/1000.
37°C with LV-1 positive serum (IF antibody titer X4096)
Processed for 30 minutes. Then, phosphate buffered saline (P
BS) for 15 minutes, and treated with fluorescein-isothiocyanate-conjugated anti-human IgG at 37°C for 30 minutes. After washing with PBS again, the number of cells emitting fluorescence, that is, expressing an HIV-specific viral antigen protein, was measured using a fluorescence microscope.

Similarly, NPF 861ASNPF-861B,
NPF 86I[A, NPF 86IIB, and N
A sulfuric acid ester of F-86I was also tested and the results were measured.

The results are shown in Table 2. 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 2, on the 6th day of infection, almost 100% of the cells in the drug-free control were positive for virus antigen, whereas in the drug-treated group, the expression of virus antigen was dependent on the concentration. significantly suppressed.

Furthermore, as is clear in the case of NF-86n-3, the antiretroviral effect was found to be greatly influenced by the sulfur content.

Similarly, NF-861S, NPF-86IA-3,
NPF 861 B 5SNPF-8611A-3
and 3 to 200μ for NPF-86IIB-3.
g/mj! When examined at the drug concentration of NF-8
Almost the same effect as 6n-3 was observed.

NF-8 is a lower molecule than the NF-86II group.
Group 61 has a slightly weaker effect.

Test Example 3 Assay of anti-HIV activity by plaque method 50μg/- on 35III plastic shear
17-L-'J gin solution l- was added dropwise and left at room temperature for 1 hour. J-L-! A petri dish coated with J-gin was prepared.

After washing each petri dish 3 times with PBS, 4 pieces of 150xIO/
- 1.5 mN of MT-4 cells were added and left at room temperature for 1 hour. After gently washing this with PBS twice, '1.
100 µl of a virus solution containing 000 PFU/- was gently added and left at room temperature for 1 hour to adsorb the virus.

Agarose overlay medium 1r111 containing drugs at various concentrations was added to each petri dish, and the mixture was incubated in a carbon dioxide ink inipator for 3 hours.
After culturing at 7°C for 3 days, 2x) 1 mj of agarose overlay medium containing Ral Red! layered. Each petri dish was further cultured at 37°C for 3 days, and the number of plaques was measured.

The results are shown in Table 3. The numbers in the table are the average number of plaques (
/dishfS, 口, n=3).

Table 3 ■) Toxicity test A) Effect on T cell proliferation In the same manner as in test example 1, HTLV-I [[B uninfected MT-
The effects of drugs on the proliferation of 4 cells were observed. The results are shown in Table 4. The numbers in the table are the number of living cells (X 10' /
mg). Although the case of NF-86-3 was shown here, other drugs also showed similar effects.

As is clear from Table 2, the effect on T cells was very weak. It was also found that the higher the sulfur content, the less the growth inhibitory effect.

On the other hand, it has already been reported that AZT at 5uM (1.3μg/ml) inhibits proliferation by about 25-50% compared to the control (Hideki Nakajima, Gann
, 78. 583 (1987)).

B) Acute toxicity An acute toxicity test was conducted using the sulfuric acid ester of the present invention by intraperitoneal administration to mice.

The results are shown in Table 5.

〔Example〕

Hereinafter, the present invention will be explained in more detail with reference to Examples, Reference Examples, and Formulation Examples.

Reference example 1 a) 100g of coarsely crushed and dried betel nut was mixed with 30g of hexane.
0- and left at room temperature for 24 hours, the hexane was removed by filtration. This operation was repeated three times to remove fat.

iv) The defatted betel nut was air-dried for 30 minutes.

c) Air-dried betel nut was immersed in methanol 300ml and extracted under boiling for 3 hours. This operation was performed three times and the extracts were collected.

2) The obtained extract was concentrated at 25° C. using an evaporator and dried under vacuum (yield: 6.86 g).

This and 150m of water! ! was added, stirred, and then filtered. The insoluble matter is an additional 100ml of water! was added and stirred, followed by filtration and the filtrate was collected.

e) 250-ethyl acetate was added to this aqueous solution for extraction. This operation was repeated three times to remove the ethyl acetate soluble portion. 1.85 g of insoluble matter remained, and 0.0 g of ethyl acetate extract remained.
61 g and 4.01 g of water extract were obtained.

F> The aqueous extract from which the non-hydrophilic organic solvent soluble portion was removed was placed in a membrane filter with a molecular weight cutoff of 1.000 (Spectra/Pore 6; manufactured by Spectrum Medical Industries), and dialyzed against water at 4°C. , and was fractionated into internal and external fluids.

g) The dialyzed fluid fractionated with a membrane filter with a molecular weight cut off of 1.000 was further put into a membrane filter with a cutoff molecular weight io, ooo (Spectra/Pore 6; manufactured by Spectrum Medical Industries), and then added to water. Te4
It was dialyzed at ℃ and fractionated into an internal solution and an external solution.

h) When fractionated in this way, the molecular weight was 1. OOO~
The target anticancer activity and 5'-nucleotidase inhibitory activity were found in the fractions of 10,000 and 10,000 or more, and both active substances could be obtained as light brown powders by freeze-drying. . Molecular weight 1.000-10.0
00 fraction (NF-861), 0.50 g can be obtained, and 50% for 5'-nucleotidase activity.
The inhibitory concentration was 124 ng/-. Molecular weight 10.0
00 or higher fraction (NF-86II), 0.75 g could be obtained and the 5'-nucleotidase activity
0% inhibitory concentration is 72ng/mj! Met.

Although we were able to obtain 2.76 g of both components with a molecular weight of 1.000 or less, the 5'-nucleotidase inhibitory activity was lower than that of NF-8.
It was very weak compared to 6I and NF-86fl.

Regarding the obtained NF-86I and NF-86II, 5
'-Nucleotidase inhibitory activity was assayed as follows.

As a substrate solution, 1.0% was added to 55mM Tris-HCl buffer (pH 8.5) containing 5.5ITIM magnesium chloride.
．． 1m! Adenosine monophosphate-sodium salt of J. (Manufactured by Sigma, Type If)
A solution of 10 mM sodium-potassium tartrate was used.

As the enzyme solution, 5'-nucleotidase derived from snake venom (manufactured by Sigma) was used.

The test was conducted as follows.

0.451nl of substrate solution and 10μ of enzyme solution! and 40 μl of the test sample were added and reacted for 20 minutes at 30°C in a hot bath. After the reaction was completed, 0.5rnI! The reaction was stopped by adding 10% trichloroacetic acid, and the resulting precipitate was centrifuged.

Take 0.5 ml of this supernatant, add 1.8 ml of 1% Triton 25μ11 distilled water and 0.251 nl of a 5N sulfuric acid aqueous solution containing 2.5% (W/V) ammonium molybdate, and after 20 minutes absorbance at 660 nm. Measured using

The results are shown in Table 6.

Table 6 In addition, the 5'-nucleotidase inhibitory activity of the extracts at each stage of the purification of the areca nut extract was assayed, and the results shown in Table 7 were obtained.

Table 7 [Reference Example 2] NPF-86I from NF-86I and NF-86II
A, NPF-861B, NPF-86IIA and N
Separation and purification of PF 86I[B was performed using high performance liquid chromatography. The conditions are as follows.

Separation column: Filled with adsorption/distribution resin (Sho
dex RS-packSDE-613: manufactured by Showa Denko) Eluent: Water: methanol = 1:9 Detector:
Ultraviolet spectroscopic detector (manufactured by JASCO Corporation) 280nm NF-86L 500mg to NPF-861A36.
7mg5NPF-861B244.2mg was obtained.

Also, from NF-86II 250mg, NPF-8
611A6 g, 8 mg, NPF-86II
68.0 mg of B was obtained.

Example I NF 861: 200 mg was suspended in 30 ml of anhydrous pyridine, 7.6 g of chlorosulfonic acid was added dropwise little by little under water cooling, and the suspension was stirred at room temperature for 2 days. Next, 80° of water was added to this under ice cooling, and after neutralization with saturated sodium bicarbonate, pyridine was removed with an equal amount of chloroform. This reaction solution was filtered through a membrane filter with a molecular weight cutoff of 1000 (
Spectra/Pore 6 (manufactured by Spectrum Medical Industries, Inc.) and dialyzed against water for 7 days, and the contents were freeze-dried to obtain 270 μl of the sodium salt NF-86I-3 of the sulfate ester of NF-86I. .

In the same manner, 254 ml of the sodium salt NF-86n-3 of its sulfuric acid ester was obtained from NF-86I.

Similarly, NPF-86IA, NPF-86IBSN
PF-86IIA and NPF-861 [B to sodium salt of their sulfate esters NPF-861A-3
, NPF-861B-3°NPF-86I[A-3 and NPF-86IIB-3 were obtained.

Next, the physical and chemical properties of NF-861-3 and NF-86It-3 will be shown.

(NF 861-3) (i) Shape: light yellowish brown powder (ii) Melting point: shows no clear melting point or decomposition point.

(iii) Elemental analysis: C32,0% H2,7% N005% 03g, 7% 86.5% Ash 15.4% (iv) Infrared absorption spectrum wax cm-'; 3436.2968.16
14.1519.1487.1440.1227.11
19.1048.832.749.707. 589.

(v) Solubility: Soluble in water, methanol, insoluble in hexane, ether, ethyl acetate, chloroform.

(vi) Stability: Stable in powder state and aqueous solution.

(NF-86n-3) (i) Shape: light yellowish brown powder (ii) Melting point: shows no clear melting point or decomposition point.

(iii) Elemental analysis: C23.2% H1.9% N O44% 0 34.8% 3 13.3% Ash 34.0% (iv) Infrared absorption spectrum: 1486.1440.1223.1048.859.7
49.588°(v) Solubility: Soluble in water, methanol, insoluble in hexane, ether, ethyl acetate, chloroform.

(vi) Stability: Stable in powder state and aqueous solution.

Formulation Example 1 (Injection/Drop) Add 5 g of powdered glucose to contain 500 mg of sulfuric acid ester or its salt, dispense into vials aseptically, seal, and fill with inert gas such as nitrogen or helium. Store in a cool, dark place. Before use, add 500 me of 0.85% physiological saline and administer as an intravenous injection once a day.
Administer 0 to 500 rnl by intravenous injection or drip depending on symptoms.

Formulation Example 2 (Injection/Drop) An intravenous injection for mild symptoms was prepared in the same manner as Formulation Example 1, except that 50 mg of the sulfuric acid ester or its salt was used.
Administer 10 to 500 rnl per day by intravenous injection or infusion depending on symptoms.

Formulation example 3 (injection, capsule) 30 mg of sulfate ester or its salt added to 1 g of refined sesame oil
and dissolved in 100 mg of aluminum stearate gel, sealed and sealed with an inert gas such as nitrogen or helium, and stored in a cool, dark place to prepare a preparation for subcutaneous injection. Depending on the symptoms, take 1 to 10 rn once a day! ! is administered by subcutaneous injection.

In addition, the above preparation is dispensed into capsules in 0.5-unit doses to prepare oral capsules, and 1 to 10 capsules are orally administered per day depending on the symptoms.

Formulation Example 4 (Enteric-coated tablet) Enteric-coated tablets for adults (A) and children (A) with the following ingredient composition.
b) 1,000 pieces of each were manufactured.

Lactose 99.4 49.7 Magnesium stearate [B] Cellulose acetate phthalate 2.0 1.0 6.0 (To) Take each component of hydroxypropyl cellulose [A] and mix well. This product was either directly pressurized or thoroughly kneaded, then granulated through the screen of an extrusion type granulator, and once thoroughly dried, the product was pressurized to produce tablets.

Next, the molded tablet is coated with a base material of well-dissolved CB) to form an enteric-coated tablet.

Regarding this tablet, please refer to the Japanese Pharmacopoeia (hereinafter referred to as "diurnal").

) Disintegration test method When an enteric-coated preparation was tested in artificial gastric fluid (pH 1, 2), it did not disintegrate even after shaking for 1 hour, and in the artificial intestinal fluid (pH 7, 5) test, it disintegrated in 5 to 6 minutes.

Formulation example 5 (enteric coated granules) Enteric-coated granules (1.000 g) were manufactured using the following ingredients.

(A) Base agent (sulfuric acid ester or its salt) 100 (1
Lactose 7
37BI Cellulose acetate phthalate 80 (after taking each component of the base [A], mixing them well, forming them into granules according to a conventional method, drying them well, sieving them, and passing them through pins, heat seal packaging, etc.) to make granules. The agent was manufactured.Next,
The granules are coated with a base material of dissolved CB while floating and flowing to form enteric-coated granules. When this granule was subjected to a disintegration test using a diurnal disintegration tester, it was found that the pH was 1,
It does not disintegrate even if it is shaken for 1 hour in the artificial gastric juice of 2. pl(7
, 5 disintegrated in 5 minutes.

Formulation Example 6 (Enteric-coated capsules) 1,000 enteric-coated capsules were manufactured using the following ingredients.

Lactose 24.6 74
．． 4 [B] Cellulose acetate phthalate 10 (g) 10
(g) Enteric-coated granules suitable for capsules were prepared using the above ingredients in the same manner as described in Formulation Example 5, and the composition was filled into capsules to obtain enteric-coated capsules.

When this capsule was subjected to a disintegration test using a diurnal disintegration tester, no disintegration or elution was observed even after shaking for 1 hour in artificial gastric fluid at pH 1 and 2, and no disintegration or elution was observed in artificial intestinal fluid at pt 17.5 for 5 minutes. Collapsed or the entire amount eluted.

[Brief explanation of the drawing]

FIG. 1 shows the infrared absorption spectrum of NPF-86IA. FIG. 2 shows the infrared absorption spectrum of NPF-861B. FIG. 3 shows the infrared absorption spectrum of NPF-86IIA. FIG. 4 shows the infrared absorption spectrum of NPF-86IIB. FIG. 5 shows the infrared absorption spectrum of NF-86I. FIG. 6 shows the infrared absorption spectrum of NF-86II. FIG. 7 shows the ultraviolet absorption spectrum of NF-861 using O, 1 normal hydrochloric acid and water solvents. FIG. 8 shows the ultraviolet absorption spectrum of NF-86n using 0.1N hydrochloric acid and water solvent. Figure 9 shows NF-86I's 0. The ultraviolet absorption spectrum using IN sodium hydroxide solvent is shown. Figure 10 shows NF-85■0. The ultraviolet absorption spectrum using IN sodium hydroxide solvent is shown. FIG. 11 shows high performance liquid chromatograms of NF-86I and NF-86II. FIG. 12 shows the infrared absorption spectrum of NF-86I-3. FIG. 13 shows the infrared absorption spectrum of NF-86I[-3. Wavelength (nm) Figure 11 (a) NF861 Yasui time (min) Yasu P! Time (minutes)

Claims (2)

[Claims] (1) NF-86 I, which has the following physical and chemical properties,
NF-86II, NPF-86 I A, NPF-86 I B
An antiretroviral agent containing as an active ingredient a substance selected from the group consisting of sulfate esters of NPF-86IIA and NPF-86IIB, and salts of the sulfate esters. (NF-86 I) (i) Shape: Light yellowish brown powder. (ii) Melting point: shows no clear melting point or decomposition point. (iii) Elemental analysis: Carbon 56.30% Hydrogen 4.61% Nitrogen 0.2% or less Ash 0.3% or less (iv) Molecular weight: 1,000-10,000 (by membrane filter) (v) Infrared absorption Spectrum: ν_m_a_xcm^-^1; 3430, 2940, 1
610, 1520, 1440, 1370, 1280, 1
110, 1060, 800 (vi) Ultraviolet absorption spectrum: Water 1% λ_m_a_xnm (E1cm) 279 (135.7)
Hydrochloric acid 1% λ_m_a_xnm (E1cm) 279 (134.5)
Sodium hydroxide 1% λ_m_a_xnm (E1cm) 290 shoulders (291.2), 420 shoulders (96.4), 500 shoulders (60.6) (vii) Solubility: Soluble in water, methanol, ethanol. Insoluble in hexane, ether, ethyl acetate, and chloroform. (viii) Color reaction: Ferric chloride reaction, ninhydrin reaction, negative p-anisidine-phthalic acid reaction, aniline-diphenylamine negative reaction, Dragendorff reaction negative (ix) Stability: Stable in powder form. (NF-86II) (i) Shape: Pale yellowish brown powder. (ii) Melting point: shows no clear melting point or decomposition point. (iii) Elemental analysis: Carbon 56.64% Hydrogen 4.59% Nitrogen 0.2% or less Ash 0.3% or less (iv) Molecular weight: 10,000 or more (by membrane filter) (v) Infrared absorption spectrum: ν ^K^B^r_m_a_xcm^-^1; 3400,
2940, 1610, 1520, 1450, 1370,
1290, 1110, 1060, 800, 500 (vi) Ultraviolet absorption spectrum: Water 1% λ_m_a_xnm (E1cm) 280 (145.3)
Hydrochloric acid 1% λ_m_a_xnm (E1cB) 279 (142.1)
Sodium hydroxide 1% λ_m_a_xnm (E1cm) 290 shoulders (306.1), 415 shoulders (100.0), 505 shoulders (61.2) (vii) Solubility: Soluble in water, methanol, ethanol. Insoluble in hexane, ether, ethyl acetate, and chloroform. (viii) Color reactions: Ferric chloride reaction, ninhydrin reaction, p-anisidine-phthalic acid reaction, aniline-diphenylamine reaction, negative Dragendorff reaction (ix) Stability: Stable in powder form. (NPF-86 IA) (i) Shape: Light yellowish brown powder. (ii) Melting point: shows no clear melting point or decomposition point. (iii) Elemental analysis: Carbon 54.82% Hydrogen 4.52% Oxygen 37.93% Nitrogen 0.2% or less Ash content 0.2% or less (iv) Molecular weight: 5,620 (gel penetration using polyethylene glycol as standard) (By chromatography.) (v) Infrared absorption spectrum: ν^K^B^r_m_a_xcm^-^1; 3400,
2940, 1610, 1520, 1440, 1380,
1280, 1260, 1210, 1160, 1110,
1060, 820, 800 (vi) Ultraviolet absorption spectrum: Water λ_m_a_xnm279 (vii) Solubility: Soluble in water, methanol, and ethanol. Insoluble in hexane, ether, ethyl acetate, and chloroform. (viii) Color reactions: Ferric chloride reaction, ninhydrin reaction, p-anisidine-phthalic acid reaction, aniline-diphenylamine reaction, negative Dragendorff reaction (ix) Stability: Stable in powder form. (NPF-86 I B) (i) Shape: Light yellowish brown powder. (ii) Melting point: shows no clear melting point or decomposition point. (iii) Elemental analysis: Carbon 57.09% Hydrogen 4.45% Oxygen 35.03% Nitrogen 0.2% or less Ash content 0.2% or less (iv) Molecular weight: 5,000 (gel with polyethylene glycol as standard) (By permeation chromatography.) (v) Infrared absorption spectrum: ν^K^B^r_m_a_xcm^-^1; 3400,
2930, 1610, 1520, 1440, 1360,
1280, 1250, 1200, 1160, 1110,
1060, (vi) Ultraviolet absorption spectrum: Water λ_m_a_xnm279 (vii) Solubility: Soluble in water, methanol, ethanol. Insoluble in hexane, ether, ethyl acetate, and chloroform. (viii) Color reactions: Ferric chloride reaction, ninhydrin reaction, p-anisidine-phthalic acid reaction, aniline-diphenylamine reaction, negative Dragendorff reaction (ix) Stability: Stable in powder form. (NPF-86IIA) (i) Shape: pale yellowish brown powder. (ii) Melting point: shows no clear melting point or decomposition point. (iii) Elemental analysis: Carbon 51.44% Hydrogen 4.44% Nitrogen 0.1% or less Ash 0.2% or less (iv) Molecular weight: 29,400 (by gel permeation chromatography using polyethylene glycol as the standard). ) (v) Infrared absorption spectrum: ν^K^B^r_m_a_xcm^-^1; 3400,
2950, 1610, 1520, 1440, 1370,
1280, 1250, 1210, 1160, 1110,
1060, 820, 800 (vi) Ultraviolet absorption spectrum: Water λ_m_a_xnm279 (vii) Solubility: Soluble in water, methanol, and ethanol. Insoluble in hexane, ether, ethyl acetate, and chloroform. (viii) Color reactions: Ferric chloride reaction, ninhydrin reaction, p-anisidine-phthalic acid reaction, aniline-diphenylamine reaction, negative Dragendorff reaction (ix) Stability: Stable in powder form. (NPF-86IIB) (i) Shape: Light yellowish brown powder. (ii) Melting point: shows no clear melting point or decomposition point. (iii) Elemental analysis: Carbon 52.46% Hydrogen 4.42% Nitrogen 0.1% or less Ash 0.2% or less (iv) Molecular weight: 8,610 (by gel permeation chromatography using polyethylene glycol as the standard). ) (v) Infrared absorption spectrum: ν_m_a_xcm^-^1; 3400, 2930, 1
610, 1520, 1440, 1370, 1280, 1
250, 1200, 1160, 1110, 1060, (
vi) Ultraviolet absorption spectrum: Water λ_m_a_xnm279 (vii) Solubility: Soluble in water, methanol, and ethanol. Insoluble in hexane, ether, ethyl acetate, and chloroform. (viii) Color reactions: Ferric chloride reaction, ninhydrin reaction, p-anisidine-phthalic acid reaction, aniline-diphenylamine reaction, negative Dragendorff reaction (ix) Stability: Stable in powder form. (2) An antiretroviral agent containing a sulfate ester of a hydrophilic solvent extract of areca nut or a salt of the sulfate ester as an active ingredient.