JPH04208264A - New alkaloid and glucosidase inhibitor comprising the same alkaloid as active ingredient - Google Patents

Abstract

NEW MATERIAL:An alkaloid shown by the formula (R1 to R3 are H or OH). EXAMPLE:2,4,5,6,7-Pentahydroxytetrahydrocyclopenta[6]pyrrolidine. USE:A glucosidase inhibitor. Usable as an antiviral agent, an immunomodulator, a cancer metastasis inhibitor, etc. PREPARATION:JIKOTSUPI, root skin of Lycium chinense MILL of the family Solanaceae, is extracted with water and/or an alcohol, a supernatant liquid obtained after removal of the extracted solution is precipitated by adding ethanol, the supernatant liquid is subjected to column chromatography or high- performance liquid chromatography for collection once or more times, eluted with water, ammonia, acetonitrile, methanol, acetone, etc., as an eluting solvent and fractioned while confirming the objective component by thin layer chromatography.

Description

DETAILED DESCRIPTION OF THE INVENTION "Industrial Application Field" The present invention relates to a luconodase inhibitor that is useful as a drug for immune disorders and the like.

U Prior Art and Problems] Glycoproteins, like proteins and nucleic acids, are widely distributed as biopolymers with extremely important functions regardless of biological species or organs. Glycoprotein sugar chains can be broadly classified into serine/threonine-linked and asparagine-linked types, and various enzymes are involved in the sugar chain biosynthesis process. α
-Glucondase can be mentioned.

Castanospermine is a known alpha-glucosidase I inhibitor that has been reported to inhibit the fusion of the AIDS virus into cells. It is thought that this is due to a change in the sugar chains of the protein.

On the other hand, it has recently become clear that sugar chains of glycoproteins are involved in various immune system reactions and cancer metastasis. It is believed that enzyme therapy can provide drugs for immune abnormalities, such as antiviral agents, immunomodulators, cancer metastasis inhibitors, etc., and the development of inhibitors of these enzymes has been desired.

[Means for Solving the Problems] In order to solve the above problems, the present inventors have conducted extensive research in search of a natural substance that has a gluconodase inhibitory effect, and as a result, they have discovered a herbal medicine that has been used in Chinese herbal medicine since ancient times. After discovering a new alkaloid, which has not been recorded in any literature, in a certain bone, and further examining the pharmacological action of this alkaloid, they discovered that it has an excellent gluconodase inhibitory action, and were able to complete the present invention. child.

That is, the present invention ('', an alkaloid (hereinafter referred to as a compound of the present invention) represented by the following formula (wherein R1, R2 and R3 are the same or different and each represents a hydrogen atom or a hydroxyl group), L It is a glucondase inhibitor containing this alkaloid as an active ingredient.

The compound of the present invention can be used, for example, in the Solanaceae family (Solanaceae).
Lycium chinens, a plant of e)
It can be obtained from the root bark (Nocoppi) of E. MILL by the following method.

That is, the bone skin is extracted with water, alcohol, or a mixed solvent of water and alcohol, and the supernatant obtained by removing the residue from the extract is precipitated by adding ethanol, and the supernatant is precipitated with diaion. Column chromatography using porous polymers such as I-(P-20), ion exchange resins such as Dowex-50 and Dowex-1, reversed-phase silica gel, polyamide, activated carbon, etc. as carriers. Graphy or hydroquinoapatite column, Asahi Bakku (8sal+i pak G5-22
0) At least once in preparative high performance liquid chromatography using ffP (=jL, water, ammonia,
Elute with one or more of acetonitrile, methanol, ethanol, chloroform, acetic acid, ethyl acetate, n-hexadi, acetone, and benzene as an elution solvent, and separate while checking the target components using thin-layer v1 chromatography. It can be obtained by drawing.

As the solvent for extraction, it is preferable to use deionized water, and as the alcohol, lower alcohols such as methanol may be used.It is recommended to heat the solvent to around the boiling point of the solvent used during extraction to improve extraction efficiency. From this point of view, it is appropriate, and if the extraction is carried out until the solvent used becomes 'F-m', it will be 51;

Furthermore, the compound of the present invention may exist as a salt due to its structure, and the present invention also includes normally pharmaceutically acceptable salts such as sulfate and hydrochloride.

Next, the fact that the compound of the present invention has a gluconodase inhibitory effect will be explained by showing experimental examples.

Experimental Example 1 The compound of the present invention obtained in the example below was added to a 0.05M sodium citrate buffer containing 5% methanol (pl-14,
5) to prepare sample solutions of various concentrations. Of these, 10 Iil was placed in a 96-well microplate, 50 enzymes and substrates were added thereto, incubated at 37°C for 15 minutes, and then added with reaction termination buffer (0.2M sodium borate buffer pH 9.8). ) was added to stop the reaction. As the enzyme, β-glucondase derived from jack bean was used in an amount of 0.025 units.

The enzyme activity was measured by using p-nitrofugo and nilu-β-D-gelcot as substrates dissolved in 005M sodium citrate buffer containing 5% methanol to give +tit/volume, and p-nitrofugo liberated by the reaction. Phenol was titrated using multi-scan, 405η〃l
Quantitative determination was made by measuring absorbance, and the inhibition rate was determined by the following formula.

The composition of the reaction solution for measuring Δ to D is summarized in Table 1.

Table 1 (however, B and D are 0.05% methanol-containing
Add 10 portions of M sodium citrate buffer. ) The results are shown in Table 2. Also, 50% of the compound of the present invention
The percent inhibitory concentration (ICAO) of compound Δ obtained in Example 1 was 6.5 ug/l11Q, and that of compound B obtained in Example 1 was 45 u9 bttQ.

As is clear from the above results in Table 2, the compounds of the present invention have excellent β-glucosidase inhibitory activity.

Next, the dosage and formulation of the compound of the present invention will be explained.

The compounds of the present invention can be administered to animals and humans either neat or with conventional pharmaceutical carriers. The dosage form is not particularly limited and may be selected and used as necessary, and may include oral preparations such as tablets, capsules, granules, fine granules, and powders, and parenteral preparations such as injections and planing preparations. It will be done.

In order to exert the desired effect as an oral agent, although it varies depending on the age, weight, and severity of the disease of the patient, it is usually necessary for an adult to administer 30M9 to 39 by weight of the compound of the present invention several times a day (J It seems appropriate to take this drug.

Oral agents include, for example, starch, lactose, sucrose, mannitol,
It is produced using carboquine methylcellulose, cornstarch, inorganic salts, etc. according to conventional methods.

In addition to the excipients mentioned above, binders, disintegrants, surfactants, lubricants, fluidity promoters, flavoring agents, coloring agents, fragrances, and the like may be used in this type of preparation as appropriate. Specific examples of each are shown below.

[Binder] Starch, dextrin, gum arabic powder, gelatin,
Hydroxypropylstarch, methylcellulose, sodium carboxymethylcellulose, hydroquinepropylcellulose, crystalline cellulose, nityl cellulose,
Polyvinyl vinyl [1 lydon, macrogol.

[Disintegrant co-starch, human loxopropyl starch, sodium carboxymethyl cellulose, carboquine methyl cellulose, carboquine methyl cellulose, low substituted hydroquine propyl cellulose.

[Surfactant] Sodium lauryl sulfate, soybean lecithin, sucrose fatty acid ester, polysorbate 80° [Lubricant] Talc, waxes, hydrogenated plant ura, sucrose fatty acid ester, magnesium stearate, carnoum stearate, stearic acid Aluminum, polyethylene glycol.

[Fluidity promoter] Light anhydrous silicic acid, dry aluminum hydroxide gel, synthetic aluminum silicate, magnesium silicate.

The compounds of the invention can also be administered as suspension solutions, emulsions, syrups, elixirs,
These various net shapes may contain flavoring agents and coloring agents.

In order to exert the desired effect as a parenteral agent, although it varies depending on the age, weight, and severity of the disease of the patient, the compound of the present invention is usually administered by intravenous injection or infusion for adults at a dose of 11"01 to 500179% by weight. Intravenous, subcutaneous, and intramuscular injections are considered appropriate.

This parenteral drug is manufactured according to a conventional method, and the diluents generally include distilled water for injection, physiological saline, aqueous glucose solution, vegetable oil for injection, sesame 41+, peanut oil, soybean ura, corn ura, and 7-pyrene glycol. , polyethylene glycol, etc. can be used. Furthermore, a bactericide, a preservative, and a stabilizer may be added as necessary. In addition, from the viewpoint of stability, this parenteral preparation may be filled into a vial or the like, then frozen, water removed by ordinary freeze-drying techniques, and a liquid preparation prepared from the freeze-dried product immediately before use. Furthermore, isotonizing agents, stabilizers, preservatives, soothing agents, etc. may be added as appropriate.

Other parenteral preparations include external solutions, liniments such as ointments, and flat preparations for intrarectal administration, and are manufactured according to conventional methods.

Next, the present invention will be explained in more detail with reference to Examples.
The present invention is not limited in any way by this.

Example 1 500 g of bone skin was put into 10 ρ of deionized water, extracted by heating at 100°C for 2 hours, and after filtration, 4 times the amount of ethanol was added to the solution to precipitate and remove other impurities. . The first supernatant was subjected to column chromatography using Diaion I-IP-20 to obtain two fractions eluted with deionized water. This water-eluted fraction was subjected to column chromatography using a cation exchange resin Dowex-50 and then an anion exchange resin Dowex-50 to obtain a deionized water-eluted fraction. Both fractions were again subjected to column chromatography using Thai Ion 1 (P-20) and eluted with 1M aqueous ammonia to obtain a fraction containing the compound of the present invention. Column, then Asahi pak
) To perform high performance liquid chromatography using a GS-220 column, Nisu, colorless crystals +5xg (referred to as compound A) and colorless crystals 65~ (referred to as compound B)
I got it.

Compound A has the physical and chemical properties shown below,
From these data the formula + 141 It, and F?
,.

is a hydroxyl group and R3 is a hydrogen atom 2, 4.5
.

6.7-pentahydrogine, tetrahydrone clopenta[6]pyrrolidine (2,4,5,6,7-pentah
ydroxy.

tetrahydrocyclopenta[6]p
yrolidine).

Specific optical rotation: [α32; x-+ 28.7° Melting point: 185
~190°C Solubility: Soluble in water, aqueous methanol, insoluble in methanol, chloroform Mass spectrum FAI3-MS: m/z I 9 2 (M -
1-1)'.

237 (M+Na)゛Ultraviolet absorption spectrum λ::':” (71〃I) Terminal absorption infrared absorption spectrum ν Ken12:Xα-1
・3250 (-01-I), '341'o (-01
-() Proton nuclear magnetic resonance spectrum (δ ppm In D20) 1.35 (I H, dd,, J = I 4.0, 1.5
l-1z).

2.4 0(II-1, dd, J=14.
0.7.0Hz).

3.02 (I H, dd, J = 9.0.9.01
(z).

3.06 (I I-I, d,, 1 = 4.5 H
z).

3 22 (IH, dd, J=8.5, 1.5Hz).

3.40 (I T-r, dd, '9.0.4.5
T-Tz).

4.15(I I-[, dd, 7.0, 3. Or-1z
) 13cm nuclear magnetic resonance spectrum (δ ppm in D20): 42.0, 65.8, 70.0, 73.8° 76.1.
77.7,92.0 Compound B has the physical and chemical properties shown below,
These data indicate that in formula I, R1 and R9 are hydrogen atoms, and R3 is a hydroxyl group.

7-Titrahydrogysi, Tetrahydrone clopenta [6
] Pyrrolidine (2,3,6,7-tetrahydro
xy.

tetrahydrocyclopenta[6]p
yrolidine).

Solubility: Soluble in water, aqueous methanol, insoluble in methanol, chloroporm Mass spectrum FΔB-MS: m/z I 76 (M+H)".

Proton nuclear magnetic resonance spectrum (δ ppm in 1) po): 1.44 (I Hlm), l, 66 (I H, m)
.

1.85 (I Hlm), 1.91 (l I (1m
).

3.21 (I H, dd), 3.25 (I ) (,
, :l).

3.3 2(I H,d), 3.4 7(I
I-1, dd) 13C-nuclear magnetic resonance spectrum (δ ppm in D20): 23.2, 30.3.57.3, 76.4.

79.2.91.9 Example 3 ■Corn starch 449 ■Crystalline cellulose 409 ■Carboxymethyl cellulose carnoum 59 ■Light silicic anhydride 0.5 g ■Magnenoum stearate 0.59 Total 100 g According to the above recipe, The mixture was mixed uniformly and compressed using a tablet machine to obtain 200 tf tablets.

Tablets - The tablets contain the compound Δ20Mg, and adults should take 5 to 15 tablets a day in several doses.

15-Example 4 ■Crystalline cellulose 84.59■Magnesium stearate O,5! il■Carboquinedyl Cellulose Calsoum 59 Total 100g According to the recipe in 1, mix ■, ■, and part of ■ uniformly, compression mold, crush, add the remaining amount of ■ and ■, and mix. A portion of the mixture was compressed and molded using a tablet machine to obtain 200 tablets.

This tablet contains the compound 1320111? Adults should take 5 to 15 tablets several times a day.

Example 5 ■ Crystalline cellulose 34.59 ■ 1O% hydroquinopropyl cellulose ethanol solution 509 ■ Carpoxymethyl cellulose 1159 ■ Magnesium stearate o, 59 ■ Compound A
+ o9 total 100@ According to the above recipe, mix ■, ■, and ■ uniformly, and then neutralize it by a conventional method, granulate it with an extrusion granulator, dry and crush it, mix ■ and ■, and pound it. A portion of the mixture was compressed using a tablet machine to obtain 200 mg tablets.

This tablet-tablet contains the compound A 2 Off9, and adults take 5 to 15 tablets a day in several doses.

Example 6 ■Corn starch 84g ■Magnesium stearate 05g ■Carpoxymedimethylulose calcium 5g ■Light silicic anhydride 059 ■Compound 8 109 Total 100y Mix ■~■ uniformly according to the above recipe and compress with a compression molding machine After molding, the mixture was crushed using a crusher and sieved to obtain granules.

This granule 19 contains compound BIO (1 g), and adults should take 1 to 3 g in several doses per day.

Example 7 ■Crystalline cellulose 559 ■10% hydroquinepropylcellulose ethanol solution 35g ■Compound A, 10g total
1009'' ① to ② were uniformly mixed and made into a paste according to the recipe in 1. After going into an extrusion granulator and granulating, the mixture was dried and sieved to obtain granules.

This granule 19 contains Compound A 10071@, and is taken by adults at 1 to 3 g per day in several doses.

Example 8 ■Corn starch 89.57■Light silicic anhydride 0.5g■Compound B
101/total 100g'' Mix ■～■ uniformly according to the recipe in 1, and add 20 O
ff9 was filled into a No. 2 capsule.

One capsule of this preparation contains compound B20π9, and adults should take 5 to 15 capsules a day in several doses.

Example 9 ■ Soybean oil 59 ■ Distilled water for injection 895 g ■ Soybean phospholipid
2.59■Glycerin 2g■Compound A 1g total
100g'' ■ was dissolved in ■ and ■ according to the recipe in 1, and the solutions of ■ and ■ were added thereto to emulsify to obtain an injection.

Patent applicant: Kitasato Research Institute (incorporated association)

Claims (2)

[Claims] (1) An alkaloid represented by the following formula I ▲There are mathematical formulas, chemical formulas, tables, etc.▼ I (In the formula, R_1, R_2 and R_3 are the same or different and represent a hydrogen atom or a hydroxyl group.) (2) The alkaloid represented by the following formula I ▲There are mathematical formulas, chemical formulas, tables, etc.▼ I (In the formula, R_1, R_2 and R_3 are the same or different and each represents a hydrogen atom or a hydroxyl group) as an active ingredient. glucosidase inhibitor.