RU2051900C1 - 5-amino-1-hydroxymethylcyclopentane-1,2,3,4-tetraol, 2- amino-4-hydroxymethyl-3a,5,6,6a-tetrahydro-4h-cyclopent- [d]-oxazole-4,5,6-triol, pharmaceutically acceptable salts thereof, and process for preparing thereof - Google Patents

Abstract

FIELD: used as biologically aceptable active materials. SUBSTANCE: the product 1: 5-amino-1- hydroxymethylcyclopentane-1,2,3,4-tetrazole of the formula: [α] $\genfrac{}{}{0ex}{}{\text{25}}{\text{D}}$ = -37^°. Reagent 1: trehazoline. Reagent 2: 4N aqueous HCl solution. Reaction conditions: at 100 C for 20 to 24 hr. Product 2: 2-amino-4-hydroxymethyl- 3a,5,6,6a-tetrahydro-4H-cyclopent [d]-oxazole-4,5,6-triol of formula: [α] $\genfrac{}{}{0ex}{}{\text{25}}{\text{D}}$ = +10^°. Reagent 1: trehazoline. Reagent 2: 0.2 N aqueous HCl solution. Reaction conditions at 100 C for 6 hr. EFFECT: improved properties of the title compounds. 8 cl, 1 dwg, 1 tbl

Description

 The invention provides two new polyoxycyclopentane derivatives that have certain valuable biological activities, and provides methods for preparing these compounds, as well as methods and compositions using them for therapeutic and prophylactic purposes.

 The compounds of the invention are 5-amino-1- (oxymethyl) -cyclopentane-1,2,3,4-tetraol and 2-amino-4- (oxymethyl) -3a, 5,6,6a-tetrahydro-4H-cyclopent- [d] -ok-sazole-4,5,6-triol, the formulas of which (I) and (II), respectively, are presented below. These compounds can be prepared by fermentation or cleavage of a compound known as triazolin, which can be represented by formula (III) shown below. However, 2-amino-4- (oxymethyl) -3a, 5,6,6a-tetrahydro-4H-cyclopent- [d] oxazole-4,5,6-triol and triazoline both undergo tautomers, and therefore they can be represented also the formulas (IIa) and (IIIa) shown in the drawing.

 It is believed that triazolin may be the same compound as the named "trialostatin" in the International Patent Cooperation Treaty (PTI) N W 0 90/10010.

 The compounds of the invention have the ability to inhibit the activity of various sugar hydrolases, and in particular β-glucosidase and β-fructofuranosidase (invertase sucrase).

 Compounds with strong inhibitory activity against β-glucosidase, such as castanospermine and deoxynojirimycin, have been reported to be useful as antidiabetic agents and as an anti-AIDS agent (Acquired Immune Deficiency Syndrome) [RAGruters et al, Nature, 330, 74-77 (1987); M.S. Humphiries et al Gancer Res, 46, 5215-5222 (1986)] It is therefore believed that compounds having the ability to inhibit β-glucosidase activity will be useful as antidiabetic or anti-AIDS agents.

 In addition, compounds with strong inhibitory activity against β-fructofuranosidase, such as AO-128 and Acarbose, have been reported to be useful as antidiabetic agents and as anti-obesity agents [Satoshi Horii et al. Journal of Medicinal Chemistry, 29, 1038-1046 (1986); T. Aida et al, Journal of the Japanese Society of Food and Nutrition, 34, (2) 134-139 (1981)] It is therefore believed that compounds having the ability to inhibit β-fructofuranosidase activity will be useful in the treatment and prevention of diabetes and obesity. .

Compounds having a certain structural similarity with the compounds of the invention are:
mannostatins described, among other things, by T. Aoyagi and others [T. Aoyagi et al, The Journal of Antibiotics, Vol. XLII N 6. 883 (1989)] which, as said, have the ability to inhibit the activity of α-D-mannosidase;
(1S, 2R, 3S, 4R, 5R) -Metyl- [2,3,4-trioxy-5- (oxymethyl) -cyclopentyl] -amine, described, inter alia, by Farrom and others [RAFarr et al, Tetrahedrom Letters, 31, 7109 (1990)] which, as said, also has the ability to inhibit the activity of α-mannosidase;
allosamidine, described, among other things, by Sakuda and others [S. Sakuda et al, Tetrahedron Letters, 27, 2475 (1986)] which is said to have the ability to inhibit insect chitinase activity;
and Kifunensin, described, among other things, by Kayakiri and others [H. Kayakiri et al, J. Org, Chem, 54, 4015 (1989)], which is said to be an immunomodulator with the ability to inhibit α-mannosidase activity.

 The aim of the invention is the provision of new compounds with inhibitory activity against certain sugar hydrolases.

 An additional objective of the invention is the provision of compounds having such activity, which therefore is supposed to be useful in the treatment and prevention of tumors (neoplasms), AIDS, obesity and diabetes.

 Other objectives and advantages will become apparent as the description of the invention.

 The compounds of the invention are 5-amino-1- (oxymethyl) -cyclopentane-1,2,3,4-tetraol, which has the formula (I) above, and 2-amino-4- (oxymethyl) -3a, 5 , 6,6a-tetrahydro-4H-cyclopent- [d] -oxazole-4,5,6-triol, which has the formula (II) above.

 5-amino-1- (oxymethyl) -cyclopentane-1,2,3,4-tetraol can be obtained by hydrolysis of triazoline or 2-amino-4- (oxymethyl) -3a, 5,6,6a-tetrahydro-4H-cyclopent - [d] -oxazole-4,5,6-triol, which is described in more detail below. 2-amino-4- (oxymethyl) -3a, 5,6,6a-tetrahydro-4H-cyclopent- [d] -oxazole-4,5,6-triol can be obtained by hydrolysis of triazoline or fermentation using a microorganism of the genus Micromonospore or Amikolatopsis.

 An example of a triazoline-producing microorganism of the genus Micromonospore is Micromonospore of the form SANK62390.

 An example of a triazoline-producing microorganism of the genus Amikolatopsis is Amikolatopsis of the form SANK60791.

 Both of these microorganisms are newly isolated strains.

 5-amino-1- (oxymethyl) -cyclopentane-1,2,3,4-tetraol, compound (I) of the present invention, and 2-amino-4- (oxymethyl) -3a, 5,6,6a-tetrahydro- 4H-cyclopent- [d] -ok-sazole -4,5,6-triol, the compound (II) according to the invention, can be obtained by hydrolysis of triazoline, obtained as described above, by culturing a triazoline-producing microorganism.

 This reaction is mainly carried out in the presence of acid and should be carried out in the presence of water. There is no particular restriction on the type of acid used, and in this case, any acid commonly used for conventional hydrolysis reactions may be equally applicable. Examples include inorganic salts such as hydrochloric or sulfuric acid, and organic acids such as acetic and propionic acid in an aqueous solution. The preferred acid is hydrochloric acid.

The reaction can take place over a wide temperature range and the precise reaction temperature is not a factor in the invention. In general, we find it convenient to carry out the reaction at a temperature from about room temperature to ^about 120 C, more preferably from 90 to 100 ^C. The time required for the reaction may also vary widely, depending on many factors, notably the reaction temperature and the nature of the reagents and solvents used, and, as explained below, from the target reaction product.

 They must pay tribute to the fact that both 5-amino-1- (oxymethyl) -cyclopentane-1,2,3,4-tetraol and 2-amino-4- (oxymethyl) -3a, 5,6,6a-tetrahydro 4H-cyclopent- [d] -oxazole-4,5,6-triol are obtained from the same starting material using the same hydrolysis reaction. Thus, the reaction product will contain a mixture of two compounds. However, it is possible to promote the production of one of these compounds more than the other by appropriate selection of reaction conditions. So, in most cases, milder conditions favor the production of 2-amino-4- (hydroxymethyl) -3a, 5,6,6a-tetrahydro-4H-cyclopent- [d] oxazole-4,5,6-triol, while as more stringent conditions contribute to the production of 5-amino-1- (oxymethyl) -cyclopent-1,2,3,4-tetraol.

 Thus, for the production of 2-amino-4- (oxymethyl) -3a, 5,6,6a-tetrahydro-4H-cyclopent- [d] oxazole-4,5,6-triol, we prefer to use a relatively milder acid reagent , for example 0.2 N. aqueous hydrochloric acid, and the reactions mainly allow to proceed over a period of time from 1 hour to 2 days, more preferably from 5 to 6 hours

 On the other hand, in order to facilitate the reaction to proceed in all ways to produce 5-amino-1- (hydroxymethyl) -cyclopentane-1,2,3,4-tetraol, a stronger acid, such as 4 N. aqueous acid, is preferred, and the reactions are preferably allowed to proceed for a period of time from 1 hour to 2 days, more preferably from 20 to 24 hours

 Additionally, 5-amino-1- (oxymethyl) -cyclopentane-1,2,3,4-tetraol can be obtained by hydrolysis of 2-amino-4- (oxymethyl) -3a, 5,6,6a-tetrahydro-4H- cyclopent- [d] oxazole-4,5,6-triol. In this case, the hydrolysis reaction of the invention can be carried out in the presence of an acid or a base. There is no particular restriction on the type of acid or base used, in which case any acid or any base commonly used in hydrolysis reactions can be used equally.

 When an acid is used for the reaction, examples of suitable acids include: inorganic acids such as hydrohalic acids (eg hydrochloric acid, hydrobromic acid or hydroiodic acid), sulfuric, perchloric, phosphoric and nitric acids; organic carboxylic acids, such as lower alkanoic acids (for example, formic, acetic, oxalic or trifluoroacetic acids); lower alkanesulfonic acids (e.g. methanesulfonic, ethanesulfonic and trifluoromethanesulfonic acids); and arylsulfonic acids (e.g., benzenesulfonic or para-toluenesulfonic acids, etc.). Inorganic acids, in particular hydrochloric acid, are preferred acids.

 The reaction is preferably carried out in the presence of a solvent. The reaction is usually preferably carried out in the presence of a solvent. There is no particular restriction on the nature (type) of the solvent used, provided that it does not adversely affect the reaction or the reagents involved in the reaction and that it can dissolve the reagents, at least to some extent. Examples of suitable solvents include alcohols such as methanol, ethanol, propanol or butanol; sulfonic acids such as dimethyl sulfoxide or sulfolane; organic acids, especially fatty acids, such as acetic or propionic acid and water. Preferred solvents include water and mixtures of water and an organic solvent.

 The amount of acid used is usually and advantageously 1 to 20 moles, more preferably 5 to 10 moles, per mole of the starting compound.

The reaction can take place over a wide range of temperatures and the precise (defined) reaction temperature is not critical to the invention. It found it convenient to carry out the reaction at a temperature from about room temperature to ^about 150 C, more preferably from 90 to 110 ^C. The time required for the reaction may also vary widely, depending on many factors, notably the reaction temperature and the nature of the reagents and solvent used. However, provided that the reaction is effected under the described preferred conditions, a period of from 1 hour to 2 days, more preferably from 20 to 30 hours, will usually suffice.

 When a base is used for the reaction, examples of suitable bases include: inorganic bases such as alkali metal hydroxides (eg lithium, sodium or potassium hydroxide); alkaline earth metal hydroxides (e.g., calcium or barium hydroxide); alkali metal carbonates (e.g. sodium or potassium carbonate); and alkali metal halides (e.g. sodium iodide, sodium bromide or potassium iodide); and organic bases such as ammonia, alkyl amines (e.g. triotylamine); and heterocyclic amines (e.g., morpholine, N-ethylpiperidine or pyridine).

 The reaction is preferably carried out in the presence of a solvent. The reaction is usually and mainly carried out in the presence of a solvent. There is no particular restriction on the nature of the solvent used, provided that it does not adversely affect the reaction or the reagents involved in the reaction and that it can dissolve the reagents, at least to some extent. Examples of suitable solvents include: alcohols, such as methanol, ethanol, propanol or butanol; ethers such as tetrahydrofuran or dioxane; sulfoxides, such as dimethyl sulfoxide or sulfolane, and water. Preferred solvents include water and mixtures of water and an organic solvent.

 The amount of base to be added is usually and advantageously from 0.01 to 10 moles, more preferably from 1 to 5 moles, per mole of the starting compound.

The reaction can take place over a wide range of temperatures and the precise (defined) reaction temperature is not critical to the invention. In general, we find it convenient to carry out the reaction at a temperature of from 0 to ¹²⁰ ° C, more preferably from about room temperature to ^about 100 C. The time required for the reaction may also vary widely, depending on many factors, notably the reaction temperature and the nature of the reagents and the solvent used. However, provided that the reaction is effected under the described preferred conditions, a period of from 0.5 hours to 2 days, more preferably from 1 to 20 hours, is usually sufficient.

 The target compounds of formulas (I) and (II) can be isolated from the reaction mixture by a conventional method commonly used to separate and isolate an organic compound, for example, by various chromatographic methods, namely column chromatography or preparative thin layer chromatography. A variety of choices are described in connection with the separation and purification of triazoline and 2-amino-4- (oxymethyl) -3a, 5,6,6a-tetrahydro-4H-cyclopent- [d] oxazole-4,5,6-triol, when they are obtained by fermentation, and these selection paths can also be used for the separation and purification of 5-amino-1- (oxymethyl) -cyclopentane-1,2,3,4-tetraol and 2-amino-4- (oxymethyl) -3a, 5,6,6a-tetrahydro-4H-cyclopent- [d] oxazole-4,5,6-triol obtained, as described above, by hydrolysis.

 The compounds of the present invention, 5-amino-1- (oxymethyl) -3a, 5,6,6a-tetrahydro-4H-cyclopent- [d] -oxazole-4,5,6-triol, each of which contains at least one the main nitrogen atom and can thus form salts by the addition of acids. There is no particular restriction on the nature (type) of these salts, provided that when they are intended for therapeutic purposes, they are pharmaceutically acceptable. When they are not intended for therapeutic applications, for example, as intermediates in the preparation of other, and possibly more active, compounds, then this limitation does not apply. Examples of such acid addition salts include: salts with inorganic acids, especially with hydrohalic acids (such as hydrofluoric acid, hydrobromic acid, hydroiodic acid or hydrochloric acid), perchloric, nitric, carbonic, sulfuric or phosphoric acids; salts with lower alkyl sulfonic acids such as methanesulfonic, trifluoromethanesulfonic or ethanesulfonic acids; salts with arylsulfonic acids such as benzenesulfonic or para-toluenesulfonic acids; salts with organic carboxylic acids, such as acetic, fumaric, tartaric, oxalic, maleic, malic, succinic or citric acids; and salts with amino acids such as clutamic acid or aspartic acid.

 The compounds of the invention necessarily contain several asymmetric carbon atoms in their molecules and, thus, can form optical isomers. Although they are all represented in the description by a single molecular formula, the invention includes both single, isolated isomers and mixtures, including their racemates. When stereospecific synthesis methods are used or optically active compounds are used as starting materials, individual isomers can be obtained directly; on the other hand, if a mixture of isomers is obtained, individual isomers can be obtained by conventional separation methods.

 5-amino-1- (oxymethyl) -cyclopentane-1,2,3,4-tetraol has the ability to inhibit β-glucosidase activity and has low toxicity, and therefore can be expected to be useful in the treatment and prevention of tumors and AIDS. 2-amino-4- (oxymethyl) -3a, 5,6,6a-tetrahydro-4H-cyclopent- [d] oxazole-4,5,6-triol has the ability to inhibit β-fructofuranosidase activity and has low toxicity, and therefore can be expected to be useful in the treatment and prevention of diabetes and obesity.

 When these compounds are intended for therapeutic use, they can be administered separately or in a suitable pharmaceutical form containing, in addition to the active compound, one or more conventional diluents, carriers or excipients. The type of dosage form depends on the proposed method of use. However, in the case of an oral route of administration, the compound is advantageously included in the dosage form in the form of powders, granules, tablets or capsules. For parenteral use, it is preferably included in the form of an injection. These drugs can be prepared by a known method by adding additives such as fillers, binders, disintegrants (substances that cause decomposition-fragmentation of the dosage form), lubricants, stabilizers and modifiers of drug substances. Although the dosage may vary depending on the symptoms and age of the patient, the nature and severity of the disease or disorder, and the method and method of administration, when administered orally to an adult human patient, the compounds of the present invention can usually be administered at a daily dose of 1-1000 mg. The compounds can be used in a single dose, or in divided doses, for example, two or three times a day.

 PRI me R 1. Obtaining triazoline.

 1 (A) Cultivation.

A full loop (100pfu1) SANK 62 390 micromonospores are used to inoculate (inoculate) each of three 500 ml Erlenmeyer flasks and equipped with all baffle plates (plates deflecting fluid flow) and each containing 80 ml of Medium 1 (having the composition below) and the inoculated flasks were incubated at ²⁸ ° C for 216 hours on a rotary vibrating conveyor, rotating at a speed of 220 rev / min, to obtain a first seed culture.

Wednesday 1, Glucose 1 Glycerin 1 Oatmeal 0.5 Sucrose 1 Soybean flour 2 Casamino acids 0.5 Pressed yeast 1 CaCO ₃ 0.1 CB 442 0.01 Water Up to 100
(before sterilization, pH 7.0)
Interest is massive, based on the final volume of the medium.

Each of four 2 liter Erlenmeyer flasks each containing 800 ml of medium 2 (having the following composition) was inoculated with 40 ml culture broth from the first flasks, and the flasks shaken at 28 ^{° C} for 96 hours on a rotary vibrating conveyor at a rate of 220 rpm to get a second seed crop. 1.5 liter of this second seed culture broth was used to inoculate each of two 30-liter fermentors between vibrating containing each 15 liters of medium 2 (having the composition described below), which is first sterilized at ¹²⁰ ° C for 35 min, and cooled to 28 ^C. The fermenters were stirred at a speed of 100 rev / min at ²⁸ ° C for 96 hours with aeration at an air flow of 15 l / min.

Wednesday 2, Glucose 2 Soluble starch 1 Pressed yeast 0.9 Meat extract (Kyokuto) 0.5 Polypenton 0.5 NaCl 0.5 CaCO ₃ 0.3 CB 442 0.01 Water Up to 100
(before sterilization, pH 7.2)
The percentages are by weight based on the final volume of the medium.

(B) Isolation
3 kg of Celite 545 (brand name for Johns Menville Products Corp. products), as a filtration aid, was added to 30 L of the total broth obtained by the described method, and the mixture was filtered. The filtrate (29 L) was passed through a column containing 6 L of Dowex SBR-P (C1- ^I ) (trademark for Dow Chemical products). The pH of the eluate was then adjusted to a value of 5.0, after which the solution was passed through a column containing 6 L of Dowex 50 WX 4 (H ⁺ ) (trademark for Dow Chemical products). Triazolin adsorbs and is thus cured by a column. The column is washed with 20 L of deionized water, and then 30 L of 0.5 N aqueous ammonia is eluted to obtain 13 L of fractions with the active compound. The whole of this eluate (13 liters) was concentrated by evaporation under reduced pressure, and lyophilized (dried at a temperature below 0 ° ^C) to obtain 43.4 g of a crude powder containing trehazolin. The crude powder is dissolved in 2 L of a 100 mM ammonium formate buffer solution (pH 6.0) and the solution is absorbed on a column containing 1.5 L of Dowex 50 WX 4 (brand), which is pre-equilibrated with a 20 mM ammonium formate buffer solution ( pH 6.0). The column is washed with 3 L of the same ammonium formate buffer solution (20 mM), followed by washing with 2 L of deionized water, after which the column is eluted with 0.2 N aqueous ammonia. The eluate is fractionated in portions with a volume of 500 ml. Fractions 2-4, which contain virtually all of the triazoline, are combined and concentrated by evaporation under reduced pressure. The reaction product is then lyophilized to give 2.55 g of a crude powder. The crude powder obtained by repeating the steps up to this point is used directly, without any further purification, in Example 3.

This crude powder is absorbed on a column packed with 200 ml Avicel (brand name for Asahi Chemical Industry X. Ltd.) using 80 vol. aqueous acetonitrile. The column is washed with 700 ml of 80 vol. aqueous acetonitrile, and then elute first with 500 ml of 75 vol. aqueous acetonitrile, and then with 700 ml of 70 vol. aqueous acetonitrile. The eluate is fractionated in parts with a volume of 19 ml. Fractions 35-50 containing triazoline are combined and concentrated by evaporation under reduced pressure. The residue was then lyophilized to give 658 mg of powder. All this powder is dissolved in 150 ml of water and the pH of the resulting solution is adjusted to a value of 6.0. Then the solution is absorbed on a column packed with 300 ml of Amberlite CG-50 (H ⁺ NH $\genfrac{}{}{0ex}{}{\text{+}}{\text{4}}$ ) 2: 3) trademark. This column is washed with 500 ml of deionized water and eluted with 0.1 N aqueous ammonia. The eluate is fractionated in portions in 20 ml. Fractions 92-121 containing triazoline are combined and concentrated by evaporation under reduced pressure. The resulting residue was then lyophilized to give 102.8 mg of powder. All this powder is dissolved in 10 ml of a 2 mM ammonium formate buffer solution (pH 6.0) and the resulting solution is absorbed on a column packed with 400 ml of Dayon CHP 20 P (trade name for Mitsubishi Kasei Co. products) using the same 2 mM ammonium formate buffer solution. The column is eluted with the same 2 mM ammonium formate buffer solution. The eluate is fractionated in portions of 5 ml. Fractions 59-73 containing triazoline are combined and concentrated by evaporation under reduced pressure. The resulting residue was then lyophilized to obtain 18 mg of powder, which was further purified by elution through Dayon CHP 20 P using the same 2 mM ammonium formate buffer solution as eluent to obtain 6.2 mg of a colorless powder.

 This powder is purified using preparative thin layer chromatography as follows. The powder (6.2 mg) is dissolved in a small amount of water and applied to 3 plates with silica gel (Merck Art 5 715, 20x20 cm). The plates are developed using a mixture of acetonitrile, acetic acid and water 6: 1: 3 in a volume ratio up to a height of 15 cm. The strip between Rf 0.42 and 0.5 is scraped from the plates and filled into a column. The column is eluted with 100 ml of deionized water. The eluate is passed through a column containing 5 ml of Dowex 50 WX4 (H +), where triazoline is absorbed and thus retained. The column is washed with deionized water and then eluted with 50 ml of 0.5 N aqueous ammonia. The eluate was concentrated by evaporation under reduced pressure, and the resulting residue was lyophilized to obtain 5.1 mg of triazoline as a colorless powder, for which high-performance liquid chromatography showed a single peak.

The reaction product has the following properties:
1) Main properties and appearance: basic colorless powder;
2) Solubility: soluble in water and methanol, insoluble in acetone and chloroform;
3) Color reaction: positive for sulfuric acid;
4) Molecular formula: C ₁₃ H ₂₂ N ₂ O ₁₀ ;
5) Molecular mass: 366 (determined by FAB mass spectrometry “FAB” means Fast Bombing of Atoms “BAB”);
6) Specific rotation: [α] $\genfrac{}{}{0ex}{}{\text{25}}{\text{D}}$ + 99.5 ^o (s 0.41, H ₂ O);
7) UV absorption spectrum: λ _max. nm (E $\genfrac{}{}{0ex}{}{\text{I\%}}{\text{Ist}}$ )

The ultraviolet absorption spectrum, measured in water, does not show any characteristic absorption maximum above 220 nm;
8) Infrared absorption spectrum: ν _max cm ^-1 (in BKr), 3367, 2938, 1663, 1552, 1384 and 1056;
9) Spectrum ¹ H-Nuclear magnetic resonance: δ ppm

Spectrum ¹ H-Nuclear Magnetic Resonance (400 MHz) is measured in deuterium oxide (deuterated water) using TMS (tetramethylsilane) as an external standard, and shows the following signals:
3.22 (1H, doublet of doublets, J = 8.98 & 10.31 Hz),
3.38 (1H, multiplet, J 2.44, 5.26 and 10.31 Hz),
3.46 (1H, doublet of doublets, J = 8.98 & 9.99 Hz),
3.53 (1H, doublet, J = 11.96 Hz),
3.55 (1 H, doublet of doublets, J = 5.26 & 12.21 Hz),
3.57 (1H, doublet of doublets, J = 5.38 & 9.99 Hz),
3.62 (1H, doublet of doublets, J = 12.44 & 12.21 Hz),
3.63 (1H, doublet, J = 11.96 Hz),
3.77 (1H, doublet, J = 4.89 Hz),
4.02 (1H, doublet of doublets, J = 2.08 & 4.89 Hz),
4.17 (1H, doublet, J = 8.55 Hz),
4.77 (1H, doublet of doublets, J = 2.08 & 8.55 Hz),
5.15 (1H, doublet, J = 5.38 Hz).

10) ¹³ C-Nuclear Magnetic Resonance Spectrum: δ ppm

Spectrum ¹³ C-Nuclear Magnetic Resonance (100 MHz) is measured in deuterium oxide using tetramethylsilane as an external standard and the following signals are shown: 60.7, 61.9, 69.6, 69.9, 72.0, 73, 0, 73.0, 80.1, 80.3, 80.6, 82.8, 87.3 and 161.1.

11) High Resolution Liquid Chromatography:
Separation column: Senshu Pak OD5-N-2151 (Senshu Science Co.), 6ph x 150 mm (5 microns),
Mobile phase 10 vol. acetonitrile of water containing 0.5% PIC B8 (products of the company Waters Inc.),
Flow rate: 1.5 ml / min,
Reference wavelength: 210 nm ultraviolet,
The peak having the retention time of 6.9 minutes was observed at ²⁵ ° C, and
12) Thin layer chromatography: Rf value: 0.44,
Adsorbent: silica gel on a glass plate (Merck Art 5715)
Developing solvent: a mixture of acetonitrile, acetic acid and water in a volume ratio of 6: 1: 3.

 PRI me R 2. Obtaining 5-amino-1- (oxymethyl) -cyclopentane-1,2,3,4-tetraol.

A solution of 19.3 mg trehazolina (prepared as described in example 1 method) dissolved in 1 ml of 4 n.vodnoy hydrochloric acid were placed in an ampoule and hydrolyzed by heating it (ampoules) at ¹⁰⁰ ° C for 24 hours. At the end of this time, the reaction mixture is mixed with water and then concentrated to dryness by evaporation under reduced pressure. The residue was again mixed with water and concentrated to dryness by evaporation under reduced pressure to distill off hydrochloric acid. The residue from this distillation is dissolved in 20 ml of water and the pH of the solution is adjusted to a value of 6.0. The resulting solution was passed through a column with 20 ml of Amberlite CG-50 (NH $\genfrac{}{}{0ex}{}{\text{+}}{\text{4}}$ The) -trademark and column are washed with 60 ml of deionized water, and then eluted with 0.2 N aqueous ammonia. The eluate was concentrated by evaporation under reduced pressure, and the residue was lyophilized to give 5.1 mg of crude 5-amino-1- (hydroxymethyl) cyclopentane-1,2,3,4-tetraol as a colorless powder.

All crude powdery 5-amino-1- (hydroxymethyl) -cyclopentane-1,2,3,4-tetraol (5.1 mg) obtained as described above is dissolved in a small amount of water and purified using preparative thin layer chromatography as follows . The solution is applied to two silica gel plates (Merck Art 5715, 20x20 cm) and developed using a mixture of acetonitrile, acetic acid and water in a volume ratio of 6: 1: 3 up to a height of 15 cm. A strip between Rf 0.36 and 0.47 is scraped out from the plate and filled into a column, which is then eluted with 50 ml of deionized water. The eluate is passed through 10 ml of Amberlite CG-50 (NH $\genfrac{}{}{0ex}{}{\text{+}}{\text{4}}$ ), while 5-amino-1- (oxymethyl) -cyclopentane-1,2,3,4-tetraol is adsorbed on the column. The column is then washed with 50 ml of deionized water and eluted with 50 ml of 0.2 N aqueous ammonia. The eluate was concentrated by evaporation under reduced pressure and lyophilized to obtain 3 mg of the desired 5-amino-1- (hydroxymethyl) -cyclopentane-1,2,3,4-tetraol as a colorless powder having the following properties:
1) color and appearance: the main colorless powder,
2) solubility: soluble in water, insoluble in acetone and chloroform,
3) color reaction: positive for ninhydrin reaction,
4) molecular formula: C ₆ H ₁₃ NO ₅ ,
5) molecular weight: 179 (determined using FAB mass spectrometry),
6) specific rotation [α] $\genfrac{}{}{0ex}{}{\text{25}}{\text{D}}$ -3.7 ^o (s0.51, H ₂ O),
7) ultraviolet absorption spectrum: λ _max nm (E $\genfrac{}{}{0ex}{}{\text{I\%}}{\text{I}}$ cm):
The ultraviolet absorption spectrum, measured in water, does not show any characteristic absorption maximum above 210 nm.

8) Infrared absorption spectrum: ν _max cm ^-1 (in KBr), 3384, 1582, 1474, 1380 and 1040.

9) Spectrum ¹ H-Nuclear magnetic resonance: δ ppm

Spectrum ¹ H-Nuclear Magnetic Resonance (400 MHz) is determined in deuterium oxide using tetramethylsilane as an external standard, and shows the following signals:
3.12 (1H, doublet, J = 7.1 Hz),
3.56 (1H, doublet, J = 11.98 Hz),
3.62 (1H, doublet, J = 12.2 Hz),
3.62 (1H, doublet, J = 6.8 Hz),
3.78 (1H, doublet of doublets, J 5.5 & 6.8 Hz),
3.90 (1H, doublet of doublets, J 5.5 & 7.1 Hz),
10) Spectrum ¹³ C-Nuclear magnetic resonance: δ, ppm

Spectrum ¹³ C-nuclear magnetic resonance (100 MHz) is determined in deuterium oxide using metramethylsilane as an external standard, and shows the following signals:
57.8, 61.0, 73.6, 79.4, 81.5 and 81.7 and
11) Thin layer chromatography:
Rf value: 0.39,
Adsorbent: silica gel on a glass plate (Merck Art 5 715),
Developing solvent: a mixture of acetonitrile, acetic acid and water in a volume ratio of 6: 1: 3.

 Example 3. Preparation of 2-amino-4- (oxymethyl) -3a, 5,6,6a-tetrahydro-4H-cyclopent- [d] -oxazole-4,5,6-triol.

100 g of the crude powder obtained according to the method described in example 1 and containing the prescribed amount (225 mg) of triazoline is dissolved in a mixture of 100 ml of water and 150 ml of 4N aqueous hydrochloric acid, and the pH of the resulting solution is adjusted to 2.5 by adding 4 N. aqueous hydrochloric acid. Then, 8 ml of concentrated hydrochloric acid and 72 ml of water are added to the solution to bring the total volume to 480 ml and to obtain a hydrochloric acid concentration of 0.2 N. The resulting solution was placed in a round bottom flask and then hydrolyzed on an oil bath maintained at 100 ^{° C} for 6 hours. At the end of this time, the reaction mixture was mixed with water and then concentrated to dryness by evaporation under reduced pressure. The sequence of mixing with water and evaporation to dryness is repeated to distill off hydrochloric acid. The residue was dissolved in 400 ml of water and the pH of the solution was adjusted to 6.0 by the addition of a 1N aqueous solution of sodium hydroxide. Then the solution is diluted with water to a volume of 8 liters. The resulting solution was passed through a column packed with 600 ml of Amberlite SG-50 (NH $\genfrac{}{}{0ex}{}{\text{+}}{\text{4}}$ ), and the column is washed with 6 L of deionized water, and then eluted with 0.5 N aqueous ammonia. The eluate was concentrated by evaporation under reduced pressure to a volume of 200 ml, and the concentrate was passed through a column packed with 800 ml of Dowex 1X2 (OH ^- ). Then the column is eluted with deionized water. After removing the first 1.5 L of eluate, the subsequent eluate is fractionated in portions of 20 ml. Each fraction was evaluated by the quantitative analysis described below to determine if it contains the target compound. Fractions 70-110 that contain this compound are combined and concentrated by evaporation under reduced pressure. The residue was lyophilized to give 30 mg of 2-amino-4- (oxymethyl) -3a, 5,6,6a-tetrahydro-4H-cyclopent- [d] oxazole-4,5,6-triol as a colorless powder having the following properties:
1) color and appearance: the main colorless powder,
2) solubility: soluble in water, insoluble in acetone and chloroform,
3) molecular formula: C ₇ H ₁₂ N ₂ O ₅ ,
4) molecular weight: 204 (determined by FAB mass spectrometry);
5) specific rotation: [α] $\genfrac{}{}{0ex}{}{\text{25}}{\text{D}}$ + 10 ^o (С0.51, Н ₂ О),
6) ultraviolet absorption spectrum: λ _max nm (E $\genfrac{}{}{0ex}{}{\text{I\%}}{\text{I}}$ cm),
The ultraviolet absorption spectrum is determined in water, and it does not show any characteristic absorption maximum above 210 nm,
7) infrared absorption spectrum: ν _max cm ^-1 (in KBr), 3358, 1668, 1528, 1398 and 1066;
8) spectrum of ¹ H-Nuclear magnetic resonance: δ, ppm

Spectrum ¹ H-Nuclear Magnetic Resonance (500 MHz) is determined in deuterium oxide using TSP (sodium trimethylsilyl propionate) as an internal standard, and shows the following signals:
3.73 (1H, doublet, J = 11.72 Hz),
3.82 (1 H, doublet, J = 12.21 Hz),
3.97 (1H, doublet, J = 4.4 Hz),
4.23 (1H, doublet of doublets, J = 4.4 & 2.44 Hz),
4.37 (1H, doublet, J = 8.79 Hz),
5.03 (1H, doublet of doublets, J = 8.79 & 2.44 Hz),
9) high resolution liquid chromatography:
Separation column: Asahi Pak E5-502C (Asahi Chemical Industry Co. Ltd.),
Mobile phase: 20 mM ammonium acetate (pH 8.5) + 50 mM aqueous sodium chloride,
Flow rate: 1 ml / min,
Reference wavelength: 210 nm ultraviolet,
Temperature: 25 ^о С,
Retention Time: 8.39 min.

 Quantification of 2-amino-4- (oxymethyl) -3a, 5,6,6a-tetrahydro-4H-cyclopentane [d] oxazole-4,5,6-triol using gas chromatography / mass spectrometry.

 The quantitative analysis referred to above is carried out as follows.

The sample is dissolved in a solvent (water) of a known volume of liquid. 10 μl of the resulting solution was placed in an ampoule and evaporated to dryness for acetylation. To the residue was added 30 .mu.l of acetic anhydride and 50 microliters of pyridine and the resulting mixture was heated at ⁶⁰ ° C for 40 min. Any excess reagents are removed by blowing a stream of nitrogen gas through the reaction mixture. The residue was mixed with a known amount of internal standard (pentaacetyl-1-amino-1-deoxy-β-D-glucose), and the mixture was dissolved in 100 μl of ethyl acetate to obtain a prototype for gas chromatographic / mass spectrometric analysis. The analysis is carried out using a capillary column with fused silica (products of G. & V. Science Co. Co. DV-5, 15 m) as a column for gas chromatography. Prototype (2 l) is injected and the column temperature was raised from 60 to 280 ^{° C} at a rate ^of 25 C / min. Negative ions are determined by chemical ionization using methane gas together with a Trio-1 quadrupole mass spectrometer (US products). Peaks of negative ions at m / z 388 (corresponding to the internal standard of the pentaacetyl compound) and at m / z 413 (corresponding to the pentaacetate 2-amino-4- (oxymethyl) -3a, 5,6,6a-tetrahydro-4H-cyclopent- [d ] oxazole-4,5,6-triol) is used for quantitative analysis. The content of 2-amino-4- (oxymethyl) -3a, 5,6,6a-tetrahydro-4H-cyclopent- [d] oxazole-4,5,6-triol is calculated using the internal standard method.

 PRI me R 4. Obtaining 5-amino-1- (oxymethyl) -cyclopentane-1,2,3,4-tetraol.

A solution of 16 mg of powdered 2-amino-4- (oxymethyl) -3a, 5,6,6a-tetrahydro-4-cyclopent- [d] oxazole-4,5,6-triol dissolved in 2 ml of 6 N. aqueous hydrochloric acid acid was sealed in an ampoule and then hydrolyzed by heating at ¹⁰⁰ ° C for 24 hours. at the end of this time, water was added to the hydrolyzate, and the resulting mixture was concentrated to dryness by evaporation under reduced pressure. The residue was redissolved in water and the resulting solution was again concentrated to dryness to remove hydrochloric acid, and then the resulting residue was dissolved in 20 ml of water and the pH of the solution was adjusted to 6.0 by adding 1 N aqueous sodium hydroxide solution. The solution is passed through 20 ml of Amberlite CG-50 (NH $\genfrac{}{}{0ex}{}{\text{+}}{\text{4}}$ ) to retain 5-amino-1- (oxymethyl) -cyclopentane-1,2,3,4-tetraol by adsorption, and the column is washed with 60 ml of deionized water, and then elute with 0.2 N. aqueous ammonia. The eluate was concentrated by evaporation under reduced pressure to obtain 5 ml of concentrate. This concentrate was placed in a column packed with 50 ml of Dowex 1X2 (OH ^- ), and the column was washed with 200 ml of deionized water, and then eluted with 20 vol. aqueous methanol. The eluate is fractionated in portions of 5 ml. Fractions 5-23, which exhibit inhibitory activity against β-glucosidase, are combined and concentrated by evaporation under reduced pressure. The concentrate was lyophilized to give 6.2 mg of 5-amino-1- (hydroxymethyl) cyclopentane-1,2,3,4-tetraol as a colorless powder having the properties described above.

 PRI me R 5. 5-amino-1- (hydroxymethyl) -cyclopentane-1,2,3,4-tetraol hydrochloride.

 8.76 mg of 5-amino-1- (hydroxymethyl) cyclopentane-1,2,3,4-tetraol was dissolved in 10 ml of water and the pKa value of the solution was measured by titration using 0.1 N standard solution of hydrochloric acid. The pKa value corresponded to 6.93. Therefore, further, 80 mg of 5-amino-1- (hydroxymethyl) cyclopentane-1,2,3,4-tetraol was dissolved in 10 ml of water and 1N was added. hydrochloric acid, then the pH was adjusted to 4.93, which was less than the pKa value of 6.93 but 2.0. The resulting solution was concentrated by evaporation under reduced pressure. The residue was lyophilized to give 101 mg of the titled compound.

Spectrum ¹ H-Nuclear Magnetic Resonance: δ ppm

The spectrum of ¹ H-nuclear magnetic resonance (400 MHz) was measured in deuterium oxide using NTSC (sodium trimethylsilyl propionate) as an internal standard and revealed the following signals:
3.68 (1H, d. J, 7.9 Hz),
3.85 (1H, d. J, 12.23 Hz),
3.86 (1H, d. J, 7.7 Hz),
3.89 (1H, d. J, 12.1 Hz),
3.97 (1 H, doublet of doublets, J = 7.6 & 7.7 Hz),
4.21 (1H, doublet of doublets, J = 7.6 & 7.9 Hz).

 PRI me R 6. 5-amino-1- (hydroxymethyl) cyclopentane-1,2,3,4-tetraolsulfate.

 Following a procedure similar to that described in example 5, but using 81 mg of 5-amino-1- (hydroxymethyl) cyclopentane-1,2,3,4-tetraol, 102 mg of the titled compound was obtained.

Spectrum ¹ H-Nuclear Magnetic Resonance: δ ppm

The measurement of the spectrum of ¹ H-Nuclear magnetic resonance (400 MHz) was carried out in deuterium oxide using NTSC (sodium trimethylsilyl propionate) as an internal standard and revealed the following signals:
3.68 (1H, d. J, 7.9 Hz),
3.85 (1H, d. J 12714 Hz),
3.86 (1H, d. J, 7.7 Hz),
3.89 (1H, d. J, 12.04 Hz),
3.97 (1H, dd. J, 7.5 and 7.7 Hz),
4.21 (1H, dd. J, 7.5 and 7.9 Hz).

 PRI me R 7. 2-Amino-4- (hydroxymethyl) -3a, 5,6,6a-tetrahydro-4H-cyclopent- [d] oxazole-4,5,6-triol hydrochloride.

 10.22 mg of 2-amino-4- (hydroxymethyl) -3a, 5,6,6a-tetrahydro-4H-cyclopent- [d] oxazole-4,5,6-triol was dissolved in 10 ml of water and the pKa value of the solution was measured titration using 0.1 N standard hydrochloric acid solution.

 The pKa value was 7.18. Therefore, further 80 mg of 2-amino-4- (hydroxymethyl) -3a, 5,6,6a-tetrahydro-4H-cyclopent- [d] oxazole-4,5,6-triol was dissolved in 10 ml of water and 1N was added. hydrochloric acid, then the pH value was adjusted to 5.18, which was 2.0 less than the pKa value of 7.18. The resulting solution was concentrated by evaporation under reduced pressure. The precipitate was lyophilized to give 102 mg of the title compound.

Spectrum ¹ H-nuclear magnetic resonance: δ ppm

The measurement of the spectrum of ¹ H-Nuclear magnetic resonance (400 MHz) was carried out in deuterium oxide using NTSC (sodium trimethylsilyl propionate) as an internal standard and showed the following signals:
3.83 (2H, d. J 1.0 Hz),
4.06 (1H, d. J 3.6 Hz),
4.39 (1H, dd. J 3.6 and 1.9 Hz),
4.56 (1H, d. J, 8.5 Hz),
5.41 (1H, doublet of doublets, 8.5 and 1.9 Hz).

PRI me R 8. 2-amino-4- (hydroxymethyl) -3a, 5,6,6a-tetrahydro-4H-cyclopent- [d] oxazole-4,5,6-triol sulfate
Following a procedure similar to that described in example 7, but using 83 mg of 2-amino-4-hydroxymethyl) -3a, 5,6,6a-tetrahydro-4H-cyclopent- [d] oxazole-4,5,6-triol, was 105 mg of the title compound are obtained.

Spectrum ¹ H-Nuclear Magnetic Resonance: δ ppm

The measurement of the spectrum of ¹ H-Nuclear magnetic resonance (400 MHz) was carried out in deuterium oxide using NTSC (sodium trimethylsilyl propionate) as an internal standard and revealed the following signals:
3.83 (2H, d. J 1.1 Hz),
4.05 (1H, d. J 3.6 Hz),
4.39 (1H, doublet of doublets, 3.6 and 1.8 Hz),
4.56 (1H, d. J, 8.6 Hz),
5.41 (1H, dd. J, 8.6 and 1.8 Hz).

 EXAMPLE 1. Biological activity.

 Inhibitory activity of 5-amino-1- (oxymethyl) -cyclopentane-1,2,3,4-tetraol against β-glucosidase.

 Samples of β-glucosidase (separated from almonds), para-nitrophenyl-β-D-glucopyranoside, deoxynojirimycin and castanospermine used in this experiment were all purchased from Sigma Chemicals Co.

 The test compounds used were 5-amino-1- (hydroxymethyl) -cyclopentane-1,2,3,4-tetraol [compound (I)] and deoxynojirimycin or castanospermine, both of which are known compounds having this type of activity.

A mixture of 0.01 units / ml of β-glucosidase and 100 .mu.l of buffer solution (pH 5.6) comprising 20 mM citric acid, 40 mM disodium phosphate and the compound under test was allowed to stand at ³⁷ ° C for 15 min. By the end of this time, the mixture was added 50 .mu.l of a buffer solution containing 3 mg / ml of p-nitrophenyl-β-D-glucopyranoside, and then this mixture was allowed to react at ³⁷ ° C for 20 min. 20 μl of 1 m glycine / sodium hydroxide buffer solution (pH 10.4) was added to the reaction mixture, and the amount of para-nitrophenol released was monitored by absorbance at 405 nm. The table lists the concentrations of each test compound required to inhibit β-glucosidase activity by 50% (IC ₅₀ ).

 EXAMPLE 2.

 Inhibitory activity of 2-amino-4- (oxymethyl) -3a, 5,6,6a-tetrahydro-4H-cyclopent- [d] -oxazole-4,5,6-triol [compound (II)] against rat β -fructofuranosidase.

 According to the method of Kessler et al. [M. Kessler et al, Biochimica et Biophisica Acta, 506, 136-154 (1978)] the enzyme solution of the rat basal border plate of the small intestine is obtained from the small intestines of three Wistar male rats and suspended in 3 ml physiological saline.

 A sample of 4-aminoantipyrine (A 4 382) was purchased from Sigma Chemicals Co. and samples of peroxidase (Grade I) and glucose oxidase (Grade I) are purchased from Behringer Mannheim Co. A buffer solution (pH 6.2) comprising 20 mM citric acid and 40 mM disodium phosphate was used as a diluent in the following experiment.

Each 96-well microtiter panel cell (Falcon Co. products) is filled with 130 μl (in all cells) of a mixture that contains 0.2 mg of bovine serum albumin (Sigma, A 7 906), 3 units of glucose oxidase, 0.132 units of peroxidase , 20 μg of 4-aminoactipyrine, 40 μg of phenol, 3 micromoles of sucrose and the test compound. 20 μl of a 100-fold diluted solution of the enzyme solution of the basal boundary plate (Bruch membrane) from rat small intestine is added to the cell. The mixture was allowed to react at 37 ^{° C} for 2 minutes and the quantity of glucose liberated was monitored by absorbance at 492 nm.

When enzymatic reactions are carried out without adding a test compound and without adding an enzyme solution, glucose concentrations are set to 0 and 100% inhibition, respectively. The concentration of compound (II) required to inhibit rat β-fructofuranosidase activity by 50% (IC ₅₀ ) was found to be 18 μg / ml.

 EXAMPLE 3.

 The inhibitory activity of triazolin against silkworm trehalase.

 Ten silkworm larvae of the fifth age stage (total mass 44 g) are homogenized using a polytron (brand) homogenizer in 120 ml of buffer solution (pH 5.6) obtained using 20 mM citric acid and 40 mM disodium phosphate for 2 min, while cooling with ice (a similar buffer solution is also used in the future). Then the homogenized mixture is centrifuged at a speed of 6000 rpm for 10 minutes and the supernatant is separated. 240 ml of acetone was added to 120 ml of the supernatant, while cooling with ice and stirring, and the resulting mixture was centrifuged at a speed of 9000 rpm for 20 minutes. The precipitate was separated and dissolved in water, and the resulting solution was lyophilized to obtain 2.0 g of a crude enzyme.

130 μl of the indicated buffer solution, 50 μl of a solution of a sample containing triazoline at various concentrations, and 50 μl of a silkworm enzyme solution prepared according to the method described above and containing 4 mg / ml of the enzyme are placed in a test tube, and the mixture is shaken in a water bath maintained at 37 ^about C for 15 minutes At the end of this time, 20 μl of a 250 mM treholase solution was added and the mixture was allowed to react for 15 minutes. The reaction mixture is then heated in a boiling water bath for 3 minutes, after which it is cooled with a mixture of ice and water. It is then centrifuged at a speed of 3000 rpm for 10 minutes, and the resulting solution, free of sediment, is used to determine the concentration of glucose.

The reaction is carried out using a Waco C-test for glucose (manufactured by Wako Pure Chemical Industries Ltd) and 10-fold amounts of a sample solution compared to the amount of this solution by a standard method. The percent inhibition is calculated from the glucose concentrations achieved by using a buffer solution instead of the sample solution and when using a buffer solution instead of a substrate solution in the form of 0 and 100% inhibition, respectively, to calculate the concentration required to inhibit the enzymatic activity by 50% (IC ₅₀ ), which is found to be 2.0 n. per ml (2.0 ng / ml).

Claims (10)

 1. 5-amino-1-hydroxymethylcyclopentane-1,2,3,4-tetraol and its pharmaceutically acceptable salts.  2. 2-amino-4-hydroxymethyl-3a, 5,6,6a-tetrahydro-4 H-cyclopent- [d] -oxazole-4,5,6-triol and its pharmaceutically acceptable salts.  3. A method of producing 5-amino-1-hydroxymethylcyclopentane-1,2,3,4-tetraol, characterized in that the hydrolysis of triazoline with strong acid is carried out for 20 to 24 hours at the boil of the reaction mixture.  4. The method according to claim 3, characterized in that the hydrolysis is 4 N. aqueous HCl for 24 hours  5. The method of obtaining 5-amino-1-hydroxymethylcyclopentane-1,2,3,4-tetraol, characterized in that the acid hydrolysis of 2-amino-4-hydroxymethyl-3a, 5,6,6a-tetrahydro-4H-cyclopent - [d] -oxazole-4,5,6-triol.  6. The method according to claim 5, characterized in that the hydrolysis is 6 N., aqueous HCl.  7. A method of producing 2-amino-4-hydroxymethyl-3a, 5,6,6a-tetrahydro-4H-cyclopent- [d] -oxazole-4,5,6-triol, characterized in that the hydrolysis of triazoline with weak acid is carried out in 5-6 hours during the boiling of the reaction mixture.  8. The method according to claim 1, characterized in that the hydrolysis is 0.2 N., aqueous HCl for 6 hours Priority by signs and points:
02/15/91 according to claim 1, except for pharmaceutically acceptable salts, PP.3 and 4.  06/04/91 according to claim 2, except for pharmaceutically acceptable salts, PP.7 and 8.  10.21.91 according to claims 5 and 6.

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