FI106026B - Process for preparing 2-amino-4-(hydroxymethyl)-3a,5,6,6a- tetrahydro-4H-cyclopent[d]oxazole-4,5,6-triol which can be used as a drug - Google Patents

Description

106026

A process for the preparation of 2-amino-4- (hydroxymethyl) -3a, 5,6,6a-tetrahydro-4H-cyclopent [d] oxazole-4.5.6-triol useful as a medicament for the preparation of 2-amino-4 - (hydroxymethyl) -3a, 5,6,6a-tetrahydro-4H-cyclopent [d] oxazole-4,5,6-triol

The present invention relates to polyhydroxycyclopentane derivatives having several valuable biological activities and to a process for the preparation of these compounds.

The present compounds are 5-amino-1- (hydroxymethyl) cyclopentane-1,2,3,4-tetraol and 2-amino-3- (hydroxymethyl) -3a, 5,6,6a-tetrahydro-4H-cyclopent [ d] oxazole-4.5.6-triol having the formulas (I) and (II) below. These compounds may be prepared by fermentation or by cleavage of a compound known as trehatoline, which may be represented by Formula (III), which is set forth below. However, tautomerism exists between 2-amino-4- (hydroxymethyl) -3a, 5,6,6a-tetrahydro-4H-cyclopent [d] -oxazole-4,5,6-triol and trehazoline and may be therefore also represented by the formulas: * [: (Ha) and (Ula), which are represented as follows: • · · «· 2 106026 HO _ΠΗ

HJN

HO ^

OH

OV-OH

H2N ^ JX ^ / ° H (Q)

OH

Hn — OH

HN = ^ o ^ V- ° H (π »)

OH

9 H „J ^>" s: «

HO OH OH

• · · '·'. '.. · HO—, H OH ^ -oh H ° ^ I ^ N = <0 ^^^ H «n«.

HO OH OH

3, 106026

It is believed that trehatolazine may be the same compound as that found in PCT International Publication No. WO 90/100100 under the name "trehalostatin".

The present compounds have the ability to inhibit the action of various sugar hydrolases, and in particular β-glucosidase and sucrase.

Compounds with potent inhibitory activity against β-glucosidase, such as chestnosperminine and deoxynojirimycin, have been shown to be useful as anti-tumor agents such as Acquired Immune Deficiency 10 Syndrome [R.A. Gruters et al., 1987, Nature 330: 74-77; MJ Humphries et al., Cancer Res., 46, 5215-5222 (1986)]. It is therefore expected that compounds having the ability to inhibit the action of β-glucosidase will be useful as anti-tumor or anti-AIDS agents.

It has also been suggested that compounds with potent inhibitory activity against sucrose (= saccharase), such as AO-128 and Acarbose, are useful as antidiabetic agents and antiobesity agents [Satoshi Horii et al., Journal of Medicinal Chemistry, 29, 1038 -1046 (1986)]; T. Aida et al., 1981, Journal of Japanese Society of Food and Nutrition, 34, (2) 134-139. Therefore, it is expected that compounds having the ability to inhibit the action of saccharase would be useful in the treatment and prevention of diabetes and obesity.

The compounds which are closely resembled to the compound prepared by the process of the present invention are: • mannostatin described, inter alia, by T. Aoyagi et al., [The Journal of Antibiotics, Vol. XLII No. 6, 883 (1989)], which are said to be able to inhibit the action of α-D- · * "· '· * mannosidase.

30 · 1S, 2R, 3S, 4R, 5R) -methyl- [2,3,4-trihydroxy-5- (hydroxymethyl) -cyclopentyl] -amine, described inter alia by R.A. Farr et al., [Tetrahedron Letters, 3J, 2109 (1990)], which is also said to inhibit the action of α-mannosidase; aalosamidine, described inter alia by S. Sakuda et al., [Tetrahedron Letters, TL, 2475 (1986)], which is said to have the ability to inhibit the action of insect chitinase; and 4 106026 5 kifunensine described, inter alia, by H. Kayakiri et al., [J. Org. Chem., 54, 4015 (1989)] which is said to be an immune modulator with the ability to inhibit the action of α-mannosidase.

It is an object of the present invention to provide novel compounds which have an inhibitory effect on a number of sugar hydrolases.

It is a further object of the present invention to provide compounds having an effect which is therefore believed to be useful in the treatment and prevention of tumors, AIDS, obesity and diabetes mellitus.

15

More particularly, the invention relates to a process for the preparation of 2-amino-4- (hydroxymethyl) -3a, 5,6,6a-tetrahydro-4H-cyclopent [d] oxazole-4,5,6-triol or for the preparation of a pharmaceutically acceptable salt thereof.

V 20 Other objects and advantages will become apparent as the explanation progresses.

The compound prepared by the process of the present invention is 2-amino-4- • * * · '· * (hydroxymethyl) -3a, 5,6,6a-tetrahydro-4H-cyclopent [d] -oxazole-4,5,6 a triol having the formula (II) given above.

The invention relates to a pharmaceutical composition comprising an effective amount of 2- «amino-4- (hydroxymethyl) -3a, 5,6,6a-tetrahydro-4H-cyclopent [d] -oxazole-4,5, 6-triol * ·: · 'in admixture with a pharmaceutically acceptable carrier, diluent or excipient.

The invention further relates to a method of treating an animal, e.g., a mammal, especially *. *: For the treatment or prevention of human, diabetes or obesity by administering 106026 5 to an effective amount of 2-amino-4- (hydroxymethyl) -3a, 5,6,6a-tetrahydro-4H-cyclopent [d] -oxazole-4,5 , 6-triol.

5-Amino-1- (hydroxymethyl) cyclopentane-1,2,3,4-tetraol can be prepared from 5-trehatolazine or 2-amino-4- (hydroxymethyl) -3a, 5,6,6a-tetrahydro-4H-cyclopent [d hydrolysis of] -oxazole-4,5,6-triol as described in more detail below. 2-Amino-4- (hydroxymethyl) -3a, 5,6,6a-tetrahydro-4H-cyclopent [d] -oxazole-4,5,6-triol can be prepared by hydrolysis or fermentation of trehatzoline using the microorganism Micromonospora or Amycolatopsis .

10

Trehatzoline, which can be used as a starting material for the preparation of a compound of the present invention, can be prepared by culturing a microorganism producing Micromonospora or a trehatsolin of Amycolatopsis, preferably a microorganism producing trehatsolin of the species Micromonospora.

Specifically, the 2-amino-4- (hydroxymethyl) -3a, 5,6,6a-tetrahydro-4H-cyclopent [d] oxazole-4,5,6-triol of formula (II) useful as a medicament for the process of the invention or a pharmaceutically acceptable salt thereof:

O

: .y h2n ^ (oh (D)

OH

25 • · * characterized by culturing the microorganism Micromonospora sp. SANK 62390, FERM BP-3521, or a mutant thereof having substantially the same morphological and biochemical properties, or the microorganism Amycolatopsis sp. SANK 60791, ··· [30 FERM BP-3513 or a mutant thereof having substantially the same morphological and biochemical * * · chemical properties capable of producing 2-amino-4- (hydroxymethyl) -3a. , 5,6,6a-tetrahydro-4H-cyclopent [d] oxazole-4,5,6-triol, and separating 2-6 106026 amino-4- (hydroxymethyl) -3a, 5,6,6a-tetrahydro-4H cyclopent [d] oxazole-4,5,6-triol from the culture medium and optionally forming a salt of the product.

An example of a microorganism producing Micromonospora trehatolazine is Micromonospora sp. SANK 62390. This microorganism was first deposited in a deposit with the domestic depository of the Fermentation Research Institute, Tsukuba-shi, Ibaraki-ken, Japan, July 26, 1990 under deposition number FERM P-11631 and then deposited under "Budapest In accordance with the Treaty, as deposited with the Fermentation Research Institute, August 10, 1991 under accession number FERM BP-3521.

An example of a trehatzoline-producing microorganism of the species Amycolatopsis is Amycolatopsis sp. SANK 60791, filed August 14, 1991, under the accession number FERM BP-3513 with the Institute of Industrial Science and Technology, Tsukuba-shi, Ibaraki-ken, Japan.

:: Both of these are recently isolated species and both form part of the present invention.

DESCRIPTION OF MICRO-ORGANISMS

• · • · * • · * • • • • • • • * Micromonospora sp. SANK 62390 25 • · · «· • * ....

The species Micromonospora sp. SANK 62390 has the following fungal properties.

1. Morphological Characteristics 30 Species SANK 62390 grows normally or slightly poorly when cultivated at a temperature of · · ·. ·: 28 ° C for 7-14 days in a conventional agar culture medium used for species identification. The fungal mycelium substrate (hyphae) elongates appropriately and spreads 7 106026 as a light orange, from orange to dark brownish gray, but without the cuts or strong twists observed in the strain of Nocardia. The mycelium in the air is rudimentary and turns white to brownish-white. Spores are observed on the mycelial substrate (hyphae) alone and form one at a time a relatively short spore colony. The spore has a spherical shape and has a smooth surface. No specific organs such as sporadic colonies, mycelial warts, eddies or the like are detected.

2. Growth in various media 10

The strain was cultured at 28 ° C for 14 days in various culture media and their properties are shown in Table 1. Color shades are detected by a color information number according to the "Guide to Color Standard" printed by Japan Color Research Institute.

15

The following abbreviations are used in the table: G: growth; AM: airborne felt; R: conversely; SP: soluble pigment.

Table 1 v ': 20 *.' · Properties of SANK 62390 * Medium Description Properties • · · 25 Sucrose G: Slightly weak, smooth,. **. nitrate agar light orange (3-9-6) • · • «· AM: not formed '' • * 30 R: light orange (3-9-6) SP: not formed 8 106026

Glucose-Asparagus G: Slight, smooth, gin agar yellow orange (12-7-7) AM: not formed 5 R: light orange (6-8-7) SP: not formed

Glycerol-Aspara-G: Slight, smooth, gin agar (ISP 5) light orange (8-7-6) AM: not formed 15 R: light brownish gray (2-6-6) SP: not formed 20

Inorganic salts- G: good, smooth, light starch agar (ISP 4) orange (8-7-6) AM: not formed 25, R: gray (N-5) SP: not formed 30 Tyrosine agar G: slightly weak, even, * ·· * (ISP 7) dim orange (6-8-6) «· AM: barely formed, rudimentary white 35: ***: R: brownish white (2-9-7) •« ·. SP: not formed 40__ 9 106026

Nutrient Agar G: Slight, Slight, (DIFCO) Yellow Orange (10-8-7) AM: Not formed 5 R: Dull Yellowish Orange (8-8-7) SP: Not formed 10

Yeast extract malt- G: good, even, dark extract agar (ISP 2) brownish gray (1-4-6) 15 AM: hardly formed, rudimentary brownish white (1-8-6) R: brownish black (1-2 -6) 20 SP: not formed

Oatmeal Agar G: Good, smooth, orange (ISP 3) (10-7-6) 25 AM: hardly formed, rudimentary, white. R: Orange (12-7-6): 30 SP: Not formed • · · • * · -—- • · · · ·

Water agar G: slightly weak, smooth, · · · * light yellowish orange 35 (2-9-9) AM: not formed ·. R: gray (N-5): ··· '40 SP: not formed 10 106026

Potato extract- G: good, even, yellowish carrot extract agar gray (1-9-10) AM: hardly formed, 5 rudimentary white R: brownish white (2-9-7) SP: not formed 10 _________ 3. Physiological properties

The physiological properties of SANK 62390 observed from day 2 to day 21 in culture medium at 28 ° C are shown in Table 2.

Table 2 20 Positive hydrolysis of starch

Gelatine thawing positive

Nitrate reduction negative,: Milk coagulation positive i V 'Milk peptonization positive 25 Production of melanoid pigments (medium 1) * negative «· i · * * ♦ *. * (Medium 2) * negative» t »• · * * · *' ( medium 3) * negative

Substrate degradation casein positive • · • · *** tyrosine negative «♦ * *! * 30 xanthine negative

* ··· * Growth temperature range (Medium 4) * 17-42 ° C

Optimal Growth Temperature (Medium 4) * 27-32 ° C

Salt tolerance 2% »· ♦ · ♦ 35 π 106026 * Media 1: Tryptone Yeast Extract Broth (ISP 1) Media 2: Peptone Yeast Extract - Iron Agar (ISP 6) Media 3: Tyrosine Agar (ISP 7) Media 4: Yeast Extract-Malt Agar (ISP 2) 5

Strain SANK 62390 was also cultured at 28 ° C using Pridham-Gottlieb agar (ISP 9) as culture medium. The assimilation of carbon sources observed for 14 days after culture is shown in Table 3.

10 Table 3 Exploited by D-glucose Exploited by D-fructose 15 Exploited by L-arabinose Not utilized by D-xylose

Sucrose did not use; ": Inositol took advantage of ': 20 Raffinose took advantage of * *.' D-mannitol did not use '* ... · Comparison did not use • ♦ ♦ ♦ ♦ ♦ ♦ ♦ ♦ 25 4. Cellular Components • The cell walls of SANK 62390 were analyzed by the method of B. Becker et al.

Microbiology, 12, 421-423 (1984)] and was found to contain meso-diaminopimelic acid. In addition, the sugar components of whole walls of SANK 62390 were analyzed at 30 M.P. Lechevalier (Journal of Laboratory & Clinical Medicine, 71, 9393 (1968)) and were found to contain arabinose and xylose but not myolic acid. The acyl-type peptide glycan on the cell wall was shown to have 12106026 glycolyl types. The major menaquinone components observed were MK-10 (H2), MK-10 (H4) and MK-10 (Hg).

It is therefore clear that this micro-organism should be classified as a novel strain belonging to the Actinomycetes family of 5 genera Micromonospora. On this basis, it was designated SANK 62390 as a species of Micromonospora.

Amycolatopsis species SANK 60791 10 Amycolatopsis SANK 60791 has the following fungal characteristics: 1. Morphological characteristics

Species SANK 60791 grows normally or poorly during culture at 28 to 15 ° C for 7 to 14 days with conventional agar culture medium used to identify the species. The fungal mycelium substrate (hyphae) elongates well and spreads evenly or irregularly from brownish white to light yellowish-brown to dull yellow. Felting in the air is rare or rudimentary and: <· has a white, light yellow to light orange color. Incubation: 'S': 20 later stages of hyphae and airborne mycelium! is divided into parts and sometimes an airborne mycelium with a basil structure is observed. The mycelium extends without the steep turns seen with the Norcar- • ·. ·. · Slide. No special organs, such as sporadic colonies, mycelium, • sweat, etc., are detected.

25 · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·

The species was cultured at 28 ° C for 14 days in various culture media and has the characteristics set forth in Table 4. The color tone is indicated by the color guide "Guide to Color:: 30 Standard '" \ n provided by Japan Color Research Institute.

I I »

The following abbreviations are used in the table: 13 106026 G: growth; AM: airborne mycelium; R: conversely; SP: soluble pigment

Table 4 5 Properties of SANK 60791 Medium Description Properties 10 Sucrose Nitrate Agar G: Good, even, yellowish gray (1-9-10) AM: slightly weak, white 15R: yellowish gray (2-9-10) SP: no formed

Glucose-Asparagine Agar G: slightly faint, smooth, 20 orange (3-9-6) AM: barely formed, yellowish (2-9-10): 25 R: light brown (3-8- 6) «· *« SP: not formed • ♦ ♦ ___ • · «· · * · 1 · 2 Glycerin-asparagine-G: good, smooth, tan: 30 agar (ISP 5) van white (2-9- 7) AM: Primary, White • · «• · • · · .3. R: light yellowish brown 35 (6-8-8): SP: not formed • · · 2 0 1 3 «m« 14 106026

Inorganic Salts - G: Slight, Slight, Starch Agar (ISP 4) Light Yellowish Brown (4-8-9) 5 AM: Primary, Light Yellow (3-9-10) R: Light Yellowish 10 Brown (6-8- 9) SP: not formed

Tyrosine agar (ISP 7) G: very good, wrinkled, 15 brownish white (2-9-6) AM: hardly formed, light orange (3-9-6) 20 R: light orange (6-8-7) SP: not formed 25 Nutrient Agar G: Good, even, light (DIFCO) yellowish brown (4-8-8) V AM: rudimentary, white * ,,! 30 R: light yellow (3-9-8) • · • * ·; Y: SP: not formed φ · • * «* · · --- # * ·

Yeast extract - malt G: very good, wrinkled 35 extract agar (ISP 2) brownish white (1-4-6) '··· • · AM: barely formed,: light orange (3-9-6) 40 * ·; · R: Light orange (6-8-7) I ”. SP: not formed • · 1 15 106026

Nutrient Agar G: Good, Cyan, Light DIFCO) Yellowish Brown (4-8-8) 5 AM: Primary, White R: Light Yellow (3-9-8) SP: Not Formulated 10 __________

Yeast Extract - Malt G: Good, Wrinkled, Frosted Extract Agar (ISP 2) Yellow (10-7-9) AM: Primary, White 15 R: Frosty Yellowish Orange (10-7-8) SP: No formed 20 _

Oatmeal Agar G: Slightly weak, even, (ISP 3) Light yellowish brown (4-8-9) 25 AM: Primary, White R: Light yellow (4-9-9) SP: Not formed 30__

Water Ag G: slightly faint, even, yellowish gray (1-9-10) «· * i · · •» · AM: slightly faint, white 35 • · · R: yellowish gray (1-9-10) ··· • · *** SP: not formed • # · I »I •« · «·« ·

| »I

• · 16 106026

Potato extract- G: slightly weak, even, carrot extract agar yellowish (1-9-10) 5 AM: rudimentary, white R: yellowish gray (2-9-11) SP: not formed 10____ 3. Physiological properties

The physiological properties of SANK 60791 observed from day 2 to day 21 after the start of culture at 28 ° C are shown in Table 5.

Table 5 20 Hydrolysis of starch negative

Gelatine thawing positive (14 l)

Nitrate reduction positive t \\ Milk coagulation positive «·« ·

Peptonization of milk negative • * • * V '. 25 Production of melanoid pigments (medium 1) * negative * · · p ·; ·, (medium 2) * negative • * · (medium 3) * negative, ···. Decomposition of casein substrate casein negative * · · «. · **; tyrosine positive ·· · 30 xanthine negative «i ·

Salt resistance (medium 4) * 3% • ·

• «« - - I I

** ··, * Medium 1: Tryptone Yeast Extract Broth (ISP 1) * · Medium 2: Peptone Yeast Extract - Iron Agar (ISP 6) 35 Medium 3: Tyrosine Agar (ISP 7) 17 106026 Medium 4: Yeast Extract Malt Extract Agar (ISP 2)

SANK 60791 was also cultured at 28 ° C using Pridham-Gottlieb agar (ISP 9) as the culture medium. The assimilation of carbon sources observed for 14 days after cultivation is shown in Table 6.

Table 6 10 D-Glucose Tapped D-Fructose Tapped L-Arabinose Slightly L-Raminose Tapped D-Xylose Tapped 15 Sucrose Tapped

Inositol did not use

Raffinose took advantage of D-mannitol took advantage of: ·! The comparison did not use:; ; The cell walls of SANK 60791 were analyzed by B. Becker. et al., 25 [.Applied Microbiology, 12, 421-423 (1984)] and was found to contain meso-diaminopiic acid. In addition, the sugar * * · ·; · 'components of all cell walls of SANK 62390 were analyzed for M.P. Lechevalier [Journal of Laboratory & Clinical

Medicine, 71, 934 (1968)] and were found to contain arabinose but not mycobenzylic acid. The acyl-type peptide glycan on the cell wall turned out to be of the 30 acetyl types. The major menaquinone component observed was MK-9 (H4).

«· • · ·« · * • · 18 106026

It is therefore reasonable for the micro-organism to be classified as a new strain belonging to the Amycolatopsis genotype of the Actinomycetes family. On this basis, it was designated Amycolatopsis species SANK 60791.

5 Identification of strains SANK 62390 and SANK 60791 was performed according to ISP standards (The International Streptomyces Project), Bergey's Manual of Systematic Bacteriology, Vol. 4, The Actinomycetes, Vol. 2 and other literature on Actinomycetes.

Strain SANK 62390 and SANK 60791 have been shown to produce trehatzoline and 2-amino-4- (hydroxymethyl) -3a, 5,6,6a-tetrahydro-4H-cyclopent [d] -oxazole-4,5,6-triol . However, as is well known, the properties of fungi in general, and of actinomycetic microorganisms in particular, can vary considerably and such fungi are highly susceptible to both natural and artificial mutations (e.g., ultraviolet radiation, radioactive radiation, chemical treatment, etc.). The present invention therefore encompasses the use of any microorganism that can be classified into the genus Micromonospora or Amycolatopsis and which, in association with species SANK 62390 and SANK 60791, shares the characteristic ability to produce trehazoline and 2-amino-4- (hydroxymethyl) -3a, 5,6, 6α-tetrahydro-4H-cyclopent [d] -oxazole--4,5,6-triol. For new microorganisms, species SANK 62390 and SANK 60791, no

I I I

'.' is expected to be exceptional and the terms "SANK 62390" and "SANK 60791" include <1 <i «t: · 1 all mutants of these species which share the characteristic of SANK 62390 and SANK 60791 the ability to produce trehatzoline and 2-amino-4- (hydroxymethyl) -? · 3a, 5,6,6a-tetrahydro-4H-cyclopent [d] -oxazole-4,5,6-triol. In addition, these mutants include those obtained by gene manipulation techniques, for example, recombination, transduction, transformation or the like. It is simple by experimenting with the · · • · '. F to determine, on the basis of the information provided herein, trehazoline and 2-amino-4- (hydroxymethyl) -3a, 5,6,6a-tetrahydro-4H-cyclopent [d] -oxazole-4,5,6-triol, whether a particular species produces or produces enough of these species to potentially become commercially interesting.

• • • • • • • 19 106026

According to the present invention, trehatolazine and 2-amino-4- (hydroxymethyl) -3a, 5,6,6a-tetrahydro-4H-cyclopent [d] -oxazole-4,5,6-triol can be prepared by culturing these fungal species in culture media which are conventionally used to produce other fermentation products from similar microorganisms. Such media necessarily contain microbiologically assimilable sources of carbon and nitrogen as well as inorganic salts as known to those skilled in the art.

Preferred examples of carbon sources include: glucose, fructose, maltose, sucrose, mannitol, glycerol, dextrin, oats, rye, corn starch, potato starch, carbon stone powder, soybean flour, cottonseed cake, cottonseed bean oil, cottonseed bean oil, cottonseed bean oil. These compounds can be used alone or in any suitable combination. Generally, the amount used will range from 1 to 10% by weight of the culture medium.

Preferred nitrogen sources are the normal proteinaceous materials commonly used in fermentation processes. Examples of such nitrogen sources include: soy flour, wheat bran, peanut flour, cottonseed cake, cottonseed oil, ammonium seed meal, casein hydrolyzates, pharmaine, fishmeal, corn extract, sodium extract, peptone, meat extract, peptone, meat extract. These nitrogen sources can be used at 4 <4 V · alone or by myself in suitable combinations. In general, it is preferable to use them at a concentration of 0.2 to 6% by weight of the culture medium.

The inorganic salts of the nutrient that can be incorporated into the culture medium, 4 · 4 * · * 25 are the conventional salts capable of supplying various ions which ... needed for growth of microorganisms such as sodium, ammonium, calcium, phosphate, sulfate, chloride and carbonate ions. In addition, the medium should contain trace amounts of essential trace elements such as potassium, calcium, cobalt, manganese, iron and magnesium.

When the method of the present invention is performed by liquid culture technique, *. *: An antifoaming agent such as silicone oil, vegetable oil or surfactant is preferably used in the culture medium. The pH of the culture medium to form trehatolazine or 2-amino-4- (hydroxymethyl) -3a, 5,6,6a-tetrahydro-4H-cyclopent [d] -oxazole-4,5,6-triol by culturing microorganisms of the genera Micromonospora and Amycolatopsis organisms, especially species SANK 52390 and SANK 60791, preferably ranges from 5.0 to 8.0, more preferably 6.5 to 7.5.

Cultivation can be carried out at any temperature in the range of 15-38 ° C, although a temperature of 22-38 ° C is preferred for good growth and a temperature of 22-28 ° C for trehazoline and 2-amino-4- (hydroxymethyl) -3a, 5,6,6a -tetrahydro-4H-cyclopent [d] -10-oxazole-4,5,6-triol.

These compounds are produced under aerobic culture conditions and by conventional aerobic culture methods such as solid culture, shake culture, and aeration mixed (immersed) culture. In the case of small-scale culture, shaking culture at 28 ° C is a typical method. In such a small-scale culture method, culturing can be initiated by one or two cell division steps that produce seed cultures, for example in Erlenmeyer flasks fitted with baffles that act as fluid flow regulators. The medium for the seed cultivation step preferably contains both carbon and nitrogen sources. In a preferred procedure for such small scale v ′ culture growth, the seed culture flasks are shaken in a constant temperature incubator at 28 ° C for 7 days until sufficient growth is obtained. The grown seed sitten · · • · * ··· * culture is then transferred to another seed medium or production medium. When an intermediate growth phase is used, substantially the same method is used for growth and a portion of the resulting intermediate is introduced into the production medium. The planted ... bottle can be kept in the bath for several days while shaking and after the incubation »· • · !!! the contents of the bottle can be centrifuged or filtered.

In the case of large-scale production, it is preferable to use a suitable «« * *; · * 30 fermentation vessels equipped with mixer and air conditioning. In this case, the nutrient medium can be prepared inside the fermenter. The medium is sterilized • · *: preferably by raising the temperature to 125 ° C; after cooling, sterilized 21 106026 medium may be implanted into previously prepared seed culture. The culture then proceeds with agitation and aeration, for example at 28 ° C. This method is suitable for obtaining large amounts of compounds of the present invention.

Cultivation Progress and Desired Volumes of Desirable Trehhatzoline and 2-Amino-4- (hydroxymethyl) -3a, 5,6,6a-tetrahydro-4H-cyclopent [d] -oxazole-4,5,6-triol as the culture proceeds can be determined by measuring the biological effects of the compounds or high performance liquid chromatography or gas chromatography-10 Ia / mass spectrometry on a purified sample of the culture broth. Trehatoline has an inhibitory effect against silkworm trehalase and its production can be accomplished by monitoring the activity of the culture broth against silkworm trehalase using the techniques described in the following Test Example 3.

On the other hand, the production of 2-amino-4- (hydroxymethyl) -3a, 5,6,6a-tetrahydro-4H-cyclopent [d] -oxazole-4,5,6-triol is best performed by high performance liquid chromatography or gas chromatography / mass spectrometry. . This can be accomplished by contacting the culture broth with a suitable adsorbing resin [e.g. 2q, sold under the trademark Amberlite IRC-50 (NH *)] 2-amino-4- (hydroxymethyl) -? 3a, 5,6,6a-tetrahydro-4H-cyclopent [d] oxazole-4,5, To adsorb 6-triol on a chromatographic column, followed by: washing with water, elution with a suitable: eluent, e.g. 0.5 N aqueous ammonia, concentration of the eluate, e.g.

• · ·: ... * by evaporation under reduced pressure; and lyophilizing the residue to produce a powder of 25- 2 -Amino-4- (hydroxymethyl) -3a, 5,6,6a-tetrahydro-4H-cyclopent [d] -oxazole-4,5,6- The amount of triol in the powder can be measured using high performance liquid chromatography, alternatively, the compound can first be acetylated and 2-amino-4- (hydroxy-4 · · · · · · · · · · · · · · · · The amount of 6α-tetrahydro-4H-cyclopent [d] -oxazole-4,5,6-triol in the powder can be measured using gas chromatography / mass spectrometry.

30 * ··· In general, the amount of trehatoline reaches a maximum between 72 and 150 hours after the onset of fermentation while 2-amino-4- (hydroxymethyl) -3a, 5,6,6a-tetrahydro-. · The amount of ro-4H-cyclopent [d] -oxazole-4,5,6-triol reaches a maximum between 96 and 168 hours after the start of fermentation of 22 106026. However, the exact time will vary with temperature and other fermentation conditions, and the exact optimum time for any combination of conditions can easily be determined by following the production of the desired compound, as suggested above.

5

When a species of the genus Micromonspora is normally used, both trehatoline and 2-amino-4- (hydroxymethyl) -3a, 5,6,6a-tetrahydro-4H-cyclopent [d] -oxazole-4,5,6-triol are formed and these separated by conventional means during the recovery process as described below. Species of the genus Amycolatopsis normally produce only 2-amino-4- (hydroxymethyl) -3a, 5,6,6a-tetrahydro-4H-cyclopent [d] -oxazole-4,5,6-triol. Both compounds are released into the liquid phase, although both are also present in the mycelium. They are most easily recovered from the liquid phase.

Upon completion of the culture, the desired trehatzoline and / or 2-amino-4- (hydroxymethyl) -15β, 5,6,6a-tetrahydro-4H-cyclopent [d] -oxazole-4,5,6-triol are present. liquid broth, may be fractionated by filtration of mycelium and other solids, preferably using diatomaceous earth as a filter aid or centrifugation. These compounds, which are then present in the filtrate or in the floating moiety, can be recovered by extraction and can then be purified by conventional means for their physicochemical properties.

: For example, these compounds may be recovered from the filtrate or float by passing through a column containing an adsorbent such as an ion exchange resin such as Amberlite IRC-50 or CG-50 or Dowex 50WX4 or SBR-P so that either the impurities remain in the resin and are thus removed or the desired compound is retained and then recovered by elution, for example with aqueous ammonia. Examples of other adsorbents include activated charcoal or other adsorbent resins such as Amberlite XAD-2 or XAD-4 (product of Rohm and Haas Co.) or Diaion HP-10, HP- 20, CHP-20, HP-50 (a product of Mitsubishi Kasei Corporation) A solution of • ♦ * ·; · * 30 trehazoline and / or 2-amino-4- (hydroxymethyl) -3a, 5,6,6a- tetrahydro-4H-cyclo * ... * pent [d] -oxazole-4,5,6-triol is passed through an adsorbent layer containing *. "one of these other adsorbents, as described above, so that either the impurities 106026 remain in the adsorbent and thus are removed or the desired compound remains in it and is then recovered by elution with, for example, aqueous methanol, aqueous acetone or the like.

The trehatzoline or 2-amino-4- (hydroxymethyl) -3a, 5,6,6a-tetrahydro-4H-cyclopent [d] -oxazole-4,5,6-triol thus obtained can be further purified by various known techniques. , for example: adsorption column chromatography using a carrier such as silica gel or florisil; split column chromatography using AviceT (product of Asahi Chemical Industry Co., Ltd) or Sephadex LH-20 (product of Pharmacia 10 Inc.); or high performance liquid chromatography using a standard, reversed phase or ion exchange column.

5-amino-1- (hydroxymethyl) cyclopenta-1,2,34-tetraol of formula (I) and 2-amino-4- (hydroxymethyl) -3a, 5,6,6a-15 of formula (II) tetrahydro-4H-cyclopent [d] -oxazole-4,5,6-triol can be prepared by hydrolyzing trehazoline prepared as described above.

This reaction is preferably carried out in the presence of an acid and must be carried out in the presence of water. There is no particular limitation on the quality of the acid used, and any acid commonly used in conventional hydrolysis reactions may be used herein. Examples are inorganic acids such as hydrochloric acid or sulfuric acid and organic acids such as acetic acid or propionic acid in aqueous solution. The preferred acid is hydrochloric acid.

The reaction can take place over a wide range of temperatures and the precise reaction temperature is not critical to the invention. In general, we find it convenient to carry out the reaction at a temperature of from about room temperature to about 120 ° C, more preferably from about 90 ° C to about 100 ° C. The time required for the reaction may also vary widely depending on many factors, such as the reaction temperature and the nature of the reagent and solvent used, and as described below for the desired product.

«· · • · · •« 24 106026

It is to be appreciated that both 5-amino-1- (hydroxymethyl) cyclopentane-1,2,3,4-tetraolo and 2-amino-4- (hydroxymethyl) -3a, 5,6,6a-tetrahydro-4H-cyclopent [d] -oxazole-4,5,6-triol is prepared from the same starting material using the same hydrolysis reaction. Therefore, the product generally contains a mixture of the two compounds. However, it is possible to favor the production of one of these compounds at the expense of the other by appropriate choice of reaction conditions. Thus, generally milder conditions favor the production of 2-amino-4- (hydroxymethyl) -3a, 5,6,6a-tetrahydro-4H-cyclopent [d] oxazole-4,5,6-triol, while more stringent conditions favor -amino-1- (hydroxymethyl) cyclopentane-1,2,3,4-tetraol.

10

Therefore, for the production of 2-amino-4- (hydroxymethyl) -3a, 5,6,6a-tetrahydro-4H-cyclopent [d] oxazole-4,5,6-triol, we prefer to use a relatively mild acidic reagent, e.g. 2N aqueous hydrochloric acid and the reaction is preferably allowed to continue for 1 hour to 2 days, more preferably 5 to 6 hours.

15

On the other hand, to ensure the complete reaction to produce 5-amino-1- (hydroxymethyl) cyclopentane-1,2,3,4-tetraol, it is preferable to use a stronger acid such as 4N aqueous hydrochloric acid and the reaction is preferably allowed to proceed for 1 hour to 2 days, more preferably 20-24 hours.

: ': 20 :: 1 In addition, 5-amino-1- (hydroxymethyl) cyclopentane-1,2,34-tetraol can be prepared

((I

: Hydrolysis of 2-amino-4- (hydroxymethyl) -3a, 5,6,6a-tetrahydro-4H-cyclopent [d] -oxazole-4,5,6- · · · · * ··· 'triol. In this case, the hydrolysis reaction of the present invention may be carried out in the presence of an acid or a base. There is no particular limitation on the quality of the acid or base used, and any acid or base which is conventionally used in hydrolysis reactions can equally be used herein.

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

106026 5 In general, the reaction is advantageously carried out in the presence of a solvent. The reaction is carried out normally and preferably in the presence of a solvent. There is no particular limitation on the nature of the solvent used, provided that it has no adverse effect on the reaction or reagents contained therein and that it is capable of dissolving the reagents, at least to some extent. Examples of suitable solvents include: alcohols such as methanol, ethanol, propanol or butanol; sulfoxides such as dimethyl sulfoxide or sulfolane; organic acids, especially fatty acids such as acetic acid or propionic acid; and water. Preferred solvents include water and mixtures of water and an organic solvent.

The amount of acid used is normally and preferably 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 reaction temperature is not critical to the invention. In general, we find it convenient to carry out the reaction at a temperature of about 20 to about 150 ° C, more preferably 90 to 110 ° C. The reaction time required for the reaction may also vary widely depending on many factors, including: * reaction temperature and the nature of the reagents and solvent used. However, provided that the reaction is carried out under the preferred conditions described above, 1 hour to 2 days, more preferably 20 hours to 30 hours, however, usually * · * * 25 is enough.

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

In general, the reaction is preferably carried out in the presence of a solvent. The reaction is carried out normally and preferably in the presence of a solvent. There is no particular limitation on the nature of the solvent used, provided that it has no adverse effect on the reaction or reagents contained therein and that it is capable of dissolving 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 added is normally and preferably 0.01 to 10 moles, more preferably 1 to 5 moles, for each mole of starting compound.

15

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

The desired compounds of formulas (I) and (II) may be recovered from the reaction mixture by conventional means, which are employed in the preparation of the compounds of formula (I) and (II). for organic compound separation and recovery, for example, various chromatographic techniques, including column chromatography or preparative thin layer chromatography. Various alternatives have been described for • · ”* trehatzoline and 2-amino-4- (hydroxymethyl) -3a, 5, 6,6a-Tetrahydro-4H-cyclopent [d] - * · 1'-oxazole-4,5,6-triol, when prepared by fermentation of · · * ··· '30 and these alternatives can also be used For the isolation and purification of 5-amino-1- (hydroxymethyl-2,4-tetraol) cyclopenta-1,3,4-tetraol obtained by hydrolysis as described above .

27 106026

The present compounds which are 5-amino-1- (hydroxymethyl) cyclopenta-1,2,34-tetraol and the 2-amino-4- (hydroxymethyl) -3a, 5,6,6a-tetrahydro-4H- cyclopentyl-oxazole-4,5,6-triol each contain at least one basic nitrogen atom and can thus form acid addition salts. There is no particular limitation on the quality of these salts, provided that, when intended for therapeutic use, they are pharmaceutically acceptable. When they are intended for non-therapeutic uses, e.g. as vehicles for the preparation of other and possibly most active compounds, this limitation does not apply either. Examples of such acid addition salts are: salts with mineral acids, in particular an acid such as hydrohalic acid (such as hydrofluoric acid, hydrobromic acid, hydroiodic acid or hydrochloric acid), perchloric acid, nitric acid, carbonic acid, sulfuric acid or phosphoric acid; salts with lower alkyl sulfonic acids such as methanesulfonic acid, trifluoromethanesulfonic acid or ethanesulfonic acid; salts with arylsulfonic acids such as benzenesulfonic acid or p-toluenesulfonic acid; salts with organic carboxylic acids such as acetic acid, fumaric acid, tartaric acid, oxalic acid, maleic acid, malic acid, succinic acid or citric acid; and salts with amino acids such as glutamic acid or aspartic acid.

The compounds of the present invention necessarily contain several asymmetric: ': 20 carbon atoms in their molecules and can thus form optical isomers. Although they;.: Are all represented herein by a single molecular formula, the present invention: includes both the isolated isolated isomers and their mixtures, including their «· · • · * ♦. · * Racemates. When stereospecific synthesis techniques or optically active compounds are used as starting materials, the individual isomers can be prepared directly; On the other hand, if a mixture of isomers is prepared, the individual isomers can be obtained by ... conventional separation techniques.

5-Amino-1- (hydroxymethyl) cyclopentane-1,2,3,4-tetraol has the ability to inhibit the action of β- * • · · · ··· * glucosidase and is of low toxicity and is therefore expected to be useful in the treatment and prevention of tumors and AIDS. 2-Amino-η 4: 4- (hydroxymethyl) -3a, 5,6,6a-tetrahydro-4H-cyclopent [d] oxazole-4,5,6-triol has a · · · · **: the ability to inhibit the action of sucrose and its low toxicity and is expected to be useful in the treatment and prevention of diabetes and obesity.

When intended for therapeutic use, these compounds may be administered alone or in a suitable pharmaceutical formulation containing, apart from the active compound, one or more conventional diluents, carriers or excipients. The quality of the formulation will, of course, depend on the intended dosing route. However, for oral administration, the compound is preferably formulated as powders, granules, tablets or capsules. For parenteral administration, it is preferably formulated as a syringe. These preparations can be prepared in a known manner by the addition of additives such as carriers, binders, disintegrants, lubricants, stabilizers and corrective agents. Although the dosage may vary according to the patient's symptoms and age, the nature and severity of the disease or disorder, and the mode and route of administration for oral administration to 15 adult human patients, the compounds of the present invention will normally be administered in a daily dose of 1-100 mg. The compounds may be administered in a single dose or in multiple doses, for example, two or three times daily.

The preparation of the compounds of the present invention is further illustrated by the following non-limiting examples.

(«(<<<« EXAMPLE IV 1 • · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · Micromonospora loop SANK 62390 was used to implant three «500 ml Erlenmeyer flasks with 80 * spacers and each containing 80 ml« · «• * * · * 30 spacers 1 (the composition of which is specified below) and inoculated bottles were incubated • «« '· ...: at 2 8 ° C for 216 hours in a rotary shaker capable of rotating at 220 • · •.' *: Rpm of the first to obtain a seed crop.

29 106026 Medium 1:

Glucose 1%

Glycerin 1% 5 Oatmeal 0,5%

Sucrose 1%

Soya bean flour 2%

Casino Acids 0.5%

Compressed Yeast 1% 10 CaCO 3 0.1% CB442 0.01%

Water up to 100% (pH 7.0 before sterilization).

Percentages are calculated by weight based on the final volume of the medium.

Each of the four 2 liter Erlenmeyer flasks, each containing 800 ml of medium 2 (the composition of which is specified below), was inoculated with 40 ml of culture broth from the first 20 bottles and shaken at 28 ° C for 96 hours with a rotary shaking on a machine rotating at 220 rpm to obtain a second seed culture, 1.5 liters of this second seed broth were used to plant each of two 30 liter • · i • · '· «· * vibratory pots each containing 15 liters of medium 2 (which the composition is • # * ♦ *** · 'specified below), which was first sterilized at 120 ° C for 35 minutes and • i »* ·' * 25 cooled to 28 ° C. The fermentation solutions were stirred at 100 rpm ... at 28 ° C for 96 hours, aired at a flow rate of 15 liters per minute

• · • · ···

• M

• • • • • • • • • • • • • • • • •

IM

• »» • *% ««) · «« «I» • * · • · 30 106026 Medium 2

Glucose 2%

Soluble starch 1% 5 Pressed yeast 0.9%

Meat extract (Kyokuto) 0,5%

Polypeptone 0.5%

NaCl 0.5%

CaCO3 0.3% 10 CB442 0.01%

Water up to 100% (pH 7.2 before sterilization).

The percentages are calculated by weight based on the final volume of the medium.

(B) Recovery 3 kg of Celite 545 Filtration Auxiliary (trademarked by Johns Manville Products Corp.;: for 20 products) was added to 30 liters of total broth obtained as described above and the filtrate was filtered. The filtrate (29 liters) was allowed to pass through a column containing 6

: * * *: liters Dowex SBR-P (Cl ') (Trademark of Dow Chemical). PH of the eluate

::: was then adjusted to 5.0, after which the solution was allowed to pass through the column,

··· I

• m · · containing 6 liters of Dowex WX4 (H) (trademark for Dow Chemicak product).

Trehatzoline was adsorbed and thus retained on the column. The column was washed with 20 liters of deionized • · * · ... · water and then eluted with 30 liters of 0.5 N aqueous ammonia to give 13 * * * • · ... * liters of active fractions. All this eluate (13 liters) was concentrated by evaporation under reduced pressure and lyophilized to give 43.3 g of crude product containing. The crude product was dissolved in 2 liters of 10 mM ammonium formate buffer (pH 6.0) and the solution was adsorbed onto a column containing 1.5 liters of Dowex · 50WX4 (trademark) previously equilibrated with 20 mM ammonium 1F formate buffer solution (pH 6.0) The column was washed with 3 liters of the same 20 mM ammonium formate buffer solution 106026 followed by 2 liters deionized water, followed by elution with 0.2 N aqueous ammonia. Fractions 2-4, which contained virtually all of the trehazoline, were combined and concentrated by evaporation under reduced pressure, and the product was lyophilized to give 2.55 g of crude pulp 5 The crude powder prepared by repeating the steps so far was used directly without any further purification, in Example 3.

This crude powder was adsorbed onto a column packed with 200 ml AviceF (trademark of Asahi Chemical Industry Co., Ltd.) using 80 t / v aqueous 10 acetonitrile. The column was washed with 700 ml of 80% v / v aqueous acetonitrile and then eluted first with 500 ml of 75% v / v aqueous acetonitrile and then with 700 ml of 75% v / v aqueous acetonitrile. The eluate was divided into 19 ml portions. Fractions 35-50 containing trehazoline were pooled and concentrated by evaporation under reduced pressure. The residue was then lyophilized to give 658 mg of powder. All this powder was dissolved in 150 ml of water and the resulting solution was adjusted to pH 6.0. The solution was then adsorbed onto a column packed with 300 ml of Amerlite CG-50 (H 2 = 3: Trademark). This column was washed with 500 ml deionized water and eluted with 0.1 N aqueous ammonia. The eluate was divided into 20 ml portions. Fractions 92-121 containing trehazoline were combined and concentrated by evaporation under reduced pressure. The resulting residue was then lyophilized to give (102.8 mg of powder. This whole powder was dissolved in 10 ml of 2 mM ammonium “: Earth buffer solution (pH 6.0) and the resulting solution were adsorbed onto a ···: V: packed with 400 ml Diaion CHP20P (trademark of Mitsubishi Kasei Co.) • · ·::: 25 using the same 2 mM ammonium formate buffer solution The column was eluted with the same 1 mM ammonium formate buffer solution The eluate was divided into 5 ml portions Fractions ♦ ♦ ·: ... · 59-73 containing threhatoline were combined and concentrated by evaporation under reduced pressure. to give 18 mg of powder which was further purified by elution through Diaion CHP20P using: 30 of the same 2 mM ammonium formate buffer as a lumen to give 6.2 mg. * * *. colorless powder.

106026 This powder was purified by preparative thin layer chromatography as follows. The powder (6.2 mg) was dissolved in a small amount of water and applied to 3 plates of silica gel (Merch Art 5715, 20 x 20 cm). The plates were developed with a 6: 1: 3 by volume mixture of acetonitrile, acetic acid and water to a height of 15 cm. The strip between Rf 5 0.42 and 0.5 was scraped off the plates and packed on a column. The column was eluted with 100 mL deionized water. The eluate was allowed to pass through a column containing 5 ml of Dowex 50WX4 (H), where the trehatzoline was adsorbed and then trapped. The column was washed with deionized water and then eluted with 50 volumes of 0.5 N aqueous ammonia. The eluate was concentrated by evaporation under reduced pressure, and the resulting residue was lyophilized to give 5.1 mg of trehatoline as a colorless powder with a single peak in high performance liquid chromatography.

The product had the following characteristics: 1) Form and appearance: colorless base powder; 2) Solubility: soluble in water and methanol, insoluble in acetone and chloroform; '; ; 20 3) Color test: positive for sulfuric acid; : * *: 4) Molecular formula; • · 5) Molecular Weight: 366 (determined by FAB Mass Spectroscopy - “FAB” is Fast • · · · Atom Bombardment) 25 6) Specific Rotation: [α] 1 → + 99.5 ° (c = 0.41, t → O ); • · • · «

Ultraviolet Absorption Spectrum: λιη & χ nm (E ^^ '' '. The ultraviolet absorption spectrum measured in water has no characteristic absorption maxima above 220 nm; • · 33 106026 δ) Infrared absorption spectrum: λmax (In KBr) 3367, 2938, 1663, 1552, 1384 and 1056; 9) 1 H-Nuclear Magnetic Resonance Spectrum: δ ppm.

The 5 H-Nuclear Magnetic Resonance Spectrum (400 MHz) was measured in deuterium oxide using TMS (tetramethylsilane) as the external standard and obtained the following signals: 3.22 (1H, two doublets, J = 8.98 & 10.31 Hz); Δ 3.38 (1H, multiplet, J = 2.44, 5.25 & 10.31 Hz); 3.46 (1H, two doublets, J = 8.98 & 9.99 Hz); 3.53 (1H, doublet, J = 11.96 Hz); 3.55 (1H, two doublets, J = 5.26 & 12.21 Hz); 3.57 (1H, two doublets, J = 5.38 & 9.99 Hz); 3.62 (1H, two doublets, J = 2.44 & 12.21 Hz); 3.63 (1H, doublet, J = 11.96 Hz); 3.77 (1H, doublet, J = 4.89 Hz); 4.02 (1H, two doublets, J = 2.08 & 4.89 Hz); , ..: 4.17 (1H, doublet, J = 8.55 Hz); &Lt; 20 4.77 (1H, doublet, J = 5.38 Hz); * 1 λ 10) C-nuclear magnetic resonance spectrum; δ ppm, · · · · 1 * 5 C nuclear magnetic resonance spectrum (100 MHz) was measured in deuterium oxide * * T using tetramethylsilane as the external standard and the following signals were obtained: O 60.7, 61.9, 69.6 , 72.0, 73.0, 73.0, 80.1, 80.3, 80.6, 82.8, 87.3 and 161.1.

«·» »« »11 11: 11) High Performance Liquid Chromatography: 4 4": Separation Column: Senshu Pak ODS-H-2151 (Senshu Scientific Co.), 6 φ (?) X 150. mm (5 μ); · ·: Mobile phase: 10% (v / v) acetonitrile water containing 0.5% PIC B8 (Waters • ·

Inc. product); 34 106026

Flow rate: 1.5 ml / min;

Set wavelength: 210 nm ultraviolet;

A peak with a retention time of 6.9 minutes at a column temperature of 25 ° C was observed; and 5 12) Thin layer chromatography:

Rf value: 0.44;

Adsorbent: silica gel on glass plate (Merck Art 5715);

Development solvent: 6: 1: 3 v / v acetonitrile / acetic acid / water.

10 EXAMPLE 2 Preparation of 5-Amino-1- (hydroxymethyl) cyclopentane-1,2,3,4-tetraol A solution of 19.3 mg of trehatzoline (prepared as described in Example 1 above) was dissolved in 1 ml. 4N aqueous hydrochloric acid was placed in an ampoule and hydrolyzed by heating at 100 ° C for 24 hours. The reaction mixture was then mixed with water and concentrated to dryness by evaporation under reduced pressure. The residue was again mixed with water and evaporated to dryness by evaporation under reduced pressure to remove the hydrochloric acid. The residue from this distillation was dissolved in 20 ml of water and the pH of the solution was adjusted to 6.0. As a result. . the resulting solution was allowed to pass through 20 mL of Amberlite CG-50 (NH 4) - Trademark -

through the column and the column was washed with 60 ml deionized water and then eluted with 0.2 N

• ·: '· *: 25 with aqueous ammonia. The eluate was concentrated by evaporation under reduced pressure and the residue lyophilized to give 5.1 mg of crude 5-amino-1- (hydroxymethyl) cyclopentane-1,2,3,4-tetraol as a colorless powder.

· »« • · ·. The crude powdery 5-amino-1- (hydroxymethyl) cyclopentane-1,2,3,4-tetraol obtained as described above was completely dissolved in a small amount of water and, ···, purified by preparative thin layer chromatography as follows. . The solvent was applied to two plates of silica gel (Merck Art 5715 20 x 20 cm) and developed using a 6: 1: 3 by volume mixture of acetnitrile, acetic acid and water at 15 cm height. Blue Rf between 0.36 and 0.47 was scraped off and packed on a column which was then eluted with 50 ml deionized water. The eluate was allowed to pass through 10 ml of Amberlite CG-50 (NH *), 5-aminoethyl) cyclopentane-1,2,3,4-tetraol adsorbed on 5 columns. The column was then washed with 50 mL deionized water and eluted with 50 mL 0.2 N aqueous ammonia. The eluate was concentrated by evaporation under reduced pressure and lyophilized to give 3 mg of the desired 5-amino-1- (hydroxymethyl) cyclopenta-n-1,2,3,4-tetraol as a colorless powder having the following characteristics: 10) Color and appearance: colorless base powder; 2) Solubility: soluble in water, insoluble in acetone and chloroform; 3) Color test: positive for ninhydrin reaction; 4) Molecular Formula; C ^ H ^ NO ^; 5) Molecular Weight: 179 (determined by FAB mass spectroscopy); 6) Specific rotation: + 3.7 ° (c = 0.51, H2O); 7) Ultraviolet Absorption Spectrum: λmax nm (λmax) The # 25 UV Ultraviolet Absorption Spectrum, measured in water, does not show any natural absorption maxima at wavelengths greater than 210 nm; 8) Infrared Absorption Spectrum: umax cm -1 (in KBr) • · · - ·· '·; 3384, 1582, 1474, 1380 and 1040.

• * ·. ·. 30 • «·. * ··. 9) 1 H-Nuclear Magnetic Resonance Spectrum: δ ppm.

1 H Nuclear Magnetic Resonance Spectrum (400 MHz) was measured in deuterium oxide using tetramethylsilane as the external standard and the following signals were obtained: 36 106026 3.13 (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); Δ 3.78 (1H, two doublets, J = 5.5 & 6.8 Hz); 3.90 (1H, two doublets, J = 5.5 & 7.1 Hz); 10) C-nuclear magnetic resonance spectrum; δ ppm, C-Nuclear Magnetic Resonance Spectrum (100 MHz) was measured in deuterium oxy-10 using tetramethylsilane as the external standard and the following signals were obtained: 57.8, 61.0, 73.6, 79.4, 81.5 and 81.7; and 11) Thin layer chromatography: 15 Rf value: 0.39;

Adsorbent: silica gel on glass plate (Merck Art 5715);

Development solvent: 6: 1: 3 v / v acetonitrile / acetic acid / water.

EXAMPLE 3 7:20. '. Preparation of 2-Amino-4- (hydroxymethyl) -3a, 5,6,6a-tetrahydro-4H-cyclopent [d] oxazole-4,5,6-t triol. 100 g of crude product prepared as described in Example 1 and estimated

225 g of trehatzoline were dissolved in a mixture of 100 ml of water and 150 ml of 4N

Aqueous hydrochloric acid and the pH of the resulting solution was adjusted to 2.5 by adding 4N aqueous hydrochloric acid. 8 ml of concentrated hydrochloric acid and 72 ml of water were then added to the solution to give a total volume of 480 ml and a concentration of hydrochloric acid:> 0.2 N was obtained. The resulting solution was placed in a round bottom flask and then hydrolysed in an oil bath maintained at 100 ° C for 6 hours. After this time, the reaction mixture was washed with water and concentrated to dryness by evaporation under reduced pressure. These sequential water mixing and evaporation operations to dry 37106026 were repeated to remove the hydrochloric acid. The residue was dissolved in 400 ml of water and the pH of the solution was adjusted to pH 6.0 by the addition of 1 N aqueous sodium hydroxide solution. The solution was then diluted in water to a volume of 8 liters. The resulting solution was allowed to pass through a column packed with 600 ml of Amberlite CG-50 (NH 4) and the column was washed with 6 liters 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 allowed to pass through a column packed with 800 ml Dowex 1X2 (OH-). The column was then eluted with deionized water. After removing the first 10 1.5 liters of eluate, the subsequent eluate was divided into 20 ml portions. Each fraction was subjected to the quantitative analysis described below to determine whether it contained the desired compound. Fractions 70-110 containing this compound were combined and concentrated by evaporation under reduced pressure. The residue was lyophilized to give 30 mg of 2-amino-4- (hydroxymethyl) -3a, 5,6,6a-tetrahydro-4H-cyclo-15 pent [d] oxazole-4,5,6-triol as a colorless powder. had the following characteristics: 1) Form and appearance: colorless base powder; 2) Solubility: soluble in water and methanol, insoluble in acetone and chloro-, - form; I (<I <(I '4 3) Molecular Formula; 4 · Molecular Weight: 204 (determined by FAB mass spectroscopy); • - • · · A -::: 25 5) Specific rotation: [α] 20 D + 10.0 ° (c = 0.51, ^ O); Ultraviolet Absorption Spectrum: Xmax nm (E ^ I 4) "· · · ··· The Ultraviolet Absorption Spectrum, measured in water, has nothing of nature. / "teenage absorption maxima above 210 nm; 38106026 7) Infrared absorption spectrum: υ" "ν cm" ^ (in KBr) IId 3358, 1668, 1528, 1398 and 1066; 8) 1 H-Nuclear Magnetic Resonance Spectrum: 6 ppm.

The 5 H-Nuclear Magnetic Resonance Spectrum (500 MHz) was measured in deuterium oxide using TSP (sodium trimethylsilyl propionate) as the external standard and obtained the following signals: 3.73 (1H, doublet, J = 11.72 Hz); Δ 3.82 (1H, doublet, J = 12.21 Hz); 3.97 (1H, doublet, J = 4.4 Hz); 4.23 (1H, two doublets, J = 4.4 & 2.44 Hz); 4.37 (1H, doublet, J = 8.79 Hz); 5.03 (1H, two doublets, J = 8.79 & 2.44 Hz); 9) High Performance Liquid Chromatography:

Separation column: Asahi Pak ES-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;

Set wavelength: 210 nm ultraviolet; : Temperature: 25 ° C; • · ·

Retention time: 8.39 minutes.

2-Amino-4- (hydroxymethyl) -3a, 5,6,6a-tetrahydro-4H-cyclopent [d] oxazole-4,5,6- *. quantitative analysis of triol using gas chromatography / mass spectrometry »c: ,,, 'The above quantitative analyzes were carried out as follows: 4 · 4 The sample was dissolved in a solvent (water) of known volume. 10 μΐ of the resulting solution was placed in a vessel and evaporated to dryness for acetylation.

30 39 106026 30 μΐ acetic anhydride and 50 μΐ pyridine were added to the residue and the resulting mixture was heated at 60 ° C for 40 minutes. Any excess of reagents was 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-5D-glucose) and the mixture was dissolved in 100 μl of ethyl acetate to make a test sample for gas chromatography / mass spectrometry. Analyzes were performed using a combined silica capillary column (product of J & W Scientific Co., DB-5 15 meters) as a gas chromatography column. A test sample (2 μΐ) was injected and the column temperature raised from 60 ° C to 280 ° C at 10 25 ° C / minute. Negative ions were recorded by a chemical ionization method using methane gas as a kuadrupol mass spectrometer Trio-1 (product of VG). Negative ion peaks at m / z 388 (corresponding to internal standard Pentaacetyl compound) and m / z 413 [corresponding to 2-amino-4- (hydroxymethyl) -3a, 5,6,6a-tetrahydro-4H-cyclopenta [d ] oxazole-4,5,6-triol Pentaacetate] was used for quantitative analysis. The content of 2-amino-4- (hydroxymethyl) -3a, 5,6,6a-tetrahydro-4H-cyclopenta [d] oxazole-4,5,6-triol was calculated by the internal standard method.

EXAMPLE 4 2-Ammo-4- (hydroxymethyl) -3a, 5,6,6a-tetrahydro-4H-cyclopent [d] oxazole. Preparation of <4,5,6-triol «Il» · *: Y; (A) Cultivation 25 stitches taken from the sloping surface of SANK 60791 species of Amycolatopsis were used to inoculate two · · · 500 ml Erlenmeyer flasks, each fitted with partitions and • containing 80 ml of medium 1 (of the composition given in Example 1 above) and the inoculated flasks were incubated at 28 ° C for 96 hours in a rotating shaker rotating at 210 rpm seed culture broth. 30 to obtain.

• · I I · 40 106026

Two 30 liter shaking starter vessels each containing 15 liters of medium 2 (the composition given in Example 1 above) were sterilized at 120 ° C for 30 minutes. They were then cooled to 28 ° C and 75 ml of seed culture broth were placed in each shaking fermentation vessel. The shake fermentation vessels were then stirred at 5 ° C at 28 ° C, adjusted to 100-400 rpm (2 ppm to maintain dissolved oxygen) for 144 hours and air-conditioned at a flow rate of 7.5 liters per minute.

(B) Recovery 10 1.5 kg of Celite 545 filter aid was added to 25 liters of whole broth [obtained as described in (A) above] and the mixture was filtered to obtain a 23 liter filtrate. The filtrate was adjusted to pH 6.0 by addition of aqueous hydrochloric acid and the solution allowed to pass through a column packed with 3 liters of Amberlite IRC-50 (NH 4) 2-amino-4- (hydroxymethyl) -3a, 5,6,6a-tetrahydro For adsorption of 4 H -cyclopent [d] oxazole-4,5,6-triol. The column was washed with 15 liters of deionized water and then eluted with 0.5 N aqueous ammonia. After the eluate had become alkaline, 4.5 liters of eluate was collected and concentrated by evaporation under reduced pressure to a volume of 200 ml. The concentrate was allowed to pass through a column packed with 450 ml Dowex 1X2 (OH ") and eluted with deionized water. The first 800 ml of eluate was removed and the subsequent eluate was split into 20 ml portions. Determined by qualitative analysis using either high performance liquid chromatography. as described later or by gas chromatography / mass spectrometry that fractions 60-90 · 25 contained the desired 2-amino-4- (hydroxymethyl) -3a, 5,6,6a-tetrahydro-4H-cyclopent [ d] oxazole-4,5,6-triol and so these fractions were combined and concentrated by evaporation under reduced pressure to lyophilize the concentrate to give 16.6 mg of 2- [beta] -amino-4- (hydroxymethyl) ) -3a, 5,6,6a-tetrahydro-4H-cyclopent [d] oxazole-4,5,6-triol; as a colorless powder having the characteristics described above.

r: 30 • «· • • I · f 4 • · •« «• · ·« 1 · • · 41 106026

Quantitative analysis using high performance liquid chromatography:

Separation column: Asahi pak ES-502C (product of Asahi Chemical Industry Co., Ltd.); Mobile phase: 20 mM ammonium acetate (pH 8.5) + 50 mM saline; Flow rate: 1 ml / minute Assay wavelength: 210 nm; Temperature: 25 ° C;

Retention time: 8.39 minutes.

10

Quantitative analysis using gas chromatography / mass spectrometry

The above quantitative analysis was performed using essentially the same procedures as described in Example 3.

EXAMPLE 5 2-Amino-4- (hydroxymethyl) -3a, 5,6,6a-tetrahydro-4H-cyclopent [d] oxazole-4,5,6-; Preparation of 20 triols

c i I

<tt (<(A) Cultivation) A portion of the sloping surface of SANK 62390 of the Micromonospora species was homogenized in 10 ml of 25 saline to make a suspension. 1 ml of the suspension was inoculated into each. : *** into two 2 liter Erlenmeyer flasks, each fitted with a baffle plate and ··· each containing 500 ml of intermediate medium 3 (of the composition given below). «*; and the inoculated flasks were incubated at 28 ° C for 96 hours on a rotary shaker.

I I I

: '' '· At a rotation speed of 210 rpm to make the first seed broth.

Ill 30 • I «« · «· I» ♦ • · ♦ • · 42 106026 Medium 3:

Glucose 2%

Yeast Extract (Difco) 0.5% 5 Polypepton 0.5%

CaCO 3 0.1% CB-442 0.01% water up to 100% (pH 7.2 before sterilization).

10

Percentages are calculated by weight based on the final volume of the medium.

30 liters of medium 3 were placed in a 60 liter shaking fermenter and sterilized at 120 ° C for 30 minutes. It was then cooled to 28 ° C and 600 ml of the first seed culture broth were inoculated. The fermenter was agitated at 165 rpm at 28 ° C for 48 hours by aerating at a rate of 15 liters per minute to produce a second seed culture.

A 20,600 liter container containing 300 liters of medium 4 (the composition given below) was sterilized at 120 ° C for 35 minutes. It was then cooled to 28 ° C and 15 liters of another seed culture broth were planted there. Container • ·. ···. was then stirred at 28 ° C at a speed adjusted to 82-142 rpm · · · · · (to maintain 2 ppm dissolved oxygen) for 144 hours by aerating at 150 liters per ***** 2 at a flow rate of 25 minutes and an internal pressure of 0.5 kg / cm.

• ♦ · • ♦ • · • · «♦ ♦ €« <* «4« 4 4 4 • 4 4 4 4 4 · · «4« · «· 4 • 4« «4« · 43 106026 Medium 4:

Glucose 8% (previously sterilized at 120 ° C for 15 minutes) 5 Lustergen FK * 2%

Compressed Yeast 1.8%

Meat extract (Kyokuto) 1%

Polypeptone 1%

NaCl 0.5% 10 CaCO3 0.3% K2HPO4 0.25% CB-442 0.02% water to 10% pH 7.2 before sterilization.

15 * Trade name for starch grade sold by Nichiden Kagaku Co. Ltd.

(B) Recovery 20 kg of Celite 545 filter aid was added to 300 liters of whole broth prepared as described above and the mixture was filtered to obtain a 290 liter filtrate.

.. # · # The pH of the 20 liter filtrate was adjusted to 6.0 by the addition of aqueous hydrochloric acid.

• ·. ···. The resulting solution was allowed to pass through a column packed with 3 liters • · «··,

Amerlite IRC-50 (NH.) And the desired 2-amino-4- (hydroxymethyl) -3a, 5,6,6a-tetrahydro-. * · * 4. ·: ·. The 4H-cyclopent [d] oxazole-4,5,6-triol was maintained on the column by adsorption. The column was washed with 15 liters of deionized water and eluted with 0.5 N aqueous ammonia. After the eluate had become alkaline, 4.5 liters of eluate were collected and concentrated under reduced pressure to a volume of 150 ml. The concentrate was allowed to pass through II »1«. . ·. 30 columns packed with 500 ml Dowex 1X2 (OH ') and eluted with deionized water. The first 1 liter was removed and the subsequent eluate was split into 20 ml portions. Each fraction was subjected to quantitative analysis as described in Example 3. Fractions 58-80 were found to contain the active compound and these fractions were combined and concentrated by evaporation under reduced pressure. The residue was lyophilized to give 9.6 mg of 2-amino-4- (hydroxymethyl) -3a, 5,6,6a-tetrahydro-4H-cyclopent [d] oxazole-4,5,6-triol as a crude powder. The powder was again purified by column chromatography using a column packed with 100 mL Dowex 1X2 (OH ') eluted with deionized water to give 4.8 mg of 2-amino-4- (hydroxymethyl) -3a, 5, 6,6a-tetrahydro-4H-cyclopent [d] oxazole-4,5,6-triol as a colorless powder having the characteristics described above.

EXAMPLE 6 10 Preparation of 5-Amino-1- (hydroxymethyl) cyclopentane-1,2,3,4-tetraol

A solution of 16 mg of powdered 2-amino-4- (hydroxymethyl) -3a, 5,6,6a-tetrahydro-4H-cyclopent [d] oxazole-4,5,6-triol was dissolved in 2 ml of 6N aqueous hydrochloric acid, sealed in ampoules and hydrolyzed by heating at 100 ° C for 24 hours. Water was then 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 addition of 1 N aqueous sodium hydroxide solution. The solution was allowed to pass through 20 mL of Amberlite CG-50 (NH 4) to retain 5-amino-1- (hydroxymethyl-1,3) -cyclopentane-1,2,3,4-tetraol by adsorption and the column was washed. • 1. · · ·. 60 ml deionized water was then eluted with 0.2 N aqueous ammonia solution. The eluate was concentrated by evaporation under reduced pressure to give 5 ml of a concentrate. This concentrate was applied to a column packed with 50 mL Dowex 1X2 (OH ") and the column was washed with 200 mL deionized water and then eluted with 20% v / v aqueous methanol. The eluate was divided into 5 mL portions. · Had inhibitory activity against β-glucosidase, pooled and concentrated by evaporation under reduced pressure, The concentrate was lyophilized to give 6.2 mg of 511, 11 ', amino-1- (hydroxymethyl) cyclopentane-1,2, 3,4-tetraol as a colorless powder having the above-described properties.

• · · · • · 45 106026

TEST EXAMPLE 1 BIOLOGICAL EFFECT

5-Ammo-1- (hydroxymethyl) cyclopentane-1,2,3,4-tetraolrain inhibitory activity against β-glucosidase (isolated from almonds), p-nitrophenyl-BD-glucopyranoside, deoxynojirimycin and castanspermine experiment, 10 were obtained 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.

15

A mixture of 0.01 units / ml β-glucosidase and 100 μΐ buffer solution (pH 5.6) comprising 20 mM citric acid, 40 mM disodium phosphate and test compound was allowed to stand at 37 ° C for 15 minutes. Subsequently, 50 μΐ of buffer solution containing 3 mg / ml of p-nitrophenyl-β-D-glucopyranoside was added to the mixture which was then reacted at 37 ° C for 20 minutes. 20 μΐ 1 M Glycine / Sodium. , the hydroxide buffer solution (pH 10.4) was added to the reaction mixture and the amount of p-nitrophenol released .... was recorded by absorbance at 405 nm. Table 7 • ·. ···. lists the concentrations of each test compound required to inhibit the β-glucosidase activity by 50% (IC ^ q).

25 • • • • • • • • * 46 106026

Table 7

Concentration required for 50% inhibition of β-glucosidase

Compound (I) 1.0 Mg (ml

Deoxynojirimycin 15 μg / ml

Chestanospermine 4.3 μg / ml 10-TEST EXAMPLE 2 2-Amino-4- (hydroxymethyl) cyclopentane-3a, 5,6,6a-tetrahydro-4H-cyclo-15 pent [d] dioxazole-4,5,6-triol [Compound (II)] Inhibitory Activity Against Rat Sucrose

According to the method of M. Kessler et al. [Biochimica et Biophysica Acta, 506 136-154 (1978)], a "brush border" (brush border = gut, the 20 surface of the fabric lankalisäkkeen surface alkalimamodifiointi) membrane enzyme solution of rat small intestiinistä male rat, three Wistar species of the small intestine and suspended in 3 ml of physiological saline.

A sample of 4-aminoantipyrin (A 4382) was obtained from Sigma Chemical Co. and peroxide · · V: 25 Dase samples (grade I) and glucose oxidase (grade I) were obtained from Boehringer.

· Mannheim Co. Buffer solution (pH 6.2) consisting of 20 mM citric acid and 40 mM

disodium phosphate, was used as diluent in the following experiment.

Each well of a microtiter plate containing 96 wells (Falcon Co.

',,,, 30 te) was filled with 130 silicon containing 0.2 mg bovine serum albumin (Sigma, A

<7906), 3 units glucose oxidase, 0.132 units peroxidase, 20 μg 4-: aminoantipyrin, 40 μg phenol, 3 μmol sucrose and test compound. The recess • «· * '. Was added to 20 μΐ 100-fold dilution of rat small intestine brush border rajamembraanientsyy- 106 026 47 min the solution. The mixture was allowed to react at 37 ° C for 20 minutes and the amount of glucose released was measured at 492 nm.

When enzyme reactions were performed without addition of test compound and addition of enzyme-5 solution, glucose concentrations were assumed to be 0-100% inhibition. The concentration of Compound (II) to 50% inhibition (IC50) of rat sucrose activity was found to be 18 pg / ml.

TEST EXAMPLE 3 10

Inhibitory effect of trachezoline against silkworm trehalase 10 fifth generation silkworm larvae (total weight 44 g) were homogenized using a Polytrone (trademark) homogenizer in 120 ml of buffer solution 15 (pH 5.6) prepared with 20 mM citric acid and 40 mM citric acid. minutes while cooling with ice (the same buffer was used thereafter). The homogenized mixture was then centrifuged at 6000 rpm for 10 minutes and the float was separated. 240 ml of acetone was added to 120 ml of the floating portion while cooling with ice and stirring, and the resulting mixture was centrifuged at 20,000 at 9000 rpm for 20 minutes. The sediment was separated and dissolved in water and the resulting solution was lyophilized to give 2.0 g of crude enzyme.

130 µl of the above buffer solution, 50 µl of the sample solution containing treazoline at various concentrations and 50 µl of the silkworm enzyme solution prepared as described above containing 4 mg / ml enzyme was placed in a test tube and the mixture was shaken in a water bath maintained at 37 ° C for 15 minutes. Subsequently, 20 μΐ of a 250 mM solution of trehalose was added and the mixture allowed to react for · 15 minutes. The reaction mixture was then heated in a boiling water bath for 3 minutes, after which it was cooled with ice water. It was then centrifuged at 3000 rpm <i r 30 for 10 minutes, and the resulting sediment-depleted solution was used. for the determination of glucose concentration.

• · • · • · «<· •« 48 106026

The reaction was carried out using glucose C-test Wako (product from Wako Pure Chemical Industries Ltd.) and 10 times the sample solution compared to the standard method. The inhibitory rate was calculated from the glucose concentrations obtained by using a buffer solution instead of a sample solution and using a buffer solution instead of a substrate-5 solution that was 0-100% inhibition to calculate the concentration required to inhibit 50% enzyme activity (IC50). 0 ng / ml.

· «• * •« «• * * * * · · · ·

• M

• • • · # # # # # # # # # # #

(I

4 I <4 »t« «II ·« · · I «·« ·

Claims (3)

A process for the preparation of 2-amino-4- (hydroxymethyl) -3a, 5,6,6a-tetrahydro-4H-cyclopent [d] oxazole-4,5,6-triol of the formula (II) ) or a pharmaceutically acceptable site thereof (D) Characterized in that the microorganism Micromonospora sp. SANK 62390, FERM BP-3521 or mutants thereof with substantially the same morphological and biochemical properties, or the microorganism Amycolatopsis sp. SANK 60791, FERM BP-3513 or mutants thereof having substantially the same morphological and biochemical properties, capable of producing said 2-amino-4- (hydroxymethyl) -3a, 5,6,6a-tetrahydro-4H cyclopent [ d] oxazole-4,5,6-triol, is grown and said 2-amino-4- (hydroxymethyl) -3a, 5,6,6a-tetrahydro-4H-cyclopent [d] oxazole-4,5,6-triol is separated from the culture medium and optionally the sage of the product is formed. 20 Method according to claim 1, characterized in that the microorganism 3 is Micromonospora sp. SANK 62390, FERM BP-3521. • • • • • • • ♦ ♦ Method according to claim 1, characterized in that the microorganism is Amycolatopsis sp. SANK 60791, FERM BP-3513. • · · «* • * • · · t« * I I t I <I • * • * «•« · * · • · * • · • · * • ·