WO2006017976A1

WO2006017976A1 - Crystalline form of (+)-(1 s,2s,3 s,4r,sr)-1-amino-s-hydroxymethyl-n-2-(1,3-dihydroxypropyl)-2,3,4-cyclohexanetriol and its preparation method

Info

Publication number: WO2006017976A1
Application number: PCT/CN2005/001169
Authority: WO
Inventors: Hongjie Chang; Jiayong Zhu; Kaidong Feng
Original assignee: Shenzhen Taitai Pharmaceutical Industry Ltd.
Priority date: 2004-08-16
Filing date: 2005-08-01
Publication date: 2006-02-23
Also published as: CN1736977B; CN1736977A

Abstract

The present invention relates to a new crystalline -form of (+)-(1 S,2S,3S,4R,SR)-1-amino-5-hydroxymethyl-N-2-(1,3-dihydroxypro pyl)-2,3,4-cyclohexanetriol, which has stake morphology and specific melting point and also has property required for industrial synthesis and formulation. The new crystalline form of the present invention has good α-glucosidase inhibitory activity.

Description

Crystalline form of 2g, 3S, 4I ₅ amino-5-hydroxymethyl-Μ-2-ί1, 3-dihydroxypropyl)-2,

3, 4-cyclohexanetriol anti-preparation method

The present invention relates to a novel crystalline form of the compound (+)-(lS, 2S, 3S, 4R, 5R)-1-amino-5-hydroxymethyl-N-2-(l,3-dihydroxypropyl)-2 , 3, 4-cyclohexyl-triol (hereinafter referred to as crystalline form _{(+) -? (lS,} 23, 3S 4R, 5R) -1- amino-5-hydroxymethyl -N-2- (l, 3- Dihydroxypropyl) -2,3,4-cyclohexanetriol).

The present invention also relates to (+)-(lS, 2S, 3S, 4R, 5R)-1-amino-5-hydroxyindenyl-N-2-(l,3-dihydroxypropyl) containing the crystalline form of the present invention. A pharmaceutical composition of -2,3, 4-cyclohexanetriol.

The present invention also relates to the preparation of crystalline forms of (+)-(lS, 2S, 3S, 4R, 5R)-1-amino-5-hydroxyindolyl-N-2-(l,3-dihydroxypropyl)-2 , 3, 4-cyclohexanetriol method. Background technique

Compound ((+)-(lS, 2S, 3S, 4R, 5R) small amino-5-hydroxyindenyl-indole-2-(1,3-dihydroxypropyl)-2,3,4-cyclohexyl Alcohol (also known as valibose) is an alpha-glucosidase inhibitor that inhibits the intestinal disaccharide hydrolase (α-glucosidase) and delays sugar after oral administration. Digestion and absorption of the class to improve postprandial hyperglycemia. Postprandial hyperglycemia can cause impaired insulin secretion and insulin action, further worsening glucose metabolism, so correcting postprandial hyperglycemia is preventing diabetes and complications from developing. At the same time, ct-glycosidase inhibitors are the most tolerated drugs in oral hypoglycemic agents. They have mild hypoglycemic effects, are less prone to hypoglycemia and have fewer side effects. They are more suitable for the elderly. Relevant information disclosures of the preparation methods are: US Patent US 4,701,559; US 4,777,294; US 4,803,303»

In U.S. Patent No. 4,803,303, the preparation of the above-mentioned compound is specifically mentioned. The method uses Validamine and dihydroxyacetone, sodium cyanoborohydride, using hydrochloric acid as a catalyst, reacting in dimercaptosulfoxide, and freezing the product to obtain Amorphous (+)-(lS, 2S, 3S, 4R, 5R)-1-amino-5-hydroxymethyl-N-2-(l,3-dihydroxypropyl)-2, 3, 4- Cyclohexanetriol powder, yield 17%. The specific method is to add 2.0 g of Validamine, 3.4 g of dihydroxyacetone, 1.5 ml of 2N hydrochloric acid solution and 2.6 g of sodium cyanoborohydride in 50 ml of dimethyl sulfoxide at 60 to 65. After stirring for 16 hours, dimethyl sulfoxide was distilled off under reduced pressure, the residue was dissolved in 100 ml of water, and the solution was passed through an Amberlite H+ type CG-50 column. Washed with water, 0.5N aqueous ammonia, the eluate was concentrated under reduced pressure, and the concentrate was subjected to Dowex i X 2 0ΪΓ chromogen speak column, eluting with ₅ water eluate was concentrated under reduced pressure and lyophilized to give a white 0.5g Valibose powder. Formulas (I) and (II) are the structural formulas of Validamine and Valibose, respectively:

In large-scale industrial production, the above amorphous compound is a raw material of the pharmaceutical industry because of low yield, high cost of separation and purification, and the preparation conditions are very demanding, and the process conditions such as lyophilization are required, which leads to costly. Moreover, the amorphous powder is extremely hygroscopic, which limits its application in industrial large-scale pharmaceutical applications.

It is an object of the present invention to provide (+)-(lS, 2S, 3S, 4R, 5R)-1-enyl-5-hydroxyindenyl-N-2-(l,3-dihydroxypropyl) in crystalline form - 2, 3 , 4-cyclohexanetriol, which has the properties required for large-scale synthesis or formulation of therapeutic agents.

Another object of the present invention is to provide a pharmaceutical composition comprising weiligose of the crystalline form of the present invention.

Still another object of the present invention is to provide a process for preparing a crystalline form of weiligose which can produce a weilipoose crystal in a high yield and which has a good purification effect and is suitable for mass production.

According to an aspect of the present invention, there is provided a crystalline form of a weiliposaccharide compound having the following physical properties: (1) a powder X-ray diffraction pattern when subjected to powder X-ray diffraction using a Cu-Kct radiation source Characteristic peaks expressed by diffraction angle 2 显示 are displayed at least at 19.5 - 19.9 and/or at 21.8 - 22.2. Further included are significant peaks at about 13.24 ~ 13.26, about 14.54 ~ 14.64, about 15.50 ~ 15.62, about 17.04 ~ 17.12, about 23.42 ~ 23.5, about 23.94 ~ 24.00, about 25.92 - 25.98, about 26.80 - 26.86, and about 29.16 ~ 29.18. (2) Its DSC endothermic transition is about 89. C; (3) The specific rotation is [ a ] _D ²⁵ + (77-79) °. The present invention also provides pharmaceutically acceptable salts of the crystalline form of weiligose, including, but not limited to, hydrochlorides, picrates, sulfates, phosphates, acetates, maleates, and the like. In one embodiment of the present invention, ₃ provides a crystalline form of weiliposaccharide hydrochloride having the following physicochemical properties: (1) when powder X-ray diffraction is carried out using a Cu-Κα radiation source, the powder X thereof - The ray diffraction pattern shows a characteristic peak expressed by a diffraction angle of 2 至少 at least at 20.01 - 20.5 and/or at 23.8 - 24.2. In addition, it has obvious peaks at about 10.94~10.98, about 13.22 ~ 13.28, about 16.14 ~ 16.16, about 23.28 ~ 23.32 and about 25.02 - 25.08; (2) its DSC endothermic transition is about 144.3 °C; (3) Degree is [ a ] _D ²⁵ +53. .

In another embodiment of the invention, a virion sugar picrate salt in crystalline form is provided. The present invention also provides a pharmaceutical composition comprising a crystalline form of weilipoose or a pharmaceutically acceptable salt thereof, which can be added to a crystalline form of weiligose or a pharmaceutically acceptable salt thereof in a pharmaceutically acceptable manner. The carrier and/or excipients are prepared as a pharmaceutical composition for treating diabetes.

According to still another aspect of the present invention, there is provided a method for preparing a crystalline form of weiligose, which is characterized by a formula (chemical name: (+)-(lS, 2S, 3S, 4R, 5R) small amino-5-hydroxyindole) -2,3,4-cyclohexanetriol, formula I) is reacted with dihydroxyacetone to form a Schiff base (Schiff base), and then reduced with sodium cyanoborohydride to obtain the virgin sugar of the present invention (Formula II) ), the reaction is as follows:

In the method of the present invention, the raw material Validamine is prepared by hydrolysis of chloramphenicol A to obtain validoxylamine A (J ^1⁄2^^ cs 2¥.' 57-58, 1971), and then by validoxylamine oxime and N-brominated The diimide (NBS) reaction gives a validamine {Chem.Lett, 1989, 725-728) with the following reaction formula:

Jinggangmycin A Validoxylamine A

The product obtained by the above reaction was separated to obtain pure Validamine (the compound of the formula I) and Valienamine, respectively.

It has been proved by in vitro and in vivo experiments that the crystalline form of the veoliose of the present invention has a good α-glucosidase inhibitor activity, while the crystalline form of weiliposaccharide hydrochloride and picrate is in vitro against α-glucosidase. The inhibition of α-amylase activity is basically consistent with the crystalline form of weiligose.

According to the large-scale preparation test, the preparation of the crystalline form of the weiligose of the present invention is convenient, the raw material has stable properties, good compressibility, easy molding, and excellent performance in the preparation process.

The method for preparing a crystalline form of weiligose of the present invention comprises the steps of:

1) Using glacial acetic acid as a catalyst and anhydrous methanol as a solvent, (+)-(lS, 2S, 3S, 4R,

5R)-1-Amino-5-hydroxyindolyl-2,3,4-cyclohexanetriol is reacted with dihydroxyacetone and sodium cyanoborohydride to give (+)-(lS, 2S, 3S, 4R, 5R) Small amino-5-hydroxymethyl-N-2-(l,3-dihydroxypropyl)-2,3,4-cyclohexanetriol;

2) after removing the solvent obtained in the above step 1), dissolved in water and acidified, and then neutralized with a base to about pH 6.0; 3) Separate ₅ by the following columns in sequence and trace the detection by TLC;

Strongly acidic cation exchange resin;

Weakly acidic cation exchange resin;

Strongly basic anion exchange resin;

4) collecting the product and concentrating it to a thousand;

5) Dissolving the product obtained in the step 4) with methanol, ethanol or a mixture thereof, and adding seed crystal (Williolose) to crystallize the crystal form (+>(1S, 2S, 3S, 4R, 5R) Small amino-5-hydroxyindenyl-N-2-(l,3-dihydroxypropyl)-2,3,4-cyclohexanetriol.

In the method of the present invention, it is preferred to use glacial acetic acid in an amount of 8 to 25 % (glacial acetic acid volume/raw material (+)-(lS, 2S, 3S, 4R, 5R) small amino-5-hydroxyindenyl-2, 3 The weight of 4-cyclohexanetriol, the same applies hereinafter, and more preferably the amount of glacial acetic acid is 20%. The reaction of the step 1) is carried out under anhydrous conditions, thereby effectively increasing the yield of the product, and the use of anhydrous methanol avoids product decomposition, yield reduction and separation difficulty caused by heating of the product under high heat for a long time. On the one hand, the post-processing operation is simplified, making the concentration operation simple and easy.

In the method of the present invention, the compound prepared by separation and purification using an ion exchange resin column can be purified by sequentially using the following ion exchange resin column:

(1) H+ type strongly acidic cation exchange resin column, eluted with water and ammonia water;

(2) NH type weakly acidic cation exchange resin column, water or ammonia water elution;

(3) OH_ type strong basic anion exchange resin column, water elution;

Wherein the H + -type strongly acidic cation exchange resin column may be selected, for example, 001 x 7 (732) or PUROLITE C100E, and the ammonia concentration is 0.5 N; the N3⁄4+ weakly acidic cation exchange resin column may be selected, for example, YD CG-50 Or HD-2 or Amberlite CG-50, the ammonia concentration is 0.1N; the column type strong alkaline anion exchange resin column can be selected, for example, Dowexl x 2 or PUROLITE A 400 or HZ-202; considering resin properties and production costs In terms of factors, separation and purification are preferably carried out using PUROLITE C100E, HD-2 and HZ-202.

The invention will be described in detail below with reference to the accompanying drawings, in which FIG. DRAWINGS

Figure 1 is a crystal form of the invention of the viscose sugar ^-NMR nuclear magnetic resonance spectrum; 2 is a ¹³ C-NMR nuclear magnetic resonance spectrum of a crystalline form of weiligose in the present invention;

Figure 3 is a powder X-ray diffraction pattern of the crystalline form of the virgin sugar of the present invention;

Figure 4 is a powder X-ray diffraction pattern of another batch of crystalline form of Wolliose of the present invention; Figure 5 is a powder X-ray diffraction pattern of another batch of crystalline form of Wolliose of the present invention; Figure 6 is a DSC chart of the crystalline form of the weiligose of the present invention;

Figure 7 is a H-MR nuclear magnetic resonance spectrum of the crystalline form of the veolipodose hydrochloride of the present invention; Figure 8 is a ¹³ C-NMR nuclear magnetic resonance spectrum of the crystalline form of the veolipodose hydrochloride of the present invention. Figure 9 is a powder X-ray diffraction pattern of the crystalline form of the veoliose hydrochloride salt of the present invention; Figure 10 is a powder X-ray diffraction pattern of another batch of crystalline form of the veoliose hydrochloride salt of the present invention; ;

Figure 11 is a powder X-ray diffraction pattern of another batch of crystalline form of weiliposaccharide hydrochloride of the present invention;

Figure 12 is a DSC spectrum of the crystalline form of weiliposaccharide hydrochloride of the present invention;

Figure 13 is a graph showing the effect of the crystalline form of Wolliose on the blood glucose profile of a normal mouse after sucrose loading;

In the figure: Nor is a normal mouse control group, Acar is a positive control drug, Baitangping group (10 mg/kg), TD-01 (0.2 mg), TD-01 (1 mg), TD-01 (5 mg) For the dose group of weibopoose (TD-01), the dose was divided into '0.0 mg/kg, 1 mg/kg, 5 mg/kg; n = 10

Figure 14 is a graph showing the effect of the crystalline form of weiligose on the area under the blood glucose curve (AUC) of normal mice after sucrose loading;

In the figure: Nor is a control group, Acar is a positive drug in the Tangtang group (10 mg/kg), TD-01 (0.2 mg)^ TD-01 (1 mg), TD-01 (5 mg) is a Wei Libo The dose of saccharide (TD-01) was 0.2 mg/kg ^ 1 mg/kg and 5 mg/kg, respectively; compared with Nor, ***p < 0.001; n = 10 Figure 15 is the crystalline form of the present invention. The effect of weilipoposaccharide on blood glucose curve after sucrose loading in alloxan hyperglycemic mice;

In the figure: Con is the control group, Acar is a positive drug, Baitangping group (10 mg/kg), TD-01 (0.2 mg), TD-01 (1 mg), TD-01 (5 mg) is Wei Libo. The dose of sugar (TD-01) was 0.2 mg/kg, 1 mg/kg and 5 mg/kg, respectively; n = 10

Figure 16 is a graph showing the effect of the crystalline form of weiligose on the area under the blood glucose curve (AUC) of sucrose-loaded mice in alloxan hyperglycemia; In the figure: Con is the control group ₅ Acar is a positive drug, Baitangping group (10 mg/kg), TD-01 (0.2 mg), TD-01 (1 mg), TD-01 (5 mg) is Wei Libo sugar (TD-01) administration groups at doses of 0.2 mg / lcg, 1 mg / kg and 5 mg / kg; ₃ ** comparison with control group ^{Con:; i p <0.001;} n = 10 DETAILED DESCRIPTION

Preparation of crystalline form 2S, 3§, 43R, amino-5-hydroxyindole 3-dihydroxypropyl)-2,3,4-cyclohexanetriol I Example 1 2 Preparation of Validamine

The starting material, Validamine, can be prepared by methods disclosed in the prior art documents, for example: J. Antibiotics 24: 57-58, 1971 and Chem. Lett, 1989, 725-728 (Harii, sj T. Iwasa, method, present In the preparation process of the invention, the raw material Validamine is prepared by hydrolysis of valentin A (purchased from Zhejiang Qianjiang Biochemical Co., Ltd., content 60%) to obtain validoxylamine A, and then by validoxylamine A and odorant uglyimine ( NBS) (Shanghai Wulian Chemical Factory, chemically pure) reacts to obtain validamine. The reaction formula is as follows:

Jinggangmycin A Validoxylamine A

Validoxylarnine A Validamine Valienamine The reaction product was passed through a CG-50 weakly acidic cation exchange resin column and eluted with 0.1 N aqueous ammonia to obtain pure Validamine and Valienamine.

[I Example 2] Preparation of crystalline form of Wolliose

The reaction formula is as follows:

Validamine Schiff base weiligose in 2600 ml of anhydrous decyl alcohol (analytical grade), adding Validamine 104 g (586.9 mmol), 1,3-dihydroxyacetone 81.8 g (FRUKA 97%, 880.8 mmol), ice 10.4 ml of acetic acid and 76.5 g of sodium cyanoborohydride (97%, 1.181 mol of Yingkou Sanzheng Chemical Co., Ltd.) were refluxed for 6 hours. After completion of the reaction, methanol was distilled off, water (500 ml) was added, acidified to pH 1.5 with a 2N hydrochloric acid solution, and then neutralized to pH 6 with a 1N sodium hydroxide solution. PUROLITE CIOOE H+ type strong acid cation exchange resin column (water washing and 0.5N ammonia water elution), YD CG-50 NH ₄ ⁺ type weakly acidic cation exchange resin column (0.1N ammonia elution) and HZ-202 OH- Type strong alkaline anion exchange resin column (water elution) separation, TLC tracking detection (chromatography plate: Merck Silica gel 60; developing agent: n-butanol: methanol: chloroform: ammonia = 4: 5: 2: 5; Toner: 0.2% ninhydrin ethanol solution; Rf = 0.46 (validamine); Rf = 0.52 (valibose). Combined with ninhydrin and positive with Valibose The single-spot fraction having the same Rf value was concentrated to dryness under reduced pressure, dissolved in ethanol, and crystallized to give 93 g. Yield: 63.1%, mp: 88-89 °C. Yield calculation:

Where: W: amount of feed or yield M: molecular weight [Example 33 Preparation of crystallized form of Wolliose

In ²⁵ 00 ml of anhydrous methanol (analytical grade), add 100 g of Validamine (564.3 亳mol), raise the temperature to 5 CTC, add 63 g of 1,3 dihydroxyacetone (: FRUKA 97%, 678.4 mmol), glacial acetic acid 20 ml 55 g of sodium cyanoborohydride (97%, 849.3 mmol) of Yingkou Sanzheng Chemical Co., Ltd., refluxing for 6 hours. After the reaction was completed, the methanol was distilled off, 500 ml of water was added, acidified to H 1.5 with a 2N hydrochloric acid solution, and neutralized to pH 6.0 with a 1 N sodium hydroxide solution, followed by a (PUROLITE C100E) H+ type strongly acidic cation exchange resin column ( Water washing and 0.5N ammonia elution), YD CG-50 H type weakly acidic cation exchange resin column (0.1N ammonia elution) and HZ-202 OH-type strong basic anion exchange resin column (water elution) separation, TLC tracking test (chromatography plate: Merck Silica gel 60; developing solvent: n-butanol: sterol: chloroform: ammonia = 4: 5: 2: 5; developer: 0.2% ninhydrin ethanol solution; Rf = 0.46 (validamine); Rf = 0.52 (valibose)). A single spot component which is positive for ninhydrin and has the same Rf value as the Valibose reference. It is concentrated to dryness under reduced pressure, and dissolved in a mixed solution of methanol and ethanol (volume ratio sterol: ethanol = 1: 2). Crystallization, get 121 grams. Yield: 85.3%, mp: 88-89 °C. [Example 4] Preparation of crystalline form of Wolliose

In 1000 ml of anhydrous methanol (analytical grade), 40 g of Validamine (225.7 mmol) was added, and the temperature was raised to 50 ° C. 25.2 g of 1,3-dihydroxyacetone (FRUKA 97%, 271.4 mmol), glacial acetic acid 8 was added. ML, sodium cyanoborohydride 22 g (Yingkou Sanzheng Chemical Co., Ltd. 97%, 339.7 mmol), reflux reaction for 6 hours. After the reaction was completed, methanol was distilled off, 200 ml of water was added, acidified to pH 1.5 with a 2N hydrochloric acid solution, and neutralized to pH 6.0 with a 1 N sodium hydroxide solution, followed by a (PUROLITE C100E) H ⁺ type strongly acidic cation exchange resin column ( Water washing and 0.5N ammonia elution :), YD CG-50 N3⁄4+ weakly acidic cation exchange resin column (water elution) and HZ-202 ΟΗ' strong Basic anion exchange resin column (water elution) separation, TLC tracking detection (chromatography plate: Merck Silica gel 60; developing solvent: n-butanol: methanol: chloroform: ammonia = 4: 5: 2: 5; chromogenic reagent : 0.2% ninhydrin ethanol solution; Rf = 0.46 (validamine); Rf = 0.52 (valibose)). The single spot component which was positive for ninhydrin and had the same Rf value as the Valibose reference was concentrated under reduced pressure to dryness _{5 and} dissolved by ethanol to crystallize ₅ crystals to obtain 47.7 g. Yield: 84.1%, mp: 88-89.

E Comparative Example 2 Preparation of amorphous weilipoose

Instruments: United States VIRTIS 2K freeze dryer

Operation: 3 g of a sample of weilipodose concentrated to dryness was diluted into a solution of 5%, 10%, 15%, 20%, 25%, and each 3 ml was placed in a lyophilized bottle having a volume of 10 ml. After pre-freezing at -55 °C for 4 hours, it was lyophilized at the same temperature and a vacuum of 40 mtorr for 24 hours or more to obtain a transparent gel. None of the five samples obtained the white powder described in the literature (US 4,803,303).

The comparative examples illustrate that it is very difficult to prepare an amorphous powder of weilipoose in accordance with the currently known methods. Physical properties of crystalline form of Wolliose

[Example 5] Determination of the physical properties of the granules of the crystalline form

First, the obtained crystalline form of the compound was subjected to nuclear magnetic resonance analysis and elemental analysis, and it was confirmed that the compound obtained by the present invention is (+)-( 1 S , 2S, 3S, 4R, 5R) small amino-5-hydroxyindenyl- Ν-2-(1 , 3-dihydroxypropyl) -2, 3 , 4-cyclohexanetriol, followed by powder X-ray diffraction analysis and differential scanning calorimetry using powder X-ray diffraction (XRD) And DSC differential thermal analysis to characterize the crystal of the compound of the present invention, and further, the obtained compound crystal is subjected to measurement of melting point and specific rotation, and subjected to mass spectrometry, infrared spectrum analysis and solubility measurement to obtain a compound related to the present invention. More information on crystals.

1. Nuclear magnetic resonance analysis

Instrument: Varian 500

Results: See Figure 1 and Figure 2

^-NMR (D ₂ 0, 500MHZ) δ

1.32(1H, ddt, J=3,1.5,15), 1.80-1.87(lH,m), 1.92(1H, dt ₅ J=3 ₃ 15), 2.78(lH,m,J=5.5), 3.15( 1H, dd, J=3.5), 3.25(1H, dd, J=10.5). 3.50-3.55(2H, m), 61 80·9 9£'P\ Ζ Li S0"9 W \ £91 60·9 ZZ£ 99·9 Z'£ll Π 89'9 W£it 9Z L9'9 9Z'£l I

°I/I 1 P QZ °I/I f P ΘΖ °I/I P

£ τ Ϊ

ΪΗ^ a 措^待 χ ^mnm ^ 11

:1土^3⁄4^ '令晷节 ® θΗΧ^^^^^ψ '

1⁄2 V6Z - 9V6Z 98"9Z ~ 08·9 B ^, 86Ή ~ Z6'9Z, 00'W ~ WiZ

9-£Z~ZV£Z ^ ZYL\ ~WL\ ^ 29^l~0S^l ^ ^9 ΐ ~^ t 9Γ£Ι ~ Ρ ίΙ

Ra~8"TZ 6·6Ι~ ·6ΐ Ding Festival Θ3 * 02

5ffi~e

/οΖ0 : Broken

. 09 - Ζ = θ Ζ '( YSO l

^W - χ νιπ-χνΐΛΐ/α ^ 3⁄4t β :n

^-i ^ ^ , ϊ

. ^ ^^ 边管υ]^1⁄2 '3⁄4^^畔 "^ X f(翁县尜 οΐ

3⁄4 ^ 卿tf if ^ ' fjf 3⁄4 ^a ^ -t 1⁄2 w ^£ Ψ ^峰^4持郑狞

'Ν -L - £'8 Ή

3⁄4ι ) H*3⁄4

IS'S ⁶ R 'Ζ1'8 Ή '·08· 1 Ό (%)

Peak ia °TM ^A

( ⁹ OM ^T 3⁄4 ^0I D ) , τ sf£' L 'IZ'PL 'WiL ^C 0 Z9 Ί£'ΐ9 ^Γ6ξ '£9'ζζ 'A6"8£ '19' Ζ

S (ΖΉΜξί '03⁄4) ΉΜΜ-Ο _ει

•(Π

69ll00/S00ZN3/X3d 9.6.Ϊ0/900Ζ OAV 3 15.50 5.71 14 4 15.62 5.67 12 3 15.52 5.70 18

4 17.04 5.20 35 4 17.12 5.17 25 4 17.06 5.19 41

6 19.66 4.51 诵 6 19.72 4.50 74 5 19,70 4.50

7 21.§8 face 7 22.0 4.04 100 2L88 4 bird S

8 23.42 3.80 20 8 23.5 3.78 18 7 23.42 3.80 23

9 23.98 3.71 24 9 23.94 3.71 37 8 24.00 3.70 30

10 25.92 3.43 12 10 25.98 3.43 9 9 25.94 3.43 12

11 26.80 3.32 16 11 26.86 3.32 14 10 26.82 3.32 17

12 29.16 3.06 11 14 29.18 3.06 26 13 29.16 3.06 12

4, differential scanning calorimetry (DSC)

Instruments: Germany NETZSCH DSC2004

Range: 40-140 ° C

Heating rate: 10 ° C / min

The crystalline form of the veoliose of the present invention has a DSC endothermic transition at about 89 ° C, and its DSC spectrum is shown as 6.

5, melting point determination

Instruments: Switzerland BRUKER BUCHI Melting Point B-545

Measurement results: melting point 88 ~ 89 ° C

6, specific rotation measurement

Instrument: Polartronic HHW5 polarimeter

Test temperature: 25+0.5 °C

Sample tube length: 100mm

Incident light wavelength: 589.3 Xia

Test solvent: water (C = l)

As a result of the measurement: [ a ] _D ²⁵ + (77-79).

7, mass spectrometry

Instrument model: UK VG company ZAB-HS double focus magnetic mass spectrometer

Determination conditions: fast atom bombardment ionization (FAB)

Atlas data: see Table 2

Table 2 Mass spectrometric analysis results of the crystalline form of Wolliose Mass-to-charge ratio (m/z) relative abundance (%) Remarks

252 100

220 26 M-CH ₂ OH

154 72

136 62

107 26

89 29

77 33

57 22

8, infrared absorption spectrum

Instruments: Germany Bruker Fourier Transform Infrared Optical-Infrared Microscope

Figure borrowing analysis: see Table 3

Table 3 Infrared Absorption Spectra of Crystalline Morphology

9, solubility determination

According to the method stipulated in the Second Edition of the Pharmacopoeia of the People's Republic of China 2000 (the case), accurately weigh the amount of the control powder (prepared according to the above method), and determine its solubility at room temperature (25 ° C). The results are shown in Table 4. . The results show that the crystalline form of the weiligose prepared by the present invention is highly soluble in water. Soluble in methanol, slightly soluble in ethanol.

Crystalline form of veoliose hydrochloride

Example 6 Preparation of the hydrochloride salt of the virgin sugar of the crystal form and determination of its physical properties 2 g of the crystal form of the virose sugar (prepared according to the method in the previous examples), dissolved in water, and added dropwise with stirring 2N hydrochloric acid solution, to pH 3, continue to stir for 2 hours, elute through an activated carbon column, elute with water, collect the eluate (TLC tracking), concentrate the obtained sample to dryness, recrystallize with methanol, filter, dry, One gram of the crystalline form of the veoliose hydrochloride was obtained. Molecular formula: C _1Q H ₂₂ N0 ₆ C1

Determination of physical properties:

1. Nuclear magnetic resonance analysis

Instrument: Varian 500

Results: See Figure 7 and Figure 8

1H-NMR (D ₂ 0, 500MHZ) δ

1.58-1.68 (2Η, m) ₃ 2.03(1H, d, J=14.4). 3.21(1H, t, J=9.3), 3.37-3.51 (2H,m), 3.55-3.59(2H, m), 3.60 -3.70 (4 m), 3.72-3.78 (2H, m);

1 ³ CN R (D ₂ 0, 75MHZ) δ

26.78, 40.69, 59.68, 60.25, 60.38, 63.97, 64.03, 72.63, 74.26, 76.43

2, elemental analysis (of^NOeCl)

Instruments: Germany ELEMENTAR vario EL Element Analyzer

Calculated values: ( % ) C, 41.74; H, 7.70; N, 4.89; CI, 12.30

Found (twice): (%) C, 41.56/41.58; H, 7.61/7.64; N, 4.55/4.60; C1,

12.20/12.23

3, powder X-ray diffraction analysis

Instrument: Japan Science Motor D/MAX-IIIAX ray powder diffractometer Target: Cu-Κα radiation (λ = 1.5405A), 2 θ = 2 - 60.

Tube pressure: 35KV

Tube flow: 25mA

Step scan: 0.027 steps

Filter: Graphite Monochromator

The three batches of the crystal form of the veoliose hydrochloride prepared by the present invention have an XRD pattern substantially as shown in FIG. 9 to FIG. 11 , and the crystal form of the veoliose crystal hydrochloride is visible from the graph. According to the diffraction angle 2Θ, the powder X-ray diffraction pattern using Cu-Kot radiation has characteristic peaks at 20.10 - 20.5 and 23.8 ~ 24.2, and their relative intensities reach 97 ~ 100 and 58 ~ 65, respectively. About 10.94~L0.98, about 13.22 ~ 13.28, about 16.14 ~ 16.16, about 23.28 ~ 23.32 and about 25.02 ~ 25.08 have obvious peaks, and the measured samples of each batch basically coincide, the results are shown in Table 5:

Table 5 Powders of crystalline form of weiliposaccharide hydrochloride X-ray diffraction pattern

Sample batch

twenty three

Peak 2 Θ d value I/Io peak 2 Θ d value I/Io peak 2 Θ d value I/Io

1 10.98 8.05 23 1 10.98 8.05 27 1 10.94 8.08 22

3 13.26 6.67 30 2 13.28 6.66 46 3 13.22 6.69 37

5 16.16 5.48 20 3 16.16 5.48 7 5 16.14 5.49 20

7 20.24 4.38 100 6 20.28 4.38 100 8 20.22 4.39 97

8 23.30 3.81 29 7 23.32 3.81 13 9 23.28 3.82 57

9 23.94 3.71 58 8 23.96 3.71 65 10 23.90 3.72 100

10 25.08 3.55 32 9 25.08 3.55 17 12 25.02 3.56 31

4, differential scanning calorimetry (DSC)

Instruments: Germany NETZSCH DSC2004

Range: 40-160Ό

Heating rate: 10 ° C / min

The crystalline form of the veoliose sugar hydrochloride of the present invention has a DSC endothermic transition at about 144.3 Å,

The DSC spectrum is shown in Figure 12.

5, melting point determination Instruments: Swiss BRU ER BUCHI Melting Point B-545

Measurement results: melting point 145~146 °C

6, specific rotation measurement

Instrument: Polartronic HHW5 polarimeter

Test temperature: 25±0.5°C

Sample tube length: 100mm

Incident light wavelength: 589.3nm

Test solvent: water ( C = 1 )

Measurement results: [a] _D ²⁵ +53. Crystalline form of weiliposaccharide picrate

实施Example 72 Preparation of the picric acid salt of the virgin sugar of crystalline form and determination of its physical properties

1. Preparation method:

Violone (1.55 g, 6.17 mmol) was dissolved in 20 ml of methanol, and 2,4,6-trinitrophenol (2.82 g: 12.31 mmol) was added. The mixture was heated to reflux for 4 hours and then stirred at room temperature. The crude product was recrystallized from 80% sterol.

Molecular formula: C ₁₆ H ₂₄ N ₄ 0 ₁₃

2. Determination of melting point

Instruments: Switzerland BRUKER BUCHI Melting Point B-545

Determination results: melting point 163.5-165.5 ° C

3. Elemental analysis ( C ₁₆ H ₂₄ N ₄ 0 ₁₃ )

Instruments: Germany ELEMENTAR company varioEL element analyzer

Calculated: (%) C, 40.00; H, 5.04; N, 11.66

Found: (%) C, 39.92; H, 5.28; N, 11.57

4. Nuclear magnetic resonance analysis

Instrument: Varian500

1H-NMR (D ₂ 0, 500MHZ) δ

1.62-1.73 (m, 2H ), 2.10 (m, 1H ), 3.20-3.30 (m, 1H),

3.38-3.48 ( m, 2H ), 3.54-3.62 ( m, 2H ), 3.62-3.84 ( m, 6H ),

8.60 (s, 2H ); "C-NMR (D ₂ 0 ₅ 75MHZ) δ

25.18, 39.10, 58.03 ₅ 58.63, 58.75, 62.32, 62.38, 70.99, 72.60 ₃ 74.80, 127.86, 128.54, 141.85, 163.21;

5. Mass spectrometry

Instrument: US API2000 mass spectrometer

ESI-MS m/z: 503 ( M ⁺ +1+Na).

6. Specific rotation measurement

Instrument: Polartronic HHW5 polarimeter

Test temperature: 25±0.5°C

Sample tube length: 100mm

Incident light wavelength: 589.3nm

Test solvent: water ( C = 1 )

Determination results: [ a ] _D ²⁵ +36 ° Crystalline morphology of the effect of vitamin Pipose

实施Example 8]

The crystalline form of the weiligose prepared by the foregoing examples of the present invention is an experimental drug, and the crystalline form of the weiligose and the a-glucosidase inhibitors Acarbose and Bexin are compared in vitro. Voglibose) inhibited a-glucosidase (sucrose, maltase) and α-amylase, and found that the crystal form of weilipoose has a significant inhibitory effect on sucrase and maltase activities, IC ₅₀ of 1.28, respectively. l (T ⁸ mol / L and 3.18 x 10- ⁷ mol / L, of α - amylase had no effect (10 ⁴ mol / L), it indicates that the force-dimensional wave form of sugar crystallization inhibitor is a disaccharide. In vivo experiments were performed with normal ICR mice and alloxan hyperglycemia mice, and Acarbose was used as a positive control drug to compare the effects of weilipoose on blood glucose elevation after sucrose loading, demonstrating the dimensionality of crystalline morphology. Liposaccharide can significantly reduce the peak blood glucose level and the area under the blood glucose curve, and shift the peak blood glucose level, indicating that the crystal form of the weiligose can inhibit the intestinal disaccharidase, ie, α-glucosidase, so that the carbon water The last part of the compound digestion process The stage is the hydrolysis of the disaccharide to a monosaccharide delay, thereby delaying the rate of glucose absorption into the blood.

First, the experimental materials

Test drug The crystalline form of Wolliose is a chemically synthesized compound prepared according to the method of the previous examples. The batch number is 20030801, the molecular weight is 251.28, and the purity is 99.8%. The experiment is carried out with deionized water to prepare the desired concentration.

2. Positive control drug

Acarbose (trade name: Bay Tang) is the world's first alpha-glucoside inhibitor introduced by Bayer Pharmaceuticals in Germany in the early 1990s. It is now one of the most commonly used oral hypoglycemic agents in the clinic. Selected as a positive control drug. Bai Tangping is the product of Bayer Company of Germany, batch number 101101, the raw material medicine (in vitro) is the gift of Bayer Company of Germany, the batch number is 264086D, and the required concentration is prepared by deionized water in the experiment.

Voglibose (trade name: xinxin) A new generation of α-glucosidase inhibitor from Japan Takeda Pharmaceutical Co., Ltd., batch number 20040304, prepared with deionized water to prepare the required concentration.

Second, experimental animals

1. Normal male ICR mice, 22-24g; normal male SD rats, 140-160g, all purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd., license number SCXK (Beijing) 2002-0003, raised in China Secondary animal house of the Institute of Medicine, Academy of Medical Sciences.

2. Alloxan hyperglycemia mice normal ICR mice intravenous injection of alloxan 70 mg / kg, 72 hours after the prediction of blood glucose (glucose oxidase method, the same below), the choice of blood glucose at 250-500 mg / The mice of dl were subjected to experimental grouping. Third, test methods and results (including experimental design, main experimental procedures and experimental results, etc.)

1. Inhibition of ex-glucosidase activity by Wei Lipoose

Sucrose and maltose are decomposed into glucose by the action of α-glucosidase. By measuring the amount of glucose in the reaction system, the inhibitory effect of weiligose on α-glucosidase activity was determined, and the IC _{50 was} calculated according to the percentage inhibition of enzyme activity at different concentrations of the drug to measure the test drug against the enzyme. Inhibitory intensity of activity. Reference methods were used to isolate invertase and maltase from the upper intestinal mucosa of normal rats for inhibitor studies.

The results in Table 6 indicate that the IC ₅ Q of the inhibitory effect of weilipoose on the activity of invertase is 1.28 X 10' ⁸ M, which is stronger than that of Baitangping and weaker than that of Baixin; the IC _5G for inhibiting maltase activity is 3.18 X 10 — ⁷ M, the effect is weaker than Bai Tang Ping and Bei Xin.

Table 6. Inhibition of α-glucosidase (sucrose, maltase) by weilipodose IC ₅₀ ( mol/L)

Test compound ——

Sucrase maltase dimensional wave force - Sugar 1.28 X 10- ⁸ 3.18 10 ^'7

Acarbose 1.35 χ 10— ⁷ 6.08 χ 10” ⁸

Voglibose 3.62 χ 10" ⁹ 3.84 10" ⁸

2. Inhibition of α-amylase activity by Wei Lipoose

Starch decomposes under the action of amylase ₅ By determining the amount of starch remaining in the reaction system (iodine-starch method), it is determined whether or not the weiligose has an inhibitory effect on amylase activity.

The results show that at a concentration of 10- ⁴ mol / L, and glucose-dimensional wave forces on Basen did not inhibit the amylase activity; Acarbose IC Inhibition of amylase activity I ι + _5. It is 3.74 xl (T ⁶ mol/L. It suggests that the side effects of Wei Libo sugar flatulence may be much smaller than that of Bai Tang Ping and similar to Bei Xin.

3. Effect of Wei Lipoose on the increase of blood glucose after sucrose load in normal mice

Five groups of normal ICR mice, 10 in each group, fasted overnight before the experiment, a group of oral sucrose solution (4.0 g kg) as a control group (Nor), a group of oral sucrose and Acarbose 10 mg/kg As a positive drug control group (Acar), the other three groups were orally administered with sucrose and different doses of weiligose (0.2 mg/kg, 1 mg/kg, 5 mg/kg) as the administration group. Blood glucose levels were measured before administration (0 min) and at 30 min, 60 min, and 120 min after administration.

Table 7. Effect of Wolliose on blood glucose after sucrose loading in normal mice (X ± SD) Dose Blood glucose (mg/dl)

Group

Mg/kg 0 min 30 min 60 min 120 min

Nor a 79.7 ± 9.5 247.9 ± 38.3 181.5 ± 34.5 122.3 + 15.7

128.5

Acar 10 76.1 + 14.2 109.3 ± 14.1

34.5

Wei Libo 131.9 ± 120.3 soil 102.1 ±

0.2 77.2 + 19.9

4 Tang 35.9*** 33.6 7.3

103.9 ± 89.7 ±

1 71.1 ± 13.0

25.7 15.0*** 1CH.9 soil 111. Soil 97, 4 people compared with Nor, *-p<0.01, ***p<0.001; n = 10; Nor is the control group ₅ Aear is the positive control drug Bai Tangping group, Wei Libo Sugar for administration group

The result is shown in Figure

Table 8. Effect of weilipodose on area under the blood glucose curve (AUC) and peak blood glucose after sucrose loading in normal mice

Dose blood glucose peak peak time group

Mg/kg (mg/dl) (min.)

Nor - 341.2 ± 37..3 247.9 ± 38.3 30

Acar 10 235.8 ± 33.9*** 131.5 + 16.7 30

Wei Libo

0.2 226.5 ± 45.3*** 131.9 ± 35.9 30

Sugar

1 197.4 soil 42.8 110.4 + 33.5 60

5 204.0 ± 29.9 111.7 ± 16.0 60

Compared with Nor, ***p<0.001; data is expressed as X ± SD, n = 10; Nor is the control group, Acar is the positive control drug, Baitang group, and Wei Lipoose is the drug-administered group.

The result is shown in Figure

The results showed that (Table 7-8, Figure 13-14), Wei Lipoose in the dose range of 0.2 mg / kg - 5 mg / kg, can make the normal blood glucose peak drop and shift right after normal sucrose load, blood sugar The area under the curve is reduced, showing a dose-effect relationship.

4. Effects of weilipox on blood glucose elevation after sucrose loading in alloxan hyperglycemic mice. Groups of alloxan hyperglycemia mice, 10 rats in each group, fasted overnight, a group of oral sucrose solution (4.0 g/ Kg) As a control group, one group of oral sucrose and Acarbose 10 mg/kg were used as the positive drug control group, and the other three groups were given sucrose and different doses of weiligose (0.2 mg/kg, 1 mg/). Kg, 5 mg/kg) was used as the administration group. Blood glucose levels were measured before and after 30 min, 60 min, and 120 min after administration.

Table 9. Effect of weiligose on blood glucose after sucrose loading in alloxan hyperglycemic mice (± SD) Dose of blood glucose (mg/dl)

Group mg/k

0 min 30 min 60 niin 120 min

Con - 145.1 ±42.3 413.6 + 38.5 410.9 + 39.5 296.9 + 77.1 Acar 10 151.6 ±35.2 253.8 ±63.9* " 246.1 ±77·6 224.9 soil 93.3

0.2 160.6 ±72.3 346.0 ± 76. Γ 339.4 ± 58.0' ^: ' 317.0 ± 73.0

1 183.8 + 61.9 265.4 ±76 250.2 ± 56. (f 246.9 ± 92.6

5 192.9 ±49.2 245.1 ±50.2^ 266.9 ±67. ^3⁄4 254.1 + 72.8 Compared with Con, *p<0.05, ** ^5!5 p<0.001; n= 10; Con is a control group of alloxan hyperglycemia mice, Acar is a positive drug in the Tang Tang group, and Wei Lipoose is a drug-administered group.

The result is shown in Figure

Table 10. Effect of weiligose on the area under the blood glucose curve (AUC) and peak blood glucose after sucrose loading in alloxan hyperglycemic mice (±SD) Dose Blood glucose peak peak time group

Mg/kg (mg - hr/dl) (mg/dl) (min.)

Con - 699.7 ±81.0 413.6 ±38.5 30

Acar 10 461.8 ±130.7*** 253.8 ±63.9 30

0.2 626.2 ± 130.1 346.0 + 76.1 30

1 489.7 ± 136.3*** 265.4 ±76.6 30

5 498.0 ± 95.2*** 266,9 ±67.1 60

Compared with Con, ***p<0.001; n=10; Con is a control group of alloxan hyperglycemia mice, Acar is a positive drug, Baitangping group, and Wei Lipoose is a drug-administered group.

See Figure 16 for results.

The results showed that weiligose significantly reduced the blood glucose level after oral administration of sucrose in alloxan hyperglycemic mice, and significantly reduced the area under the blood glucose curve (Table 9-10, Figure 15-16).

The above results indicate that (+)-(lS, 2S, 3S, 4R, 5R)-1-amino-5-hydroxyindenyl-N-2-(l,3-dihydroxypropyl) crystal form of the present invention -2,3,4-cyclohexanetriol is a preferred alpha-glucosidase inhibitor which lowers blood glucose by inhibiting the metabolism of carbohydrates in the body. 刍Efficacy experiment of crystalline form of Wei Libo sugar salt

[Example 2: Crystallization morphology of different acid bismuths] Preparation of sucrose for α-glucosidase activity Samples

1. A crystalline form of weiliposaccharide hydrochloride (barrel TD-01 hydrochloride) was prepared according to the method of Example 6. Molecular weight 287.74 ₅ Appearance is a white powder. The experiment is carried out with deionized water to prepare the desired concentration.

2. Crystalline form of vegetalpicuric acid salt (abbreviated as TD-01 picrate), prepared according to the method of Example 7, having a molecular weight of 480.38, and having a yellow powder appearance. The test was carried out using deionized water to prepare a desired concentration.

3. The crystal form of the veoliose (abbreviated as TD-01) was prepared as described above. The molecular weight is 251.28, and the appearance is white powder. The experiment is carried out with deionized water to prepare the desired concentration.

4. Voglibose (trade name: xinxin): produced by Japan Takeda Pharmaceutical Co., Ltd., batch number 20040304, molecular weight 267.28, white powder, used in deionized water to prepare the desired concentration.

5. Acarbose (trade name: Bay Tang): produced by Bayer, Germany, molecular weight 645, white powder, prepared with deionized water to achieve the desired concentration. Second, test methods and results

1. Inhibition of invertase and maltase activities by different acid crystals

Sucrose and maltose are decomposed into glucose by the action of α-glucosidase (sucrose, maltase). By measuring the amount of glucose in the reaction system, the inhibition of the α-glucosidase activity of the crystal form of different acid radicals and the morphine wave of vivitose can be determined, and the percentage of inhibition of enzyme activity at different concentrations of the drug is determined. The IC _{50 was} calculated to measure the inhibitory intensity of the sample on enzyme activity. Reference methods were used to isolate invertase and maltase from the upper intestinal mucosa of normal rats for inhibitor studies.

The results in Table 11 indicate that the hydrochloride and picrate of TD-01 have the same effect as TD-01 and inhibit the activity of invertase IC ₅ . Respectively 8.59xl0- ^{^8,} 7.63X 10- ⁸ and 7.32xlO- ⁸ M, Acarbose and slightly stronger than weaker than Basen; inhibition of maltase activity IC _5G is 1.13xl0- ^{^6,} 1.08x10- ⁶ and 1.14χ10- ⁶ Μ, are less effective in Glucobay and Basen. Table 11. Inhibition of α-glucosidase (sucrose, glucosidase) by TD-01 with different acid radicals

IC ₅₀ (mol/L)

: test compound

Sucrase maltase

TD-01 hydrochloride 8.59xl0" ⁸ 1.13x10- ⁶

TD-01 picrate 7.63 lO" ⁸ l.OSxlO" ⁶

TD-01 7.32χ10" ⁸ 1.14x 1ο- ⁶

Voglibose 4.30xl0" ⁸ 1.45X 10- ⁷

Acarbose 5.98χ10 ^-7 1.43xl0" ⁷

2. Inhibition of α-amylase activity by the crystal form of different acid groups

Starch is decomposed by amylase. By measuring the amount of starch remaining in the reaction system (iodine-starch method), it is determined whether the crystal form of different acid roots has an inhibitory effect on amylase activity.

The results show that at a concentration of ^{10- 5 mol / L, TD-} 01 hydrochloride, TD-01 picrate, TD-01 and Basen on amylase activity was no inhibition; inhibition of amylase activity Acarbose IC ₅₀ of 2.61 X 10- ⁶ mol / L. Third, the conclusion

TD-01 hydrochloride, TD-01 picrate and TD-01 have almost no difference in the inhibition of α-glucosidase and α-amylase activity in vitro, all in the same order of magnitude.

Oral pharmaceutical preparation

[Embodiment 10]

I prescription 1000 tablets

Wei Libo Sugar 2.5g

Lactose 112.5g

Hydroxypropyl fluorenyl cellulose

Crospovidone 2.4g

Magnesium stearate 1.2g

Opada The raw material drug weilipoose is a crystalline form of weilipoose prepared according to the above method, and other excipients are commercially available products.

Tanning method

Each raw and auxiliary materials are passed through an 80 mesh sieve and accurately weighed according to the prescription; hydroxypropyl fluorenyl cellulose solution is prepared as a binder with 75% ethanol; double the weilipoose and some lactose in a suitable container Dilute the method to mix evenly, and then mix with the remaining amount of lactose, the prescribed amount of crospovidone, and then add the binder, and then pass through 20 mesh sieve wet particles; the wet particles are placed in a hot air circulation oven, the temperature is controlled at 55- 65 ° C, drying to a moisture content of 1.5-2.0%, using a 20 mesh sieve to complete the granules; the prepared granules, plus the prescription of magnesium stearate mixed and compressed; with the coating material Oubad to prepare the coating liquid, The tablets are coated. Industrial applicability

The invention solves the problem that it is very difficult to prepare the weibopoose which has not been solved in the field for many years, and obtains the crystal form of the weiligose and its salt, which has a stable morphology and a determined melting point, and has good chemical stability. The new form of weilipoose and its salt have the properties required for large-scale synthesis or formulation of drugs. They are compressible and easy to form during preparation, making it a large-scale industrial preparation of drugs containing weilipoose. The cost of raw materials is greatly reduced, the raw materials are convenient to store, the production operation is more convenient, and the quality is easier to control.

It has been demonstrated in vitro and in vivo that the novel form of the viscose compound of the present invention has a good α-glucosidase inhibitor activity, and the carbohydrate digestive process is inhibited by inhibiting the intestinal disaccharidase, c-glucosidase. In the final stage, the hydrolysis of the disaccharide to the monosaccharide delay delays the rate of absorption of glucose into the blood, and thus the crystalline form of the virgin sugar of the present invention has a good prospect as a drug for treating or preventing diabetes.

The method of the present invention can obtain high-yield crystalline form of weiligose, and the preparation step and the separation and purification step fully consider the needs of large-scale industrialization, the operation is simple and easy, the cost is low, and the separation effect is good.

The above description of the preferred embodiments of the present invention is not intended to limit the scope of the present invention, and various modifications and changes can be made by those skilled in the art without departing from the spirit and scope of the invention.

Claims

Rights request

1. A crystalline form of (+)-(lS, 2S, 3S, 4R ₅ 5R)-1-amino-5-hydroxyindenyl-N- 2-(1,3-dihydroxypropyl)-2, 3, 4-cyclohexanetriol ₅ When powder X-ray diffraction is carried out with a Cu-Κα radiation source, the powder X-ray diffraction pattern is at least 19.5 to 19.9 and/or at 21.8 to 22.2, which is represented by a diffraction angle of 2Θ Characteristic peak.

2. The crystalline form of (+)-(lS, 2S, 3S, 4R, 5R)-1-amino-5-hydroxymethyl-N-2-(l,3-dihydroxypropyl) according to claim 1. -2,3, 4-cyclohexanetriol, characterized in that when powder X-ray diffraction is carried out using a Cu-Ka radiation source, the powder X-ray diffraction pattern is further included in about 13.24 ~ 13.26, about 14.54 ~ 14.64, about 15.50 ~ 15.62, about 17.04 - 17.12, about 23.42 ~ 23.5, about 23.94 ~ 24,00, about 25.92 ~ 25.98, about 26.80 ~ 26.86 and about 29.16 ~ 29.18 show the characteristic peak represented by the diffraction angle 2 。.

3. (+)-(lS, 2S, 3S, 4R, 5R) small amino-5-hydroxyindenyl-indole-2-(1,3-dihydroxypropyl) in the crystalline form according to claim 1 or 2. - 2, 3 , 4-cyclohexanetriol, characterized by a DSC endothermic transition at about 89 °C.

4. A pharmaceutical composition comprising (+)-(lS, 2S, in the crystalline form according to claim 1;

3S, 4R, 5R)-1-amino-5-hydroxyindenyl-N-2-(l,3-dihydroxypropyl)-2,3,4-cyclohexanetriol or a pharmaceutically acceptable salt thereof .

5. The pharmaceutical composition according to claim 4, which is characterized in that it further contains a drug

A pharmaceutically acceptable carrier and/or excipient.

The pharmaceutical composition according to claim 4, wherein the pharmaceutically acceptable salt is (+)-(lS, 2S, 3S, 4R, 5R) small amino- 5-oxindole in a crystalline form. Base-N-2-(l,3-dihydroxypropyl)-2,3,4-cyclohexanetriol hydrochloride.

The pharmaceutical composition according to claim 6, characterized by (+H1S, 2S, 3S, 4R, 5R) small amino-5-hydroxymethyl-N-2-(l, 3- Dihydroxypropyl) -2,3,4-cyclohexanetriol hydrochloride When powder X-ray diffraction is carried out with a Cu-Ka radiation source, the powder X-ray diffraction pattern is at least 20.01 ~ 20.5 and / or Characteristic peaks expressed by the diffraction angle 2Θ are shown at 23.8 to 24.2.

The pharmaceutical composition according to claim 7, characterized by (+)-(lS, 2S, 3S, 4R, 5R)-1-enyl-5-hydroxyindenyl-N-2 in the crystalline form -(l,3-dihydroxypropyl)-2,3,4-cyclohexanetriol hydrochloride When powder X-ray diffraction is carried out with a Cu-Ka radiation source, its powder X-ray diffraction pattern is further 10.94~10.98, about 13.22 ~ 13.28, about 16, 14 ~ 16,16, about 23.28 ~ 23.32 Characteristic peaks expressed by diffraction angle 2Θ are shown at about 25.02 to 25.08.

9. The pharmaceutical composition according to claim 8, characterized in that the crystalline form of (+)-(lS, 2S, 3S, 4R, 5R)-amino-5-hydroxyindenyl-N-2-( l, 3-Dihydroxypropyl)-2,3,4-cyclohexanetriol hydrochloride has a DSC endothermic transition of about 144.3. C.

The pharmaceutical composition according to claim 4, wherein the pharmaceutically acceptable salt is (+)-(lS ₅ 23, 33, 4R, 5R)-1-amino-5-hydroxy in crystalline form. Methyl-indole-2-(1,3-dihydroxypropyl)-2,3,4-cyclohexanetriol picrate.

11. Preparation of crystalline form of (+)-(lS ₃ 2S, 3S, 4R, 5R)-1-amino-5-hydroxymethyl-N-2-(l,

A method of 3-dihydroxypropyl)-2,3,4-cyclohexanetriol comprising the steps of:

5R) Small amino-5-hydroxymethyl-2,3,4-cyclohexanetriol is reacted with dihydroxyacetone and sodium cyanoborohydride to give (+)-(lS, 2S, 3S, 4R, 5R)-1 -amino-5-hydroxyindenyl-N-2-(l,3-dihydroxypropyl)-2,3

4-cyclohexanetriol;

2) after removing the solvent obtained in the above step 1), dissolving in water and acidifying, and then neutralizing with a base to about pH 6.0;

3) Separate the column by the following column and trace it by TLC:

Strongly acidic cation exchange resin;

Weakly acidic cation exchange resin;

Strongly basic anion exchange resin;

4) Collect the product and concentrate it to dryness;

5) Dissolving the product obtained in the step 4) with methanol, ethanol or a mixed solution thereof, and adding seed crystals to form crystals of (+)-(lS, 2S, 3S, 4R, 5R)-1-amino -5-Hydroxymethyl-N-2-(l,3-dihydroxypropyl)-2,3,4-cyclohexanetriol.

The process according to claim 11, wherein the amount of glacial acetic acid added is glacial acetic acid volume/raw material (+)-(lS, 2S, 3S, 4R, 5R)-1-amino-5-hydroxymethyl group. -2, 3, 4-cyclohexanetriol weight, 8 ~ 25 %.

The process according to claim 12, wherein the amount of glacial acetic acid added is glacial acetic acid volume/raw material (+)-(lS, 2S, 3S, 4R, 5R) small amino-5-hydroxymethyl-2. , 3, 4-cyclohexanetriol weight, 20%.

14. The production method according to claim 11, wherein the strongly acidic cation exchange resin is 001 x 7 or PUROLITE C100E H ⁺ type strongly acidic cation exchange resin, the eluent is water and ammonia; the weakly acidic cation exchange resin is YD CG-50 or HD-2 or Amberlite CG-50 N3⁄4 ⁺ weak An acidic cation exchange resin, the eluent is water or ammonia; the strong base anion exchange resin is Dowexl X 2 or PUROLITE A 400 or HZ-202 OH-type strongly basic anion exchange resin, and the eluent is water.

The preparation method according to claim 14, wherein the resin column separation step comprises: a PUROLITE C100E H+ type strongly acidic cation exchange resin column, water and 0.5 N ammonia water elution; YD CG-50 H ₄ + type weakly acidic cation Exchange resin column, 0.1N ammonia water elution;

HZ-202 Off strong basic anion exchange resin column, water elution.