CN102659813A - 2-((2-(3-aminopiperidine-1)-4-oxythiophene (3, 2-d) pyrimidine-3(4H)-methyl) polymorphic benzonitrile, and preparation method and pharmacological applications thereof - Google Patents

Abstract

The invention discloses a 2-((2-(3-aminopiperidine-1)-4-oxythieno[3,2-d]pyrimidine-3(4H)-methyl)benzonitrile polymorph , its preparation method and its pharmacological use. Compared with the prior art, the polymorphic form of the compound has the advantages of strong DPP4 inhibitory activity, strong hypoglycemic activity in vivo, and good pharmacokinetic properties, and is particularly suitable for use in 2 type diabetes treatment.

Description

2-((2-(3-aminopiperidine-1)-4-oxythiophene[3,2-d]pyrimidine-3(4H)-methyl)benzonitrile polymorph, its preparation method and its Pharmacological use

technical field

The present invention relates to crystals of pharmaceutical compounds, in particular, the present invention relates to 2-((2-(3-aminopiperidine-1)-4-oxythieno[3,2-d]pyrimidine-3(4H)-methanol Base) benzonitrile polymorph, its preparation method, crystal composition, pharmaceutical composition and use. 

Background technique

Diabetes is a common metabolic disease. It is characterized by abnormally high plasma glucose concentrations and hyperglycemia in the fasted state or following an oral glucose tolerance test. The World Health Organization divides diabetes into type 1 and type 2 according to the clinical form, and most diabetic patients have type 2 diabetes mellitus (T2DM). Type 2 diabetes, also known as non-insulin-dependent diabetes, has a high risk of vascular complications, such as coronary artery disease, stroke, hypertension, nephropathy, peripheral vascular disease, neurological disease, and retinopathy. This metabolic disease has become a public health problem. According to the World Health Organization, there are nearly 194 million diabetics in the world, which will increase to 366 million by 2030. Promoting the secretion of insulin by pancreatic β-cells is the main method for the treatment of T2DM. However, in addition to being able to play a therapeutic role, it will also cause certain side effects, such as: hypoglycemia, weight gain, cardiovascular morbidity, and β-cell death. the

Studies have shown that for every 1% reduction in glycated hemoglobin A1c (HbA1c), complications in T2DM patients are reduced by 35%. Therefore, how to reduce complications and adverse side effects has become the main direction for the treatment of type 2 diabetes. Clinically validated targets for the treatment of diabetes include peroxisome proliferator-activated receptor (PPAR) α/γ, glucagon-like peptidase-1 (GLP-1) and dipeptidyl peptidase IV (Dipeptidyl peptidase IV). , DPP4) etc. DPP4 inhibitors have become a new option for the treatment of type 2 diabetes, and DPP4 inhibitors are relatively safe and effective drugs so far, whether they are administered alone or in combination. the

Dipeptidyl peptidase IV (DPP4; also known as T-cell antigen CD26), is a highly specific serine protease in the form of a dimer. It contains two states, one is a transmembrane protease, which contains 766 amino acids, and is widely distributed in kidneys, intestinal fluffy walls, cell membranes, liver cells, vascular endothelium, T cells, B cells and suicide cells. The other exists in the plasma in a dissolved state. The most important enzymatic function of DPP4 is to hydrolyze polypeptides containing alanine or proline at the N-terminal of the peptide chain, such as hydrolyzing GLP-1. If the activity of DPP4 is inhibited, the content of GLP-1 in the body will be increased indirectly, thereby causing a series of physiological effects in the body and achieving the purpose of treating type 2 diabetes. the

At present, the most clinically used drugs are anti-type 2 diabetes drugs related to GLP-1, such as: (a) DPP4-resistant GLP-1 analogs; (b) small molecule GLP-1 receptor agonists; ( c) DPP4 inhibitors. As a new oral antidiabetic drug, DPP4 inhibitor can prevent the rapid degradation of incretin hormones, increase the level of GLP-1 after meals, and has less toxic and side effects and good effect. Recent studies have shown that whether it is treated alone or Combined with other antidiabetic drugs, DPP4 inhibitors can reduce the level of glycated hemoglobin A1c. Moreover, the risk of hypoglycemia is very small, and has been favored by more and more pharmaceutical companies. the

The research on DPP4 inhibitors has made great breakthroughs. The currently marketed drugs include Merck’s Sitagliptin, Novartis’ Vildagliptin and Bristol-Myers Squibb’s Saxagliptin. Ting (Saxagliptin) and so on. Since there are other families of serine proteases, the design of DPP4 inhibitors should first consider its selectivity to avoid causing other toxic and side effects. Therefore, the research on DPP4 inhibitors is still a big challenge. the

2-((2-(3-aminopiperidine-1)-4-oxythieno[3,2-d]pyrimidine-3(4H)-methyl)benzonitrile (formula I) is a novel DPP4 inhibitor, It is a new type of anti-diabetic active molecule. The DPP4 inhibitory activity of this compound has reached the nanomolar level, which is equivalent to that of the listed drug Sitagliptin. It has no inhibitory activity on other enzymes of the DPP4 family, and its DPP4 selectivity is better than that of the listed drug. The drug Sitagliptin has guaranteed its safety at the molecular level. In terms of pharmacodynamics research, the acute hypoglycemic effect of a single administration on ICR mice has been carried out, and its hypoglycemic effect is better than that of marketed drugs Vildagliptin; at the same time, the drug efficacy evaluation on the female BKS mouse model and the drug efficacy evaluation on the male BKS mouse model were also carried out. The experimental results showed that the hypoglycemic effect of the compound in vivo was better than that of the marketed drugs Vildagliptin (Vildagliptin). Pharmacokinetic experimental studies show that this compound has good pharmacokinetic properties in vivo, and the half-life is better than the listed drug Alogliptin (Alogliptin). Safety experiments show that this compound is safe. Wherein rat acute toxicity test shows, 1g/kg administration group does not see animal death; According to the results of pharmacological evaluation, pharmacokinetic research, and safety evaluation, the hypoglycemic effect of this compound in vivo is better than that of DPP4 inhibitors currently used clinically. Therefore, this compound is expected to be developed as a new drug for the treatment of type 2 diabetes. 

Polymorphism refers to the phenomenon that elements or compounds form solid states with different physical and chemical properties in different crystal forms. Different polymorphs differ in structure and properties. Various physical properties, such as solubility, density, stability, melting point, etc., vary with different crystal forms. Pharmaceutical solids usually form molecular crystals and polymorphic variants are readily available. At the same time, in the field of pharmaceutical research, polymorphism also includes multi-component crystal forms such as organic solvates and hydrates. The different physical and chemical properties or processability of these different crystal forms sometimes directly affect the safety and effectiveness of drugs. Therefore, crystal form research and control has become an important research content in the process of drug development. Actual blood levels of a drug can be favorably influenced by selecting polymorphic forms with different solubility and inherent dissolution rates. the

Contents of the invention

One aspect of the present invention provides the A crystal form of the compound shown in formula I

处有衍射峰，进一步典型地在约 

处有衍射峰，更进一步典型地在约9.28 

处有衍生峰。  The XRPD (X-ray powder diffraction) spectrum of the A crystal form using Cu-K _α radiation is shown in Figure 1, and the value of the crystal plane distance d is expressed at about Diffraction peaks at , typically around

There are diffraction peaks at , further typically around

There is a diffraction peak at about 9.28

There are derived peaks.

The IR (infrared absorption) spectrum of the crystal form A is shown in Figure 2, and there are absorption peaks at approximately 3431, 2953, 2222, 1681, 1550, ^{and 785 cm −1} .

The DSC (differential scanning calorimetry) spectrum of the crystal form A is shown in FIG. 3 , wherein the initial value of the endothermic transition temperature is about 154.32°C. the

处有衍射峰，典型地在约 

处有衍射峰，进一步典型地在约 

处有衍射峰，更进一步典型地在约 

处有衍生峰。  Another aspect of the present invention provides the B crystal form of the compound shown in formula I, the XRPD (X-ray powder diffraction) spectrum of the B crystal form using Cu-K _α radiation is shown in Figure 6, with the crystal plane distance d Values are expressed in approx.

Diffraction peaks at , typically around

There are diffraction peaks at , further typically around

There are diffraction peaks at , further typically around

There are derived peaks.

The IR (infrared absorption) spectrum of the B crystal form is shown in Figure 7, and there are absorption peaks at approximately 2939, 2864, 1664, 1552, 1518, 1373, 1350, 1230, 787, 771, and 663 cm ⁻¹ .

The DSC (differential scanning calorimetry) spectrum of the crystal form B is shown in Fig. 8, wherein the initial value of the endothermic transition temperature is about 147.57°C. the

Another aspect of the present invention provides a crystal form composition, wherein the above-mentioned crystal form A accounts for more than 60%, preferably more than 90%, more preferably more than 95%, most preferably more than 99% of the weight of the composition. In addition to the A crystal form, the A crystal form composition may also contain a small amount of other crystal forms and amorphous 2-((2-(3-aminopiperidine-1)-4-oxythiophene[3,2- d] pyrimidine-3(4H)-methyl)benzonitrile. 

Another aspect of the present invention provides a crystal form B composition, wherein the above-mentioned crystal form B accounts for more than 60%, preferably more than 90%, more preferably more than 95%, most preferably more than 99% of the weight of the composition. In addition to the B crystal form, the B crystal form composition may also contain a small amount of other crystal forms and amorphous 2-((2-(3-aminopiperidine-1)-4-oxythiophene[3,2- d] pyrimidine-3(4H)-methyl)benzonitrile. 

Another aspect of the present invention provides a pharmaceutical composition, which includes a therapeutically effective amount of the above crystal form A or B, or the above crystal form A composition or B crystal composition and a pharmaceutically acceptable excipient. Wherein, the excipients include conventional fillers, disintegrants, binders, lubricants, etc., and the fillers are selected from starch, lactose, microcrystalline cellulose, dextrin, mannitol, magnesium oxide, sulfuric acid Calcium and its mixture, the disintegrating agent is selected from carboxymethyl cellulose and its salt, croscarmellose and its salt, crospovidone, sodium carboxymethyl starch, low-substituted hydroxypropyl fiber Vegetables and mixtures thereof; the binding agent is selected from povidone, hydroxypropyl methylcellulose, starch slurry and mixtures thereof; the lubricant is selected from magnesium stearate, calcium stearate and mixtures thereof. the

Another aspect of the present invention provides a method for preventing or treating type II diabetes and/or its complications, which comprises administering a therapeutically effective amount of the above-mentioned crystal form A or B, the above-mentioned crystal form A composition to a patient in need or B crystal composition. the

Another aspect of the present invention provides the above-mentioned crystal form A or B, and the use of the above-mentioned crystal form A composition or crystal form B composition in the preparation of a medicament for preventing or treating type II diabetes and its complications. the

The aforementioned complications of type II diabetes include coronary artery disease, stroke, hypertension, nephropathy, peripheral vascular disease, neurological disease, retinopathy, and the like. the

2-((2-(3-aminopiperidine-1)-4-oxythieno[3,2-d]pyrimidine-3(4H)-methyl)benzonitrile A crystal form of the present invention can be as follows The method is prepared to obtain:

method one:

(1) Dissolve 2-((2-(3-aminopiperidine-1)-4-oxothieno[3,2-d]pyrimidine-3(4H)-methyl)benzonitrile in esters selected from In one of solvents and alcoholic solvents;

(2) Add a solvent selected from ethers and alkane solvents under stirring;

(3) crystallization;

(4) The solid obtained in step (3) is vacuum-dried for 30-100 hours at 20-80° C. and 0-10 KPa. the

Further, the ester solvent described in the present invention is selected from methyl acetate, ethyl acetate, ethyl formate, isopropyl acetate, butyl acetate, isobutyl acetate, propyl acetate or mixtures thereof, more preferably ethyl acetate ester. the

Further, the alcohol solvent of the present invention is selected from ethanol, methanol, n-butanol, isopropanol, n-propanol or mixtures thereof. More preferred is ethanol. the

2-((2-(3-aminopiperidine-1)-4-oxythieno[3,2-d]pyrimidine-3(4H)-methyl)benzonitrile B crystal form of the present invention can be as follows The method is prepared to obtain:

Method Two:

Except that the solvent used in step (1) is a ketone solvent, obtain 2-((2-(3-aminopiperidine-1)-4-oxythiophene[3,2-d]pyrimidine in the same way as method one -3(4H)-methyl)benzonitrile B crystal form.

The ketone solvent is selected from acetone, butanone, methyl isobutyl ketone, methyl butyl ketone or mixtures thereof, preferably acetone. the

Furthermore, the ether solvent described in step (2) of the method of the present invention is selected from petroleum ether, tert-butyl methyl ether, diethyl ether, isopropyl ether, diethyl ether or mixtures thereof, preferably petroleum ether. the

Further, the alkane solvent in step (2) of the method of the present invention is selected from n-heptane, n-hexane, n-pentane or a mixture thereof, more preferably n-heptane.

Further, the step (3) specifically adopts the following method for crystallization: control the crystallization temperature at 0-50° C., and stand or stir to crystallize until the crystals are completely precipitated. Preferably, the crystallization temperature is controlled at 0-20°C. the

Further, the step (3) specifically adopts the following method for crystallization: evaporating the solvent to dryness at 30-80° C. under reduced pressure or normal pressure for crystallization. Preferably, the solvent is evaporated to dryness at 35-50° C. under reduced pressure or normal pressure for crystallization. the

Further, the step (4) is dried under the following conditions to obtain 2-((2-(3-aminopiperidine-1)-4-oxythiophene[3,2-d]pyrimidine-3(4H)-methanol Base) Benzonitrile Form A: Vacuum dry at 25-40°C and 0-10KPa for 60-80 hours. 

Description of drawings

Fig. 1 is an X-ray diffraction powder diffraction spectrum diagram of the crystal (form A) of Example 1 of the present invention. the

Fig. 2 is an infrared spectrogram of the crystal (form A) of Example 1 of the present invention. the

Fig. 3 is a differential thermal scanning analysis (DSC) diagram of the crystal (form A) of Example 1 of the present invention. the

Fig. 4 is a thermogravimetric analysis (TGA) diagram of the crystal (form A) of Example 1 of the present invention. the

Fig. 5 is an X-ray diffraction powder diffraction spectrum diagram of the crystal (form A) of Example 2 of the present invention. the

Fig. 6 is an X-ray diffraction powder diffraction spectrum diagram of the crystal (form B) of Example 5 of the present invention. the

Fig. 7 is an infrared spectrogram of the crystal (form B) of Example 5 of the present invention. the

Fig. 8 is a differential thermal scanning analysis (DSC) diagram of the crystal (crystal form B) of Example 5 of the present invention. the

Fig. 9 is a thermogravimetric analysis (TGA) diagram of the crystal (crystal form B) of Example 5 of the present invention. the

Fig. 10 is an amorphous X-ray diffraction powder diffraction spectrum diagram of Example 6 of the present invention. the

Fig. 11 is an infrared spectrogram of the amorphous form of Example 6 of the present invention. the

FIG. 12 is a thermogravimetric analysis (TGA) chart of the amorphous state of Example 6 of the present invention. the

Fig. 13 is an amorphous differential thermal scanning analysis (DSC) chart of Example 6 of the present invention. the

Fig. 14 is an experiment of DPP4 activity in plasma of SD rats with polymorphic forms. the

Figure 15 shows the effect of polymorphism on the oral glucose tolerance of normal ICR mice. the

Detailed ways

），θ为衍射角。对于同种化合物的同种晶型，其XRPD谱图在整体上具有相似性，表征峰位置的d值误差一般在±2%之内，大部分误差不超过±1%；相对强度误差可较大，但变化趋势一致。另外，判断晶型是否一样时应注意保持整体观念，因为并不是一条衍射线代表一个物相，而是一套特定的“d-I/I₁”数据才代表某一物相。还应指出的是，在混合物的鉴定中，由于含量下降等因素会造成部分衍射线的缺失，此时，无需依赖高纯试样中观察到的全部谱带，甚至一条谱带也可能对给定的晶体是特征性的。  It should be noted that in XRPD, the diffraction pattern obtained from a crystalline compound is often characteristic of a particular crystal form, where the relative intensity of the bands (especially at low angles) may vary due to crystallization conditions, particle size, and other factors. The dominance orientation effect varies with the test conditions. Therefore, the relative intensities of the diffraction peaks are not characteristic of the crystal in question, and when judging whether it is the same as a known crystal form, more attention should be paid to the positions of the peaks rather than their relative intensities. In the XRPD spectrum, the 2θ angle or the crystal plane distance d is usually used to represent the peak position. Since the 2θ angle is related to the wavelength of the incident X-ray, it is more representative to use the crystal plane distance d. There is a simple conversion relationship between the two: d=λ/2sinθ, where d represents the distance between crystal planes, and λ represents the wavelength of incident X-rays (for Cu-K _α ,

), θ is the diffraction angle. For the same crystal form of the same compound, their XRPD spectra are similar on the whole, and the error of the d value representing the peak position is generally within ±2%, and most of the errors do not exceed ±1%; the relative intensity error can be compared with large, but the trend is the same. In addition, when judging whether the crystal form is the same, you should pay attention to maintaining the overall concept, because not a diffraction line represents a phase, but a set of specific "dI/I ₁ " data represents a certain phase. It should also be pointed out that in the identification of mixtures, due to factors such as content decline, some diffraction lines will be missing. At this time, it is not necessary to rely on all the bands observed in the high-purity sample, and even one band may affect the given Certain crystals are characteristic.

DSC measures the transition temperature when a crystal absorbs or releases heat as a result of a change in its crystal structure or as the crystal melts. Thermal transition temperatures and melting points are typically within about ±5°C, usually within about ±3°C, in successive analyzes of the same crystal form of the same compound. DSC provides an auxiliary method to distinguish different crystal forms. Different crystal forms can be identified by their different transition temperature characteristics. It should be pointed out that for mixtures, the DSC peak or melting point may vary in a larger range. In addition, since the melting process of the substance is accompanied by decomposition, the melting temperature is closely related to the heating rate. the

IR measures infrared absorption caused by specific chemical bonds associated with groups in molecules that vibrate in response to light. Due to the different electrical environment of the covalent bond of molecules in different crystal forms, the strength of the covalent bond may also change, and the change in the strength of the covalent bond will inevitably lead to the difference in the IR spectrum of different crystal forms. the

The present invention is further illustrated below with examples, but the present invention is not limited thereto. the

Example

2-((2-(3-aminopiperidine-1)-4-oxythiophene[3,2-d]pyrimidine-3(4H)-methyl)benzonitrile Reference: European Journal of Medicinal Chemistry 46( 2011) 71-76 prepared. ¹ HNMR (CDCl ₃ ): δ7.71(d, J=5.2HZ, 1H), 7.62(d, J=8HZ, 1H), 7.45(t, J=8HZ, 1H), 7.31(t,J=8HZ,1H),7.19(d,J=5.2HZ,1H),7.03(d,J=8HZ,1H),5.51(s,2H),3.24(m,1H),3.01( m,2H),2.80(m,1H),2.67(m,1H),1.91(m,1H),1.72(m,1H),1.60(m,1H),1.25(m,1H); m/z 366.1[M+H] ⁺ . The compound 2-((2-(3-aminopiperidine-1)-4-oxythiophene[3,2-d]pyrimidine-3 Amorphous powder of (4H)-methyl)benzonitrile.

Example 1: Preparation of Form A of 2-((2-(3-aminopiperidine-1)-4-oxythieno[3,2-d]pyrimidine-3(4H)-methyl)benzonitrile

Dissolve 200 mg of 2-((2-(3-aminopiperidine-1)-4-oxothieno[3,2-d]pyrimidine-3(4H)-methyl)benzonitrile in 1 mL of ethyl acetate , add 3mL petroleum ether, crystallize under stirring at room temperature, until there is no more solid precipitation, filter, put the obtained solid material in a vacuum drying oven, and vacuum dry at 25°C and 5KPa for 70 hours to obtain 2-(( 2-(3-aminopiperidine-1)-4-oxythieno[3,2-d]pyrimidine-3(4H)-methyl)benzonitrile A crystal 100mg.

Example 2: Preparation of Form A of 2-((2-(3-aminopiperidine-1)-4-oxythieno[3,2-d]pyrimidine-3(4H)-methyl)benzonitrile

Dissolve 200 mg of 2-((2-(3-aminopiperidine-1)-4-oxothiophene[3,2-d]pyrimidine-3(4H)-methyl)benzonitrile in 1 mL of ethanol, add 12mL of petroleum ether was crystallized under stirring at room temperature until no solid precipitated out, filtered, and the obtained solid material was placed in a vacuum drying oven, and vacuum-dried at 25°C and 5KPa for 70 hours to obtain 2-((2- (3-aminopiperidine-1)-4-oxythieno[3,2-d]pyrimidine-3(4H)-methyl)benzonitrile A crystal 110mg. 

Example 3: Preparation of Form A of 2-((2-(3-aminopiperidine-1)-4-oxythieno[3,2-d]pyrimidine-3(4H)-methyl)benzonitrile

Dissolve 200 mg of 2-((2-(3-aminopiperidine-1)-4-oxothieno[3,2-d]pyrimidine-3(4H)-methyl)benzonitrile in 1 mL of ethyl acetate , add 12mL of n-heptane, crystallize under stirring at room temperature, until no solid precipitates out, filter, place the resulting solid material in a vacuum drying oven, and vacuum dry for 70 hours at 25°C and 5KPa to obtain 2-( (2-(3-aminopiperidine-1)-4-oxythieno[3,2-d]pyrimidine-3(4H)-methyl)benzonitrile A crystal 100mg.

Example 4: Preparation of Form A of 2-((2-(3-aminopiperidine-1)-4-oxythieno[3,2-d]pyrimidine-3(4H)-methyl)benzonitrile

Dissolve 200 mg of 2-((2-(3-aminopiperidine-1)-4-oxothieno[3,2-d]pyrimidine-3(4H)-methyl)benzonitrile in 1 mL of ethyl acetate , add 8mL of n-heptane, mix evenly, let it stand for 24 hours to crystallize, filter, place the obtained solid material in a vacuum drying oven, and dry it in vacuum for 70 hours at 25°C and 5KPa to obtain 2-((2- (3-aminopiperidine-1)-4-oxythieno[3,2-d]pyrimidine-3(4H)-methyl)benzonitrile A crystal 110mg. 

Example 5: Preparation of Form B of 2-((2-(3-aminopiperidine-1)-4-oxythieno[3,2-d]pyrimidine-3(4H)-methyl)benzonitrile

Dissolve 200 mg of 2-((2-(3-aminopiperidine-1)-4-oxothiophene[3,2-d]pyrimidine-3(4H)-methyl)benzonitrile in 1 mL of acetone, add 15mL of petroleum ether, mixed evenly, left to stand for 24 hours to crystallize, filtered, the resulting solid material was placed in a vacuum drying oven, and vacuum-dried for 70 hours at 25°C and 5KPa to obtain 2-((2-(3- Aminopiperidine-1)-4-oxothieno[3,2-d]pyrimidine-3(4H)-methyl)benzonitrile B crystal 90mg. 

Example 6: Preparation of amorphous 2-((2-(3-aminopiperidine-1)-4-oxythieno[3,2-d]pyrimidine-3(4H)-methyl)benzonitrile

Dissolve 200 mg of 2-((2-(3-aminopiperidine-1)-4-oxothieno[3,2-d]pyrimidine-3(4H)-methyl)benzonitrile in 5 mL of dichloromethane , after complete dissolution, the solvent was evaporated under reduced pressure at 39°C, placed in a vacuum drying oven, and dried in vacuum at 25°C and 5KPa for 70 hours to obtain 2-((2-(3-aminopiperidine-1) -4-oxothiophene[3,2-d]pyrimidine-3(4H)-methyl)benzonitrile amorphous 200mg. 

Embodiment 7: the test of crystal

Sample: the crystal of Example 1 (Form A)

X-ray powder diffraction test: instrument model: Shimadzu XRD-6000; light source: Cu-K _α 40kV40mA; graphite monochromator; divergence slit (DS): 1°; scanning mode: 2θ/θ, continuous scanning; scanning Range: 3°~40°, scanning speed 4°/min, the results are shown in Figure 1.

Infrared absorption spectrum test: US NICOLET company 670FI-IR spectrometer, KBr tablet method, scanning range 400~4000cm ^-1 , the results are shown in Figure 2.

DSC test: Instrument: Mettler-Toledo ST; measurement conditions are as follows: use an alumina crucible, scan from 30°C to 400°C at a heating rate of 20°C/min under air purging, and the results are shown in Figure 3. the

Thermogravimetric analysis (TGA) test: NETZSCH TG 209F1 is used, and the measurement conditions are as follows: use an alumina crucible, scan from 25°C to 400°C at a heating rate of 10°C/min under nitrogen purge, and the results are shown in Figure 4. the

Embodiment 8: the test of crystal

Sample: the crystal of Example 2 (form A)

X-ray powder diffraction test: instrument model: Shimadzu XRD-6000; light source: Cu-K _α 40kV40mA; graphite monochromator; divergence slit (DS): 1°; scanning mode: 2θ/θ, continuous scanning; scanning Range: 3°~40°, scanning speed 4°/min, the results are shown in Figure 5.

Embodiment 9: the test of crystal

Sample: the crystal of Example 5 (Crystal Form B)

X-ray powder diffraction test: instrument model: Shimadzu XRD-6000; light source: Cu-K _α 40kV40mA; graphite monochromator; divergence slit (DS): 1°; scanning mode: 2θ/θ, continuous scanning; scanning Range: 3°～40°, scanning speed 4°/min, the results are shown in Figure 6.

Infrared absorption spectrum test: 670FI-IR spectrometer from American NICOLET company, KBr tablet method, scanning range 400~4000cm ^-1 , the results are shown in Figure 7.

DSC test: Instrument: Mettler-Toledo ST; measurement conditions are as follows: use an alumina crucible, scan from 30°C to 400°C at a heating rate of 20°C/min under air purging, and the results are shown in Figure 8. the

Thermogravimetric analysis (TGA) test: NETZSCH TG 209F1 is used, and the measurement conditions are as follows: use an alumina crucible, scan from 25°C to 400°C at a heating rate of 10°C/min under nitrogen purge, and the results are shown in Figure 9. the

Embodiment 10: the test of amorphous matter

Sample: the amorphous substance of embodiment 6

X-ray powder diffraction test: instrument model: Shimadzu XRD-6000; light source: Cu-K _α 40kV 40mA; graphite monochromator; divergence slit (DS): 1°; scanning mode: 2θ/θ, continuous scanning; Scanning range: 3°～40°, scanning speed 4°/min, the results are shown in Figure 10.

Infrared absorption spectrum test: 670FI-IR spectrometer from American NICOLET company, KBr tablet method, scanning range 400~4000cm ^-1 , the results are shown in Figure 11.

Thermogravimetric analysis (TGA) test: NETZSCH TG 209F1 is used, and the measurement conditions are as follows: use an alumina crucible, scan from 25°C to 400°C at a heating rate of 10°C/min under nitrogen purge, and the results are shown in Figure 12. the

DSC test: Instrument: Mettler-Toledo ST; measurement conditions are as follows: use an alumina crucible, scan from 30°C to 400°C at a heating rate of 20°C/min under air purging, and the results are shown in Figure 13. the

Embodiment 11: in vitro activity test

Screening method: dipeptidyl peptidase IV activity screening (DPP4 activity assay)

Instrument: microplate reader, Envision (PerkinElmer, USA)

Material: human DPP4 (dipeptidyl peptidase IV), the extracellular fragment of protease DPP4 (aa29 -766). Substrate: Ala-Pro-AMC was synthesized by Jill Biochemical (Shanghai) Company. the

Process: DPP4 can specifically hydrolyze the substrate Ala-Pro-AMC to generate the product AMC. AMC is excited by 355nm ultraviolet light to generate 460nm emission light. The linear change of fluorescence value at 460nm wavelength per unit time is dynamically measured, and the DPP4 activity is calculated. In the experiment, sitagliptin (CAS: 486460-32-6, synthesized according to the existing technology and conventional experimental techniques, reference: Journal of Medicinal Chemistry 48(2005) 141-151.) was used as the control compound. the

Sample treatment: The sample was dissolved in DMSO and stored at low temperature. The concentration of DMSO in the final system was controlled within the range that did not affect the detection activity. the

For the primary screening, the activity of the sample is tested under the condition of a single concentration, such as 20 μg/ml. For samples that exhibit activity under certain conditions, for example, the inhibition rate (Inhibition) is greater than 50, and the dose-dependent relationship of the test activity, that is, the IC ₅₀ /EC ₅₀ value, is obtained by nonlinear fitting of the sample activity to the sample concentration, and the calculation method used The software was Graphpad Prism 4, and the model used for fitting was sigmoidaldose-response (variable slope). For most inhibitor screening models, the bottom and top of the fitting curve were set to 0 and 100. In general, multiple holes (n≥2) are set for each sample in the test, and the results are represented by standard deviation (Standard Deviation, SD) or standard error (Standard Error, SE).

Experimental results:

The activity results of the compounds are shown in Table 1, which shows that the compounds of the present invention have good DPP4 inhibitory activity and selectivity. the

Table 1. DPP4 inhibitory activity and selectivity of polymorphic forms

^a NI: no inhibition

Example 12: Research on Inhibiting DPP4 Activity in Vivo

Animal: SD rat (8-10 weeks old, sex: male, body weight 250g-300g, purchased from Shanghai Slack Experimental Animal Center). the

Steps: SD rats were starved overnight, and the test compound A crystal form (prepared in the above-mentioned preparation example 1, 3 mg/kg), B crystal form (prepared in the above-mentioned preparation example 5, 3 mg/kg), amorphous (prepared in the above-mentioned Example 6 preparation, 3mg/kg), positive drug alogliptin control group (3mg/kg, CAS: 850649-62-6 synthesized according to the prior art according to conventional experimental techniques, references: Journal of Medicinal Chemistry 50 (2007 ) 2297-2300.), the positive drug LAF237 control group (15mg/kg, vildagliptin, CAS: 274901-16-5 was synthesized according to the prior art and conventional experimental techniques Reference: Journal of Medicinal Chemistry 46(2003) 2774 -2789.); with the solvent group as the blank control, blood was taken from the orbital plexus of rats before oral administration, 30, 60, 120 and 240 minutes after administration, and added to the anticoagulant-treated EPPENDORF tube, centrifuged Afterwards, plasma was collected to measure DPP4 activity. the

Results: A crystal form, B crystal form, amorphous, alogliptin (CAS: 850649-62-6 were synthesized according to the existing technology and conventional experimental techniques, reference: Journal of Medicinal Chemistry 50(2007) 2297-2300. ) and LAF237 (vildagliptin, CAS: 274901-16-5) were synthesized according to the existing technology and conventional experimental techniques. Reference: Journal of Medicinal Chemistry 46(2003) 2774-2789.) Absorbed into plasma after oral administration by mice Both can significantly inhibit the DPP4 enzyme activity in normal SD rats, and the best inhibitory effect is achieved in 60 minutes, and then the DPP4 inhibition trend is alleviated (Figure 14). Between 0-240min, crystal form B is superior to crystal form A and amorphous, but the inhibitory effect of crystal form B on DPP4 in plasma is slightly lower than that of positive control drugs alogliptin and LAF237. the

Embodiment 13: In vivo activity test

Acute hypoglycemic effect of single administration in ICR mice

Animals: ICR mice (8-10 weeks old, male, weighing 25g-30g, purchased from Shanghai Slack Experimental Animal Center)

steps:

1. Record the food intake of the animals two days before the experiment, and calculate the daily food intake of each animal;

2. The animals were starved overnight (1/3 of the daily food intake, about 2g);

3. The blood glucose was measured the next day, and the animals were randomly divided into 4 groups according to the blood glucose, 8 animals in each group. Respectively solvent control group (0.5% methylcellulose MC): A crystal form (prepared in the above-mentioned preparation example 1, 3mg/kg), B crystal form (prepared in the above-mentioned preparation example 5, 3mg/kg), amorphous ( Prepared in Example 6 above, 3 mg/kg), the positive drug alogliptin control group (3 mg/kg, CAS: 850649-62-6 was synthesized according to conventional experimental techniques according to the prior art, reference: Journal of Medicinal Chemistry 50 (2007) 2297-2300.), the positive drug LAF237 control group (15mg/kg, vildagliptin, CAS: 274901-16-5 was synthesized according to conventional experimental techniques according to the prior art, reference: Journal of Medicinal Chemistry 46( 2003) 2774-2789.);

4. Oral administration (1 mouse/min), measure blood glucose 4 hours later, orally administer glucose (2.5g/kg), measure blood glucose values at 15min, 30min, 60min, 90min, and 120min. the

Results: Crystal form B was superior to crystal form A and amorphous; among them, crystal form B, alogliptin and vildagliptin could significantly improve the oral glucose tolerance of ICR mice. The effect of crystal form B at 3 mg/kg was equivalent to that of alogliptin at 3 mg/kg and LAF237 at 15 mg/kg, and neither crystal form B, alogliptin nor LAF237 would cause hypoglycemia (Figure 15). the

Embodiment 14: pharmacokinetic test

Animals: 12 healthy rats, male, weighing 150-200g,

Randomly divided into 2 groups, 6 in each group. The crystal form A was administered by intragastric administration and intravenous injection respectively, the administration volume was 10mL/kg, and the drug was prepared with DMSO/Tween 80/normal saline (5:5:90, v/v/v). Fasting 12h before the test, free to drink water. Eat uniformly 2 hours after administration. the

Take 0.3 mL of blood from the retroocular venous plexus of rats, put it in a heparinized test tube, centrifuge at 11,000 rpm for 5 min, separate the plasma, and freeze it in a refrigerator at 20°C. the

result:

Crystal form A: After intragastric administration of 20 mg/kg of crystal form A to rats, the time to peak plasma concentration T _max was 0.25 hours, and the peak concentration C _max was 212.14 ng/ml; the area under the drug-time curve AUC _0-t was 278.211 ng h/ml; terminal elimination half-life t _1/2 is 2.47h. After intravenous injection of 10 mg/kg of crystal form A, the AUC _0-t was 271.17 ng h/ml; after dose standardization, the absolute bioavailability of rats given 20 mg/kg of crystal form A was 51.3%.

Crystal form B: After intragastric administration of 20 mg/kg of crystal form B to rats, the time to peak plasma concentration T _max was 0.25 hours, and the peak concentration C _max was 556.95 ng/ml; the area under the drug-time curve AUC _0-t was 962.51 ng h/ml; terminal elimination half-life t _1/2 is 4.38h. After intravenous administration of 10 mg/kg of crystal form B, the AUC _0-t was 906.51 ng h/ml; after dose standardization, the absolute bioavailability of 20 mg/kg of crystal form B in rats was 53.09%.

Among the two crystal forms, the pharmacokinetic test results of crystal form B are better than that of crystal form A; the half-life of crystal form B is 4.38 hours, and the bioavailability is 53.09%; the half-life of crystal form A is 2.47 hours, and the bioavailability is 51.30% ; AUC _0-t of crystal form B is greater than AUC _0-t of crystal form A.

From the above experimental results, it can be seen that compared with the prior art, the polymorphic form of Compound I has the advantages of strong DPP4 inhibitory activity, strong hypoglycemic activity in vivo, and good pharmacokinetic properties: (1) plasma of the B crystal form The inhibitory activity of DPP IV is better than that of A crystal form and amorphous form, and is equivalent to the hypoglycemic activity of marketed drugs alogliptin and vildagliptin (LAF-237); (2) The in vivo hypoglycemic activity of B crystal form is better than that of A crystal (3) The half-life of crystal form B is longer than that of crystal form A and amorphous, and its bioavailability is also better than that of A crystal form and amorphous. Especially suitable for the treatment of type 2 diabetes. the

Claims (10)

1. A 2-((2-(3-aminopiperidine-1)-4-oxythieno[3,2-d]pyrimidine-3(4H)-methyl)benzonitrile crystal, which is the following crystal One of the types: A crystal form, the A crystal form uses the X-ray powder diffraction method of Cu-K _α radiation, and is represented by the value of the crystal plane distance d at about

There are diffraction peaks; B crystal form, the B crystal form uses the X-ray powder diffraction method of Cu-K _α radiation, and the value of the crystal plane distance d is expressed at about

There are diffraction peaks. 2. 2-((2-(3-aminopiperidine-1)-4-oxythieno[3,2-d]pyrimidine-3(4H)-methyl)benzonitrile crystal according to claim 1 , characterized by: The A crystal form uses the X-ray powder diffraction method of Cu-K _α radiation, and the value of the crystal plane distance d is expressed at about

There are diffraction peaks; The B crystal form uses the X-ray powder diffraction method of Cu-K _α radiation, and the value of the crystal plane distance d is expressed at about

There are diffraction peaks. 3. 2-((2-(3-aminopiperidine-1)-4-oxythieno[3,2-d]pyrimidine-3(4H)-methyl)benzyl according to claim 1 or 2 Nitrile crystals, characterized in that: The A crystal form has a powder X-ray diffraction pattern substantially as shown in Figure 1; The Form B has a powder X-ray diffraction pattern substantially as shown in FIG. 6 . 4. 2-((2-(3-aminopiperidine-1)-4-oxythieno[3,2-d]pyrimidine-3(4H)-methyl)benzyl according to claim 1 or 2 Nitrile crystals, characterized in that: The A crystal form has infrared spectrum and DSC spectrum as shown in Figure 2 and 3, respectively; The crystal form B has an infrared spectrum and a DSC spectrum substantially as shown in Figs. 7 and 8, respectively. 5. A 2-((2-(3-aminopiperidine-1)-4-oxythieno[3,2-d]pyrimidine-3(4H)-methyl)benzonitrile crystal composition, which is A crystal form composition or B crystal form composition, In the crystal form A composition, the crystal form A according to any one of claims 1-4 accounts for more than 60% of the weight of the composition; In the crystal form B composition, the crystal form B according to any one of claims 1-4 accounts for more than 60% by weight of the composition. 6. 2-((2-(3-aminopiperidine-1)-4-oxythieno[3,2-d]pyrimidine-3(4H)-methyl described in any one of claims 1-4 ) the preparation method of benzonitrile crystal, wherein: The A crystal form is prepared according to the following method: method one: (1) Dissolve 2-((2-(3-aminopiperidine-1)-4-oxothieno[3,2-d]pyrimidine-3(4H)-methyl)benzonitrile in esters selected from In one of solvents and alcoholic solvents; (2) adding a solvent selected from ethers and alkane solvents under stirring; (3) crystallization; (4) The solid obtained in step (3) is vacuum-dried for 30-100 hours at 20-80°C and 0-10KPa; The B crystal form is prepared according to the following method: Method Two: A. Dissolving 2-((2-(3-aminopiperidine-1)-4-oxythiophene[3,2-d]pyrimidine-3(4H)-methyl)benzonitrile in a ketone solvent; B. adding a solvent selected from ethers and alkane solvents under stirring; C. Crystallization; D. The solid obtained in step C is vacuum-dried for 30-100 hours at 20-80° C. and 0-10 KPa. 7. The method according to claim 6, characterized in that: The ester solvent described in step (1) of method one is methyl acetate, ethyl acetate, ethyl formate, isopropyl acetate, butyl acetate, isobutyl acetate or propyl acetate; and/or The alcoholic solvent described in step (1) of method one is ethanol, methanol, n-butanol, isopropanol or n-propanol, preferably ethanol; and/or The ketone solvent described in step A of method two is acetone, butanone, methyl isobutyl ketone or methyl butyl ketone; and/or The ether solvent described in step (2) of method 1 and step B of method 2 is petroleum ether, tert-butyl methyl ether, diethyl ether, isopropyl ether or diethyl ether; and/or The alkane solvent described in step (2) of method one and step B of method two is n-heptane, n-hexane or n-pentane; and/or The crystallization conditions of step (3) of method 1 and step C of method 2 are: the crystallization temperature is 0-50°C; and/or The first step (4) of the method is carried out under the following conditions: vacuum drying at 25-40° C. and 0-10 KPa for 60-80 hours. 8. A pharmaceutical composition comprising the 2-((2-(3-aminopiperidine-1)-4-oxythiophene[3,2 -d] pyrimidine-3(4H)-methyl)benzonitrile crystals and pharmaceutically acceptable excipients. 9. The pharmaceutical composition according to claim 8, wherein the excipients include conventional fillers, disintegrants, binding agents and/or lubricants, and the fillers are selected from the group consisting of starch, lactose, starch Crystalline cellulose, dextrin, mannitol, magnesium oxide, calcium sulfate and mixtures thereof, the disintegrating agent being selected from carboxymethyl cellulose and its salts, cross-linked carboxymethyl cellulose and its salts, cross-linked polyvinyl chloride Ketone, sodium carboxymethyl starch, low-substituted hydroxypropyl cellulose and mixtures thereof; the binder is selected from povidone, hydroxypropyl methylcellulose, starch slurry and mixtures thereof; the lubricant is selected from Magnesium stearate, calcium stearate and mixtures thereof. 10. 2-((2-(3-aminopiperidine-1)-4-oxythieno[3,2-d]pyrimidine-3(4H)-methyl described in any one of claims 1-4 ) Use of benzonitrile crystals in the preparation of medicines for preventing or treating type II diabetes and its complications.

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