阅读说明：本技术 一种天维菌素b的晶型及其制备方法和用途 (Crystal form of ivermectin B and preparation method and application thereof ) 是由 王继栋 李建宋 尹明星 张辉 张灵坚 黄隽 白骅 于 2018-05-28 设计创作，主要内容包括：本发明涉及一种天维菌素B晶型I,它可以通过X-射线粉末衍射(XRPD)谱图、红外(IR)吸收谱图和差示扫描量热(DSC)谱图等来表征。同时,本发明还涉及该天维菌素B晶型I的制备方法和用途。(The invention relates to a crystal form I of ivermectin B, which can be characterized by an X-ray powder diffraction (XRPD) spectrogram, an Infrared (IR) absorption spectrogram, a Differential Scanning Calorimetry (DSC) spectrogram and the like. Meanwhile, the invention also relates to a preparation method and application of the crystal form I of the tianweimycin B.)

1. Crystalline form I of ivermectin B, characterized by an X-ray powder diffraction pattern using Cu-Ka radiation expressed in 2 theta angles having characteristic peaks at the following positions: 9.62 +/-0.20 degrees, 11.33 +/-0.20 degrees, 11.79 +/-0.20 degrees, 12.48 +/-0.20 degrees, 13.48 +/-0.20 degrees, 21.12 +/-0.20 degrees and 23.70 +/-0.20 degrees.

2. Crystalline form I of ivermectin B according to claim 1, characterized by an X-ray powder diffraction pattern using Cu-ka radiation expressed in 2 Θ angles further having characteristic peaks at the following positions: 6.71 +/-0.20 degrees, 9.22 +/-0.20 degrees, 12.02 +/-0.20 degrees, 14.95 +/-0.20 degrees, 17.39 +/-0.20 degrees, 18.33 +/-0.20 degrees, 22.97 +/-0.20 degrees, 26.53 +/-0.20 degrees and 27.16 +/-0.20 degrees.

3. Crystalline form I of ivermectin B according to claim 2, characterized in that the X-ray powder diffraction pattern expressed in 2 Θ angles using Cu-ka radiation further has characteristic peaks at the following positions: 4.63 +/-0.20 degrees, 15.45 +/-0.20 degrees, 15.80 +/-0.20 degrees, 16.64 +/-0.20 degrees, 17.74 +/-0.20 degrees, 19.20 +/-0.20 degrees, 19.75 +/-0.20 degrees, 22.14 +/-0.20 degrees, 22.52 +/-0.20 degrees, 25.01 +/-0.20 degrees, 25.54 +/-0.20 degrees and 29.60 +/-0.20 degrees.

4. A process for preparing form I of ivermectin B according to any one of claims 1 to 3, comprising the step of precipitating form I of ivermectin B from a solvent system comprising formamide.

5. The method of claim 4, wherein the formamide-containing solvent system is a combination of a lower alcohol, formamide and water, or a combination of a lower ketone, formamide and water; wherein the lower alcohol is preferably selected from methanol, ethanol or isopropanol; the lower ketone is preferably acetone.

6. The method according to any one of claims 4 to 5, wherein the solvent system comprising formamide is a combination of ethanol, formamide and water.

7. The method according to any one of claims 4-6, comprising: dissolving the tianweisu B in ethanol, sequentially adding formamide and water, stirring, and crystallizing to obtain the tianweisu B.

8. The method according to claim 7, wherein the ratio of the mass g of ivermectin B, the volume ml of ethanol, the volume ml of formamide and the volume ml of water is 1: 2: 4 to 5: 2.

9. A composition comprising the crystalline form I of ivermectin B according to any one of claims 1 to 3.

10. Use of the crystalline form I of ivermectin B according to any of claims 1 to 3 for the preparation of a medicament for controlling parasites and harmful insects.

Technical Field

The invention relates to the field of pharmacy. More specifically, the invention relates to a crystal form of ivermectin B, and a preparation method and application thereof.

Background

The hexadecamacrolide compound produced by streptomyces has the characteristics of high activity and broad spectrum. In addition, the compounds are tightly combined with soil in a natural environment and are not easy to be washed and seeped; under the condition of illumination or under the action of soil microorganisms, the molecular fragments of the compound are rapidly degraded into inactive compounds, are finally used as carbon sources to be decomposed and utilized by plants and microorganisms, have no residual toxicity, and become a high-efficiency biological source pesticide for agriculture and veterinary use.

Due to the remarkable characteristics of the compounds, the homologues thereof are widely researched at home and abroad. On one hand, the molecular structure of the compound is modified by adopting a synthesis means, and on the other hand, the producing strain of the compound is mutated by adopting a genetic improvement method so as to discover a new compound with higher activity. A group of Haiyang pharmaceutical industry Co., Ltd applies a large-fragment DNA seamless splicing technology to integrate a milbemycins PKS gene to an avermectin producing bacterium Streptomyces avermitilis starting module to obtain a genetic engineering bacterium MA220 for producing the oryzanol (see WO 2015135242). The main components of ivermectin are ivermectin A and B (the structures of which are shown below). CN201410208660.9 and WO2015135467 disclose that the tianweimycin A and B can prevent and control pests and mites of agricultural and forestry crops such as tetranychus cinnabarinus, tetranychus urticae koch, beet armyworm, prodenia litura, cotton bollworm, black cutworm, wireworm, armyworm, pine caterpillar, pine wood nematode disease, rice stem borer and the like.

Avermectin A: r ═ CH 3; and (3) ivermectin B: r ═ C2H5

In addition, patent CN201410208660.9 and WO2015135467 disclose a method for preparing tianweimycin a and B, which comprises filtering fermentation liquid of genetically engineered bacterium MA220 with filter cloth to obtain filter cake, and extracting the filter cake with ethanol twice to obtain ethanol extract. The ethanol extract is subjected to vacuum concentration to dryness, is segmented by silica gel column chromatography, is separated and purified by a semi-preparative high pressure liquid phase (eluent: methanol/acetonitrile/water: 46/46/8), and the obtained fraction is directly concentrated to dryness to obtain the tianweimycin A and the tianweimycin B. Patent DE4031039 discloses a process for the preparation of ivermectin B. The method obtains a crude product of the ivermectin B through synthesis, and then obtains the ivermectin B through silica gel purification (ethyl acetate/hexene 2: 1). Finally, neither of the two methods for the preparation of ivermectin B have investigated the resulting crystalline forms of ivermectin B.

Through a large number of experiments, firstly, amorphous powder of the ivermectin B is obtained by repeating the two preparation methods; secondly, in the research process, the amorphous powder is found to be very unstable, so that the cost of subsequent product development is increased; finally, we found a crystal form of ivermectin B (named crystal form I), which is not easy to degrade and is more stable, and lays a solid foundation for the subsequent development of ivermectin B.

Disclosure of Invention

One of the purposes of the invention is to provide a crystal form B of the ivermectin with good chemical and physical stability, which is named as a crystal form I. The crystal form is not easy to degrade and is more stable, thereby being more beneficial to the subsequent development of products.

The crystal form I of the ivermectin B has characteristic peaks at the following positions by using Cu-Kalpha radiation and an X-ray powder diffraction pattern expressed by a2 theta angle: 9.62 +/-0.20 degrees, 11.33 +/-0.20 degrees, 11.79 +/-0.20 degrees, 12.48 +/-0.20 degrees, 13.48 +/-0.20 degrees, 21.12 +/-0.20 degrees and 23.70 +/-0.20 degrees.

Preferably, the X-ray powder diffraction pattern of the skyhawin B crystal form I of the present invention expressed in 2 θ angle further has characteristic peaks at the following positions using Cu-ka radiation: 6.71 +/-0.20 degrees, 9.22 +/-0.20 degrees, 12.02 +/-0.20 degrees, 14.95 +/-0.20 degrees, 17.39 +/-0.20 degrees, 18.33 +/-0.20 degrees, 22.97 +/-0.20 degrees, 26.53 +/-0.20 degrees and 27.16 +/-0.20 degrees.

More preferably, the X-ray powder diffraction pattern of the skyhawin B form I of the present invention expressed in 2 θ angle further has characteristic peaks at the following positions using Cu-ka radiation: 4.63 +/-0.20 degrees, 15.45 +/-0.20 degrees, 15.80 +/-0.20 degrees, 16.64 +/-0.20 degrees, 17.74 +/-0.20 degrees, 19.20 +/-0.20 degrees, 19.75 +/-0.20 degrees, 22.14 +/-0.20 degrees, 22.52 +/-0.20 degrees, 25.01 +/-0.20 degrees, 25.54 +/-0.20 degrees and 29.60 +/-0.20 degrees.

Further, the X-ray powder diffraction pattern of the crystalline form I of ivermectin B according to the present invention has 2 θ angle, d value and relative intensity data as shown in table 1 below.

TABLE 12 theta Angle, d-value and relative Strength of form I of Avermectin B

Peak numbering	2θ(°)	d (interplanar spacing)	Relative Strength (%)
				1	4.63	19.0699	5.9
2	6.71	13.1625	29.7
				3	9.22	9.5837	28.4
4	9.62	9.1862	100.0
				5	11.33	7.8034	42.3
6	11.79	7.4998	43.8
				7	12.02	7.3569	31.4
8	12.48	7.0867	49.7
				9	13.48	6.5631	90.6
10	14.95	5.9209	34.0
				11	15.45	5.7304	10.9
12	15.80	5.6043	14.0
				13	16.64	5.3232	6.1
14	17.39	5.0953	26.6
				15	17.74	4.9956	15.6
16	18.33	4.8360	29.4
				17	19.20	4.6188	16.8
18	19.75	4.9515	12.5
				19	21.12	4.2031	42.4
20	22.14	4.0117	18.5
				21	22.52	3.9449	16.9
22	22.97	3.8686	20.4
				23	23.70	3.7511	42.2
24	25.01	3.5575	11.5
				25	25.54	3.4848	4.7
26	26.53	3.3570	22.4
				27	27.16	3.2805	21.7
28	29.60	3.0154	10.5

The XRPD pattern of the crystal form I of the ivermectin B is shown in figure 1.

In addition, the crystal form I of the ivermectin B can be characterized by an infrared absorption spectrum measured by KBr tabletting, and according to the spectrum, the crystal form I of the ivermectin B has characteristic peaks at the following positions: 3481cm-1, 2930cm-1, 1732cm-1, 1678cm-1, 1371cm-1, 1183cm-1, 1124cm-1, 984cm-1, 877cm-1 and 761 cm-1.

The infrared spectrogram of the crystal form I of the ivermectin B is shown in figure 2.

The Differential Scanning Calorimetry (DSC) pattern of the crystal form I of the ivermectin B has an exothermic peak at 160.8 +/-2 ℃.

The DSC pattern of the cevicin B crystal form I is shown in figure 3.

The TGA spectrum of the crystalline form I of the ivermectin B is shown in figure 4.

It is also an object of the present invention to provide a process for preparing crystalline form I of ivermectin B comprising the step of precipitating form I of ivermectin B from a solvent system comprising formamide.

Preferably, the solvent system containing formamide is a combination of a lower alcohol, formamide and water, or a combination of a lower ketone, formamide and water; wherein the lower alcohol is preferably selected from methanol, ethanol or isopropanol; the lower ketone is preferably acetone. More preferably, the formamide-containing solvent system is preferably a combination of ethanol, formamide and water.

Preferably, the process for preparing form I of ivermectin B of the present invention comprises: dissolving the tianweisu B in ethanol, sequentially adding formamide and water, stirring, and crystallizing to obtain the tianweisu B. Wherein, more preferably, the ratio of the mass g of the ivermectin B, the volume ml of the ethanol, the volume ml of the formamide and the volume ml of the water is 1: 2: 4-5: 2.

It is also an object of the present invention to provide a composition comprising crystalline form I of ivermectin B, further comprising a pharmaceutically acceptable carrier, excipient or combination thereof.

Another object of the present invention is to provide the use of crystalline modification I of ivermectin B for the preparation of a medicament for controlling parasites and harmful insects. The control object of the pesticide composition can control parasites and harmful insects in crops, human, animals, livestock, aquatic products and other aspects. Reference is made in particular to patents CN201610213645.2 (use of tianweicin for controlling parasites on humans or animals) and CN201610211064.5 (use of tianweicin for controlling harmful insects in agricultural and forestry crops).

The invention also relates to application of the crude drug of the tianweimycin containing the tianweimycin B crystal form I in preparing the drug preparation for resisting parasites and harmful insects. The preparation comprises pour-on agent, tablets, injection, dry suspension and the like.

In one embodiment, the tianweicin B is dissolved by ethanol, then water is added, and the mixture is stirred and crystallized to obtain amorphous powder of the tianweicin B. In another embodiment, the ivermectin B is dissolved by using ethanol, then formamide and water are sequentially added, and the crystal form I of the ivermectin B is obtained by stirring and crystallization. In another example, the stability of form I and form B amorphous form of ivermectin are compared and it is found that the stability of form I of ivermectin B is much better than the stability of form I of ivermectin B.

The crystal form I of the tianweimycin B provided by the invention is the only crystal form of the tianweimycin B found at present, and fills the blank of the prior art. The crystal form product has higher purity, more uniform crystals and more controllable subsequent drying process, and in addition, the stability experiment of the crystal form product does not find the crystal transformation phenomenon, and the product does not find degradation.

Drawings

FIG. 1 is an X-ray powder diffraction pattern of crystalline form I of ivermectin B obtained in example 1

FIG. 2 is an infrared absorption spectrum of crystalline form I of ivermectin B obtained in example 1

FIG. 3 is a DSC pattern of the crystal form I of the ivermectin B obtained in example 1

FIG. 4 is a TGA profile of crystalline form I of ivermectin B obtained in example 1

FIG. 5 is an X-ray powder diffraction pattern of amorphous powder of ivermectin B obtained in comparative example 1

Detailed Description

The following examples further illustrate the invention and are not to be construed as limiting the invention.

The raw material of the tianweimycin B used in the invention is prepared according to example 1 in Chinese invention patent CN201410208660.9, and is determined to be amorphous tianweimycin B by X-ray powder diffraction detection.

The X-ray powder diffraction instrument and the test conditions related by the invention are as follows: x-ray diffraction apparatus model Rigaku D/max-2200Cu target; the operation method comprises the following steps: the scanning speed is 4 degrees/min, and the scanning step width is 0.01 degrees.

The infrared spectrophotometer and the test conditions related by the invention are as follows: the infrared spectrophotometer model: BRWKER VECTOR 22; the operation method comprises the following steps: a KBr tabletting method is adopted, and the scanning range is 400-4000 cm < -1 >.

The DSC detector and the test conditions related by the invention are as follows: the DSC detector has the model as follows: PERKIN ELMER DSC8000, 8000; the operation method comprises the following steps: the heating rate is 10 ℃/min, and the temperature range is as follows: 20-280 ℃.

The TGA detector and the test conditions related to the invention are as follows: TGA detector model: PerkinElmer TGA 400; the operation method comprises the following steps: the heating rate is 10 ℃/min, and the temperature range is as follows: 30-300 ℃.

The liquid phase (HPLC) test conditions involved in the present invention are: the chromatographic column is as follows: c18, 4.6mm × 250 mm; the mobile phase is as follows: acetonitrile: 0.1% aqueous phosphoric acid 65:35(V: V); detection wavelength: 240 nm; flow rate: 1.0 ml/min; column temperature: at 25 ℃.

The dissolution and crystallization steps involved in the present invention generally require stirring unless otherwise specified, and may be carried out in a known manner, for example, by stirring with a magnetic stirrer, mechanical stirring, etc.