CN114478679A

CN114478679A - Dihydroartemisinin-ursodesoxycholic acid conjugate polymorph and preparation method and application thereof

Info

Publication number: CN114478679A
Application number: CN202210121134.3A
Authority: CN
Inventors: 沈征武; 边泓竹
Original assignee: Yunbaiyao Zhengwu Technology Shanghai Co ltd
Current assignee: Yunbaiyao Zhengwu Technology Shanghai Co ltd
Priority date: 2022-02-09
Filing date: 2022-02-09
Publication date: 2022-05-13
Also published as: CN116003505A

Abstract

The invention relates to a polymorphic substance of dihydroartemisinin-ursodesoxycholic acid conjugate (I), a preparation method thereof and application thereof as an anti-tumor and immunosuppressant drug,

the multiple crystal forms of the conjugate (I) are characterized by means of X-ray powder diffraction, differential scanning calorimetry, thermogravimetric analysis and the like. The preparation methods of the three crystal forms are not only used for purifying raw material medicines, but also have important application value in pharmaceutical preparations.

Description

Dihydroartemisinin-ursodesoxycholic acid conjugate polymorph and preparation method and application thereof

Technical Field

The invention belongs to the field of medicinal chemistry, and particularly relates to a dihydroartemisinin-ursodeoxycholic acid conjugate (I) in crystal forms of A, D and G, a preparation method and application thereof.

Background

Polymorphism of a drug is that the same drug forms different polymorphs due to different crystal structures. In the field of pharmaceutical research, in addition to anhydrate polymorphs, multicomponent crystalline forms such as organic solvates, hydrates, and the like are included. The polymorphism of the drug widely exists in the process of drug development and is an inherent characteristic of organic small molecular compounds. Different crystal forms have different melting points, solubilities, dissolution properties, chemical stabilities, flowability, mechanical stabilities and the like, and the physical and chemical properties or the processability sometimes directly influence the bioavailability or the effective performance of the medicine. Therefore, the research and control of the crystal form become important research content in the process of drug research and development.

In patents CN201710294320.6(US10881636B2, EP18790816.5) and CN201910705451.8, conjugates (formula II) obtained by condensation of dihydroartemisinin with the steroid hydroxyl group at position 3 are disclosed:

wherein, the 10-hydroxyl of dihydroartemisinin is condensed with various steroid 3-hydroxyl to form new dihydroartemisinin derivative;

the dihydroartemisinin structure is (III):

the hydroxyl at the 10-position of the dihydroartemisinin can be in S or R configuration;

wherein, the 10-hydroxyl of dihydroartemisinin and the 3-hydroxyl of steroid compound are condensed to form ether bond. The 10-position of the dihydroartemisinin can be in S or R configuration; the steroid 3-position may be in the alpha or beta configuration;

in the general formula (II), n is 0-5;

in the general formula (II), R₁H atom at position 6 or 7, or hydroxyl, alkoxy or amino and amino, or ketocarbonyl, or a double bond formed at position 6 and position 7; if the 6-or 7-position is hydroxyl or amino and amino, it can be in the alpha or beta configuration;

in the general formula (II), R₂Can be a H atom or a hydroxyl, alkoxy, amino or amino group, or a ketocarbonyl group; r₂If it is hydroxyl, alkoxy, amino or amine, it may be in alpha or beta configuration;

in the general formula (II), R₃Can be hydrogen, carboxyl and derivatives thereof, hydroxyl and derivatives thereof, amino and derivatives thereof, halogen, alkyl and alkenes or alkynes having 1 to 10 carbon atoms, alcohols or polyols having 1 to 10 carbon atoms and derivatives thereof formed from the alcoholic hydroxyl group, carboxylic or polycarboxylic acids having 1 to 10 carbon atoms and derivatives thereof formed from the carboxyl group, alkylsulfonic acids having 1 to 10 carbon atoms and salts thereof, amines having 1 to 10 carbon atoms and derivatives thereof formed from the amine group, alkylimines and alkoxyimines having 1 to 10 carbon atoms, hydroxylamine sulfonates and salts thereof, tri-to eight-membered cyclic or heterocyclic hydrocarbons with or without heteroatoms, and also includes R₃And the 16-carbon is connected to form a ring.

In the general formula (II), the isomers of the conjugate obtained by condensing the 10-hydroxyl of dihydroartemisinin and the 3-hydroxyl of steroid compound include all isomers, such as position isomers, stereoisomers and optical isomers.

Such compounds include the following preferred compounds:

wherein R is₁H atom at position 6 or 7, or hydroxyl, alkoxy or amino and amino, or ketocarbonyl, or a double bond formed at position 6 and position 7; if the 6-or 7-position is hydroxyl or amino and amino, it can be in the alpha or beta configuration.

Patent CN201710294320.6(US20200046679, EP18790816.5) discloses the structure of dihydroartemisinin-ursodeoxycholic acid conjugate (I) and the use for cancer treatment. In addition, the patent CN201910705451.8 further discloses that the conjugate is a novel immunosuppressant, which can effectively inhibit hyperfunction of immunity, and can be used for treating various autoimmune diseases by being used singly or being combined with other medicines.

The dihydroartemisinin-ursodesoxycholic acid conjugate (I) has the following structural formula:

in vitro, the compound inhibited CD4+ T cell proliferation and CD8+ T cell proliferation in a dose-dependent manner, with an IC50 of 9nM and 19nM, respectively. The compound can also strongly inhibit IFN-gamma secretion of T cells, and is dose-dependent, and IC50 is 8 nM. The compound not only strongly inhibits concanavalin A-induced mouse spleen T lymphocyte proliferation (IC50 is 15nM), but also strongly inhibits bacterial lipopolysaccharide LPS-induced normal mouse spleen B lymphocyte proliferation (IC50 is 15 nM). The inhibition test of the compound on PHA-stimulated human PBMC proliferation in vitro shows that the compound can inhibit the proliferation of PBMC in a dose-related manner, and the IC50 is 141 nM.

The compound can obviously inhibit DNFB (2, 4-dinitrofluorobenzene) -induced ear swelling on an ear swelling model of delayed hypersensitivity of mice and has dose correlation. The compound can obviously inhibit the plantar swelling caused by sheep red blood cells in a dose-related manner on a plantar swelling model of delayed hypersensitivity caused by sheep red blood cells.

In conclusion, in vitro and in vivo experiments of dihydroartemisinin-ursodeoxycholic acid conjugate (I) showed good immunosuppressive activity.

Disclosure of Invention

The invention aims to provide a crystal form A, a crystal form D and a crystal form G of a dihydroartemisinin-ursodeoxycholic acid conjugate (I), a preparation method and application thereof, and a preparation method and application thereof, wherein the crystal forms are compared with other crystal forms.

The polymorphism research of the compound shows that the compound not only has a plurality of amorphous states, but also has a plurality of different types of polymorphism, and the crystal forms have important significance for the research of the compound used for pharmaceutical dosage forms.

The invention provides a crystalline form a of a dihydroartemisinin-ursodeoxycholic acid conjugate (I) based on X-ray powder diffraction of copper ka 1 radiation, comprising at least the following 11 diffraction angles, calculated in 2 theta ± 0.2 °:

3.72±0.2°,6.28±0.2°,7.71±0.2°,8.45±0.2°,10.60±0.2°,11.24±0.2°,12.64±0.2°,14.98±0.2°,15.57±0.2°,17.00±0.2°,18.35±0.2°。

differential scanning calorimetry analysis of the crystal form A shows that the crystal form A has a characteristic exothermic peak at 166-174 ℃, thermogravimetric analysis shows that no obvious weight loss and no obvious solvent residue exist when the crystal form A is heated to 145-165 ℃, and the crystal form A is proved to be an anhydrous crystal form.

Further, the water activity study of the crystal form A shows that the crystal form A is stable when the water activity is less than or equal to 0.641; dynamic solubility research shows that the dynamic solubility of the crystal form A in water is less than 0.01mg/ml within 24 hours, and the crystal form is not changed; hygroscopicity studies show that form a is slightly hygroscopic; one week stability studies showed that no solid morphology change or significant HPLC purity reduction of form a was observed after one week of standing at 25 ℃/60% RH and 40 ℃/75% RH. These results all indicate that form a has good physical and chemical stability.

Form a can be prepared by one of two methods:

1. the method comprises the following steps: recrystallizing or pulping the conjugate (I) by using an ester solvent;

2. the second method comprises the following steps: recrystallizing or pulping the conjugate (I) by using an aqueous or non-aqueous alcohol solvent;

wherein the ester solvent is any one of methyl acetate, ethyl acetate, n-propyl acetate, isopropyl acetate, n-butyl acetate, isobutyl acetate, sec-butyl acetate, methyl propionate, ethyl propionate, n-propyl propionate and isopropyl propionate. The alcohol solvent is any one of methanol, ethanol, n-propanol, isopropanol, n-butanol, isobutanol, sec-butanol or tert-butanol.

In the above operation, the recrystallization temperature is in the range of 0 to 120 ℃, and the beating temperature is in the range of-20 to 50 ℃.

The invention provides a dihydroartemisinin-ursodeoxycholic acid conjugate (I) in a crystal form D, which is based on X-ray powder diffraction of copper K alpha 1 radiation and at least comprises the following 13 diffraction angles in terms of 2 theta +/-0.2 degrees:

7.98±0.2°,8.46±0.2°,10.05±0.2°,10.97±0.2°,11.37±0.2°,11.65±0.2°,12.56±0.2°,13.89±0.2°,14.86±0.2°,15.84±0.2°,17.03±0.2°,17.38±0.2°,18.61±0.2°。

and the crystal form D has a characteristic exothermic peak at 150-171 ℃ by differential scanning calorimetry, and the sample loses 3.3% weight when the temperature is raised to 135-145 ℃ by thermogravimetric analysis.¹A small amount of solvent residue was observed by H NMR, and form D was anhydrous.

The preparation method of the crystal form D can be used for preparing the following steps:

dissolving the conjugate (I) in 1, 4-dioxane, performing liquid-liquid or vapor-liquid diffusion with a low-polarity solvent, and collecting the precipitated product.

Wherein the low-polarity solvent is any one of n-pentane, cyclopentane, n-hexane, cyclohexane, heptane, octane or petroleum ether.

The invention provides a crystal form G of dihydroartemisinin-ursodeoxycholic acid conjugate (I), which is based on X-ray powder diffraction of copper K alpha 1 radiation and at least comprises the following 14 diffraction angles in terms of 2 theta +/-0.2 degrees:

6.00±0.2°,7.44±0.2°,8.20±0.2°,9.97±0.2°,10.48±0.2°，11.16±0.2°,12.23±0.2°,12.70±0.2°,13.44±0.2°,14.92±0.2°,15.35±0.2°,15.51±0.2°,16.87±0.2°,18.20±0.2°。

and the crystal form G has a characteristic exothermic peak at 167-173 ℃ by differential scanning calorimetry, and has no characteristic weight loss peak when the temperature is raised to 130-160 ℃ by thermogravimetric analysis.¹Little solvent residue was observed by HNMR, and form G was anhydrous.

A process for the preparation of form G, characterized in that it can be prepared by:

the conjugate (I) was suspended in cyclopentyl methyl ether/acetonitrile and stirring was continued until the XRPD pattern showed the characteristic form G.

Wherein, the volume ratio of the cyclopentyl methyl ether to the acetonitrile can be 1/0.2-1/4 with equal proportion.

The invention also provides other polymorphic forms, and provides their characteristic diffraction angles (2 theta) by X-ray powder diffraction. Specifically, crystal forms B, C, E, F, H, I, J and K are included. Researches find that the crystal form C, the crystal form E and the crystal form H are hydrates, the crystal form B and the crystal form I are solvates, the crystal form F, the crystal form J and the crystal form K are metastable crystal forms, and only three of the crystal form A, the crystal form D and the crystal form G are anhydrous crystal forms. The metastable crystal form is unstable at room temperature, and the solvate and the hydrate are easy to lose the solvent and generate crystal transformation when heated; the three anhydrous crystal forms of the crystal form A, the crystal form D and the crystal form G have better thermodynamic stability than other crystal forms, so that the crystal form A, the crystal form D and the crystal form G have better practical value in the aspects of raw material medicine process and pharmaceutical preparation development.

The invention provides application of a polymorphic substance of dihydroartemisinin-ursodesoxycholic acid conjugate (I) in preparing a medicament for treating cancer or autoimmune diseases.

The present invention provides a pharmaceutical composition comprising a therapeutically effective amount of any of the crystalline forms of the conjugate (I) as described above, together with a pharmaceutically acceptable carrier or excipient.

The acceptable carrier is non-toxic, can be administered adjunctively, and does not adversely affect the therapeutic efficacy of the composition. Such carriers can be any solid, liquid, semi-solid or gaseous vehicle commonly available to those skilled in the art in aerosol compositions. Solid pharmaceutical excipients include starch, cellulose, talc, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, magnesium stearate, sodium stearate, glyceryl stearyl ester, sodium chloride, anhydrous skim milk, and the like. The liquid and semi-solid excipients may be selected from glycerol, propylene glycol, water, ethanol and various oils, including those of petroleum, animal, vegetable or synthetic origin, e.g., peanut oil, soybean oil, mineral oil, sesame oil and the like, preferably liquid carriers, particularly for injectable solutions, including water, saline, aqueous dextrose and glycols. Other adjuvants such as flavoring agent, sweetener, etc. can also be added into the composition.

The pharmaceutical composition of the present invention can be administered orally or by other administration means, such as injection, transdermal administration, spray administration, rectal administration, vaginal administration, etc. The preferred mode of administration is oral, which may be modulated by the extent of the disease.

Various dosage forms of the pharmaceutical composition of the present invention can be prepared according to conventional methods in the pharmaceutical field. For example, the compounds can be combined with one or more carriers and then formulated into the desired dosage form, e.g., tablets, pills, capsules, semisolids, powders, sustained release formulations, solutions, suspensions, formulations, aerosols, and the like.

The pharmaceutical composition of the invention can be used in combination with other anticancer drugs including traditional Chinese medicine anticancer drugs, such as adriamycin, bleomycin, vinblastine, taxanes, etoposide, 5-fluorouracil, cyclophosphamide, methotrexate, cisplatin, tretinoin, temozolomide, actinomycin and targeted drugs such as imatinib, gefitinib, sorafenib, erlotinib, sunitinib, rituximab, cetuximab, trastuzumab and the like.

The pharmaceutical compositions of the present invention may also be used in combination with other cancer treatment methods, such as surgical treatment, radiation therapy, bone marrow transplantation, and the like.

The pharmaceutical compositions of the invention may be administered in combination with other immunosuppressive agents including antimetabolites: azathioprine (Aza), methotrexate, Mycophenolate Mofetil (MMF), and the like; a alkylating agent: cyclophosphamide, etc.; corticosteroids: prednisone, dexamethasone, and the like; antibiotics: CsA, FK506, rapamycin, etc.; antibodies: anti-lymphocyte globulin (ALG), monoclonal T lymphocyte antibody (OKT3), and the like; chinese herbal medicines: tripterygium glycosides, Cordyceps preparation, etc.

The cancer includes brain cancer, brain glioma, endometrial cancer, ovarian cancer, cervical cancer, breast cancer, colon cancer, lung cancer, prostate cancer, liver cancer, leukemia, lymphoma, skin cancer, basal cell tumor, hemangioma, uterine cancer, laryngeal cancer, stomach cancer, lip cancer, esophageal cancer, nasopharyngeal cancer, gallbladder cancer, pancreatic cancer, kidney cancer, tongue cancer, bladder cancer, melanoma, lipoma, thyroid cancer, thymus cancer, or bone cancer.

The autoimmune diseases comprise lupus erythematosus, rheumatoid arthritis, ankylosing spondylitis, type I diabetes, nephrotic syndrome, sjogren's syndrome, multiple sclerosis, psoriasis, dermatomyositis, allergic rhinitis, intractable urticaria, autoimmune hemolytic anemia, idiopathic thrombocytopenic purpura, nonspecific enteritis, scleroderma, rejection reaction after organ transplantation of human body, etc.

Description of the drawings:

figure 1 is an XRPD pattern of form a.

Figure 2 is a DSC diagram of form a.

Figure 3 is a TGA profile of form a.

Figure 4 is a 1HNMR picture of form a.

Fig. 5 is a PLM diagram of form a.

Figure 6 is an XRPD pattern of form D.

Figure 7 is a DSC diagram of form D.

Figure 8 is a TGA profile of form D.

Figure 9 is a 1HNMR map of form D.

Fig. 10 is a PLM diagram of form D.

Figure 11 is an XRPD pattern of form G.

Figure 12 is a DSC diagram of form G.

Figure 13 is a TGA profile of form G.

Figure 14 is a 1HNMR picture of form G.

Fig. 15 is a PLM diagram of form G.

Figure 16 is an XRPD pattern of form C.

Figure 17 is an XRPD pattern of form E.

Figure 18 is an XRPD pattern of form H.

Figure 19 is an XRPD pattern of form B.

Figure 20 is an XRPD pattern of form I.

Figure 21 is an XRPD pattern of form F.

Figure 22 is an XRPD pattern of form J.

Figure 23 is an XRPD pattern of form K.

Detailed Description

The technical solutions in the embodiments of the present invention are clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

1. The main apparatus is as follows:

1.1X-ray powder diffractometer (XRPD)

1.2 thermogravimetric analysis (TGA) and Differential Scanning Calorimetry (DSC)

2. Preparation of dihydroartemisinin-ursodeoxycholic acid conjugate (I):

boron trifluoride diethyl etherate (1 ml) was slowly added dropwise to a solution of dihydroartemisinin (569 mg, 2.0 mmol) and ursodeoxycholic acid (785 mg, 2.0 mmol) in diethyl ether (50 ml) at-78 ℃, and the reaction was allowed to warm to room temperature naturally and stirred overnight. After the reaction is finished by monitoring through thin-layer silica gel chromatography, slowly quenching the reaction by using saturated sodium bicarbonate aqueous solution (30 ml), extracting by using ethyl acetate (30 ml multiplied by 3), combining organic phases, washing by using water (50 ml) for 1 time, washing by using saturated saline solution (50 ml) for 1 time, drying by using anhydrous magnesium sulfate, filtering, and carrying out reduced pressure spin-drying on filtrate to obtain a crude product. The crude product was purified by column on silica gel (eluent: petroleum ether/ethyl acetate 1/1) to give white solid dihydroartemisinin-ursodeoxycholic acid conjugate (I)749 mg, yield: 56.8 percent.

¹H NMR(400MHz,CDCl₃)：δ5.47(s,1H),4.93(d,J＝3.3Hz,1H),4.00(s,1H),3.88(s,1H),3.51(t,J＝11.0Hz,1H),2.64–2.56(m,1H),2.47–2.21(m,4H),2.10–2.02(m,2H),1.99–1.85(m,5H),1.72(m,8H),1.53(m,5H),1.45(m,6H),1.40–1.30(m,5H),1.31–1.23(m,4H),1.16(m,2H),0.99(m,7H),0.89(m,8H),0.71(s,3H)。

¹³C NMR(100MHz,CDCl₃)：δ104.03,100.14,88.08,81.29,77.24,68.67,52.65,46.86,46.51,44.56,42.21,41.33,39.32,37.47,36.51,35.58,35.29,35.22,34.80,30.25,29.72,28.71,27.58,26.70,26.30,24.70,23.22,22.60,20.41,17.24,13.08,12.46。

MS(+ESI):(m/z)[M+H]⁺＝659.9

In addition, according to a similar synthesis method of dihydroartemisinin-ursodeoxycholic acid conjugate (I), reference patent CN201710294320.6(US20200046679, EP18790816.5) and CN201910705451.8 can obtain other derivatives of similar structure as shown in the following table:

3. preparation of dihydroartemisinin-ursodeoxycholic acid conjugate (I) crystal form:

3.1 form A

Form a can be prepared by one of the following two methods.

The first method is that 1 g dihydroartemisinin-ursodesoxycholic acid conjugate (I) and 30 ml ethyl acetate are added into a round-bottom flask, heating reflux is carried out, when the solid is basically dissolved, the solid is filtered when the solid is hot, the clear filtrate is placed at room temperature for overnight, and the precipitated white solid product is collected by filtration and dried in vacuum to obtain 750 mg of the target product.

The second method comprises the following steps: 1 g of dihydroartemisinin-ursodesoxycholic acid conjugate (I) and 10 ml of methanol are added into a 50 ml round-bottom flask, and after the sample is completely dissolved, 2-8 ml of pure water is dripped. The hot filtrate was filtered, the clear filtrate was left at room temperature overnight, and the precipitated white solid product was collected by filtration and dried under vacuum to give 898mg of the title product.

The XRPD pattern of the product is shown in fig. 1, with the main peak data shown in the following table:

the DSC chart of the product is shown in figure 2, the product has a characteristic exothermic peak (single decomposition exothermic peak, peak temperature is 172.37 ℃) at 166-174 ℃, the TGA chart is shown in figure 3, no obvious weight loss is caused when the product is heated to 145-165 ℃, and¹HNMR is shown in fig. 4 as having no significant solvent residue, which is in the anhydrous crystalline form.

The PLM image of the product, shown in FIG. 5, is a thin rod-like crystal, and birefringence was observed.

3.2 form D

20mg of dihydroartemisinin-ursodeoxycholic acid conjugate (I) was dissolved in 0.2 ml of 1, 4-dioxane at room temperature, filtered and the clear solution was contained in a 1 ml vial. The vial was then placed into a 20 ml vial containing 3 ml of n-hexane. The 20 ml vial was sealed with a bottle cap and allowed to stand at room temperature to allow the organic vapor to interact with the solution. After 11 days, a large amount of solid had precipitated from the 1 ml vial and the solid product was collected by filtration.

The XRPD pattern of the product is shown in fig. 6, with the main peak data shown in the table below:

the DSC chart of the product is shown in figure 7, the product has a characteristic exothermic peak (single decomposition exothermic peak, peak temperature is 164.86 ℃) at 150-171 ℃, the TGA chart is shown in figure 8, the temperature is raised to 135-145 ℃, the weight loss of the sample is 3.3 percent, and¹a small amount of solvent residue was observed in the HNMR pattern as shown in fig. 9, which is an anhydrous crystalline form.

The product PLM is shown in FIG. 10, and is a fine needle crystal, and birefringence is observed.

3.3 form G

100 mg of dihydroartemisinin-ursodeoxycholic acid conjugate (I) was suspended in 2.5 ml of a mixed solvent of cyclopentyl methyl ether/acetonitrile (v/v ═ 1/1). The suspension was magnetically stirred at room temperature for about 3 days and the solid was collected by filtration.

The XRPD pattern of the product is shown in fig. 11, with the main peak data shown in the table below:

2-Theta	Height	Height％
			6.00	325	34.4
7.44	673	71.1
			8.20	395	41.8
9.97	242	25.6
			10.48	467	49.4
11.16	946	100.0
			12.23	242	25.6
12.70	263	27.8
			13.44	457	48.3
14.92	400	42.3
			15.35	549	58.0
15.51	346	36.6
			16.87	494	52.2
18.20	293	31.0

the DSC chart of the product is shown in figure 12, the product has a characteristic exothermic peak (an exothermic decomposition peak is present, the peak temperature is 171.11 ℃) at 167-173 ℃, the TGA chart of the product is shown in figure 13, the temperature is raised to 130-160 ℃, the sample has no obvious weight loss and the product has no obvious weight loss¹The HNMR pattern is shown in fig. 14, and a small amount of solvent residue was observed, which is an anhydrous crystalline form.

The PLM pattern of the product, shown in FIG. 15, was irregular crystals and birefringence was observed.

3.4 form C

About 20mg of dihydroartemisinin-ursodeoxycholic acid conjugate (I) was weighed out and dissolved in a mixed solvent of 0.3 ml of ethanol/methyl ethyl ketone (v/v-1/1), and the resulting clear solution was filtered and contained in a 1 ml vial. The vial was then placed into a 20 ml vial containing 3 ml of n-hexane. The 20 ml vial was sealed with a bottle cap and the organic vapor allowed to interact with the solution at room temperature. After 11 days, a large amount of solid precipitated and the solid product was collected by filtration.

The XRPD pattern of the product is shown in fig. 16, with the main peak data shown in the table below:

2-Theta	Height	Height％
			3.11	2496	45.9
6.23	5441	100.0
			7.63	4783	87.9
11.28	1969	36.2
			12.51	4813	88.5
14.92	2295	42.2
			15.43	3952	72.6
18.20	1874	34.4
			19.42	3026	55.6

the research shows that the TGA analysis of the product shows 3.08 percent of weight loss before 150-160 ℃, and¹no significant solvent residue was observed with HNMR, heating to 150 ℃ for 3 minutes transformed form C to form a, which was a hydrate.

3.5 form E

About 20mg of dihydroartemisinin-ursodeoxycholic acid conjugate (I) was weighed out and dissolved in 0.4 ml of a mixed solvent of ethanol/ethyl acetate (v/v-1/1), and filtered. Acetonitrile was added dropwise to the filtrate with stirring until the total amount of the solvent reached 5.0 ml. Stirring at room temperature for 1 day, maintaining the system in a clear solution state, turning the cover to volatilize, precipitating fine rod-shaped crystals after 3 days, filtering and collecting the solid product.

The XRPD pattern of the product is shown in fig. 17, with the main peak data shown in the table below:

2-Theta	Height	Height％
			5.30	2939	6.9
10.66	42558	100.0
			13.85	11092	26.1
20.11	5243	12.3

the DSC chart has an endothermic peak at 47.48 deg.C (peak temperature); one exothermic decomposition peak, located at 164.87 ℃ (peak temperature). The temperature is raised to 140-150 ℃ in a TGA chart, the sample loses 2.73 percent of weight¹HNMR no significant solvent residue was observed, heating to 120 ℃ for 3 minutesForm E is transformed into form F, form E being a hydrate.

3.6 form H

Excess dihydroartemisinin-ursodeoxycholic acid conjugate (I) was suspended in methyl tert-butyl ether and stirring was continued for 2 hours at room temperature. Filtering to obtain saturated solution of dihydroartemisinin-ursodesoxycholic acid conjugate (I) at room temperature, and adding solid mixture of crystal form A and crystal form D into the saturated solution to obtain suspension. After magnetic stirring of the suspension at room temperature for about 2 days, the solid product was collected by filtration.

The XRPD pattern of the product is shown in fig. 18, with the main peak data shown in the table below:

2-Theta	Height	Height％
			5.05	416	48.5
5.55	857	100.0
			5.90	699	81.6
7.03	833	97.2
			9.86	372	43.4
11.16	424	49.5
			12.70	745	86.9
15.70	224	26.1

the DSC chart has an exothermic decomposition peak, and the peak temperature is 166.52 ℃. The temperature is raised to 150-160 ℃ in a TGA chart, the weight of the sample is reduced by 6.3 percent¹HNMR observed a small amount of solvent residue, heating to 100 ℃ for 10 minutes, and the form H transformed to form J, which was a hydrate.

3.7 form B

About 20mg of dihydroartemisinin-ursodeoxycholic acid conjugate (I) was weighed out and dissolved in 1.5 ml of acetone. Filtering, filling the filtrate into a 5 ml small bottle, sealing with a sealing film, and standing at room temperature to slowly volatilize through a pinhole on the sealing film. After 11 days, a large amount of solid was precipitated from the system, and the solid product was collected by filtration.

The XRPD pattern of the product is shown in fig. 19, with the main peak data shown in the table below:

2-Theta	Height	Height％
			5.06	6616	15.3
6.67	43141	100.0
			10.75	7494	17.4
13.41	33904	78.6
			13.95	8917	20.7
15.49	15666	36.3
			20.19	10338	24.0

the DSC chart has a wide endothermic peak at 143.20 deg.C (peak temperature); one exothermic decomposition peak, located at 162.41 ℃ (peak temperature). The TGA plot increases from 130 ℃ to 160 ℃, the sample loses 5.5% weight¹HNMR detected that the sample contained 5.58% acetone. Heating to 140 deg.C for 60 min to convert form B intoAnd the crystal form B becomes amorphous, and is a solvate of acetone.

3.8 form I

100 mg of dihydroartemisinin-ursodeoxycholic acid conjugate (I) was weighed and dissolved in 1.0 ml of 1, 4-dioxane, filtered, and the filtrate was collected in a 5 ml vial. The vial was then placed in a 100 ml jar containing 15 ml of n-hexane. The vial was sealed with a cap and left at room temperature for 4 days to allow the organic vapor to interact with the solution. After 4 days, a large amount of solid precipitated from the vial and the solid product was collected by filtration.

The XRPD pattern of the product is shown in fig. 20, with the main peak data shown in the table below:

2-Theta	Height	Height％
			5.02	2655	17.5
6.46	15138	100.0
			10.48	2163	14.3
13.00	15036	99.3
			15.16	5303	35.0
16.60	1982	13.1
			18.06	1508	10.0
19.57	14085	93.0

the DSC chart has an endothermic peak at 136.43 deg.C (peak temperature); one exothermic peak, located at 161.85 ℃ (peak temperature). The TGA chart shows that the temperature is raised to 120-130 ℃, and the sample has no obvious weight loss; the temperature of 130 ℃ is raised to 160 ℃, the weight loss of the sample is 9.7 percent, and¹HNMR finds that the content of residual 1, 4-dioxane is 11.69 percent, heating to 135-145 ℃ for 20 minutes enables the crystal form I to be converted into amorphous form, and the crystal form I is a solvate of 1, 4-dioxane.

3.9 form F

And heating the crystal form E to 110-120 ℃ for 3 minutes to obtain a crystal form F.

The XRPD pattern of the product is shown in fig. 21, with the main peak data shown in the table below:

2-Theta	Height	Height％
			5.100	1677	14.4
10.462	11657	100.0
			13.680	1921	16.5
13.810	1794	15.4
			19.949	793	6.8

the study found that form F converted to form E when the sample was allowed to stand at room temperature for 1 day. Form F is a metastable form.

3.10 form J

And heating the crystal form H to 80-110 ℃ and continuing for 10 minutes to obtain a crystal form J.

The XRPD pattern of the product is shown in fig. 22, with the main peak data shown in the table below:

researches show that when a sample is placed for 1 day at room temperature in an open manner, the crystal form is unchanged, but the crystallinity is obviously weakened; the sample was left open for 3 days at room temperature, with the crystal form unchanged but the crystallinity again diminished. After an extended time, form J gradually turns amorphous. Polymorph J is a metastable crystalline form.

3.11 form K

50 mg of dihydroartemisinin-ursodeoxycholic acid conjugate (I) were weighed and suspended in 2.0 ml of methyl tert-butyl ether. After the suspension was magnetically stirred at room temperature for about 3 days, the solid product was collected by filtration to give form K.

The XRPD pattern of the product is shown in fig. 23, with the main peak data shown in the table below:

2-Theta	Height	Height％
			5.24	439	35.1
5.63	1249	100.0
			7.23	367	29.4
10.13	356	28.5
			11.53	377	30.2
12.84	610	48.8
			13.32	276	22.1

the research shows that the crystal form K is converted into the crystal form H when the sample is placed for 1 day at room temperature in an open way. Form K is a metastable form.

4. Study of Water Activity:

water activity research can confirm the mutual transformation relation among the crystal form A, the crystal form C, the crystal form E and the crystal form H. Firstly, filtering a methanol/water mixed solvent suspension of the crystal form A with different water contents, which is balanced for 2 hours at room temperature, to obtain a saturated solution of the conjugate (I) at room temperature, and then adding a solid mixture of the crystal form A, the crystal form C, the crystal form E and the crystal form H into the saturated solution. The results are shown in the following table. The result shows that the crystal form A is more stable when the water activity is less than or equal to 0.641 and the crystal form E is more stable when the water activity is more than or equal to 0.888 under the room temperature condition.

5. Dynamic solubility study of form a:

form a was formulated as a supersaturated suspension (about 10mg/mL) in water at 37 ℃. The suspensions were sampled at 1 hour, 2 hours, 4 hours and 24 hours, respectively, and the supernatants were filtered, the filtrates tested for concentration, and the solids tested for crystalline form. The result shows that the dynamic solubility of the crystal form A in water is less than 0.01mg/mL within 24 hours, and the crystal form is not changed.

6. One week stability study of form a:

after the crystal form A is respectively placed under the conditions of 25 ℃/60% RH and 40 ℃/75% RH for one week, the HPLC purity and the crystal form change are tested. As a result, no solid morphology change or significant HPLC purity reduction of form a was observed, indicating that form a has good physical and chemical stability.

7. Hygroscopicity study of form a:

and evaluating the stability risk of the sample along with the change of humidity at 25 ℃, and performing a water vapor adsorption test on the crystal form A. As a result, the water absorption in the free state was 0.29% under the condition of 80% RH, and the sample was slightly hygroscopic. The crystal form of the sample is not changed before and after the hygroscopicity test.

8. Comparative study of thermal stability of forms A-K:

each polymorph was treated with an open air at room temperature for 5 days and heated at 150 ℃ for 20 minutes, and characterized by X-ray powder diffraction, with the results shown in the following Table:

the results show that form a, form D and form G are more stable than the other forms under the two experimental conditions.

The above-mentioned embodiments only express the embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims

1. A crystal form A of dihydroartemisinin-ursodesoxycholic acid conjugate (I),

x-ray powder diffraction based on copper K α 1 radiation, comprising at least the following 11 diffraction angles in terms of 2 θ ± 0.2 °:

2. crystalline form a of dihydroartemisinin-ursodeoxycholic acid conjugate (I) according to claim 1, characterized in that it has an X-ray powder diffraction pattern as shown in figure 1.

3. Crystalline form a of dihydroartemisinin-ursodeoxycholic acid conjugate (I) according to claim 1, characterized in that it has a differential scanning calorimetry trace as shown in figure 2, with a characteristic exothermic peak at 166-174 ℃.

4. The crystalline form A of dihydroartemisinin-ursodeoxycholic acid conjugate (I) according to claim 1, which has a thermogravimetric analysis diagram as shown in figure 3, without significant weight loss upon heating to 145-165 ℃.

5. A process for the preparation of dihydroartemisinin-ursodeoxycholic acid conjugate (I) form a according to claim 1, characterized in that:

recrystallizing or pulping the conjugate (I) by using an ester solvent or an aqueous or non-aqueous alcohol solvent;

wherein the ester solvent is any one of methyl acetate, ethyl acetate, n-propyl acetate, isopropyl acetate, n-butyl acetate, isobutyl acetate, sec-butyl acetate, methyl propionate, ethyl propionate, n-propyl propionate or isopropyl propionate; the alcohol solvent is any one of methanol, ethanol, n-propanol, isopropanol, n-butanol, isobutanol, sec-butanol or tert-butanol.

6. A crystal form D of the dihydroartemisinin-ursodeoxycholic acid conjugate (I),

x-ray powder diffraction based on copper K α 1 radiation, comprising at least the following 13 diffraction angles in terms of 2 θ ± 0.2 °:

7. crystalline form D of dihydroartemisinin-ursodeoxycholic acid conjugate (I) according to claim 6, characterized in that it has an X-ray powder diffraction pattern as shown in figure 6.

8. Crystalline form D of a dihydroartemisinin-ursodeoxycholic acid conjugate (I) according to claim 6, characterized in that it has a differential scanning calorimetry trace as shown in figure 7, with a characteristic exothermic peak at 150-171 ℃.

9. The crystalline form D of dihydroartemisinin-ursodeoxycholic acid conjugate (I) according to claim 6, which has a thermogravimetric analysis chart as shown in figure 8, and the sample loses 3.3% of weight when the temperature is raised to 135-145 ℃.

10. A process for the preparation of crystalline form D of dihydroartemisinin-ursodeoxycholic acid conjugate (I) as claimed in claim 6, characterized in that:

the conjugate (I) was dissolved in 1, 4-dioxane, diffused with a low polarity solvent, and the precipitated product was collected. Wherein the low-polarity solvent is any one of n-pentane, cyclopentane, n-hexane, cyclohexane, heptane, octane or petroleum ether.

11. A crystal form G of the dihydroartemisinin-ursodeoxycholic acid conjugate (I),

x-ray powder diffraction based on copper K α 1 radiation, comprising at least the following 14 diffraction angles in terms of 2 θ ± 0.2 °:

12. crystalline form G of dihydroartemisinin-ursodeoxycholic acid conjugate (I) according to claim 11, characterized in that it has an X-ray powder diffraction pattern as shown in figure 11.

13. Crystalline form G of a dihydroartemisinin-ursodeoxycholic acid conjugate (I) according to claim 11, characterized in that it has a differential scanning calorimetry trace as shown in figure 12, with a characteristic exothermic peak at 167-173 ℃.

14. The crystalline form G of dihydroartemisinin-ursodeoxycholic acid conjugate (I) according to claim 11, which has a thermogravimetric analysis chart as shown in figure 13, and has no characteristic weight loss peak when heated to 130-160 ℃.

15. A process for the preparation of said crystalline form G of dihydroartemisinin-ursodeoxycholic acid conjugate (I) of claim 11, characterized in that:

the conjugate (I) was suspended in a mixed solvent of cyclopentyl methyl ether/acetonitrile and stirring was continued until the XRPD pattern showed the characteristic form G.

16. Further transformation of the dihydroartemisinin-ursodeoxycholic acid conjugate (I) in crystalline form a, in crystalline form D and in crystalline form G into other crystalline forms by physical or chemical means.

17. Use of any one of the crystalline forms of dihydroartemisinin-ursodeoxycholic acid conjugate (I) as claimed in any one of claims 1, 6, 11, 16 for the preparation of a medicament for the treatment of cancer or autoimmune diseases.

18. A pharmaceutical composition comprising a therapeutically effective amount of any one of the crystalline forms of conjugate (I) as claimed in claims 1, 6, 11, 16 and a pharmaceutically acceptable carrier, diluent or excipient.

19. Use according to claim 17, characterized in that: the cancer is any one of brain cancer, brain glioma, endometrial cancer, ovarian cancer, cervical cancer, breast cancer, colon cancer, lung cancer, prostate cancer, liver cancer, leukemia, lymph cancer, skin cancer, basal cell tumor, hemangioma, uterine cancer, laryngeal cancer, stomach cancer, lip cancer, esophageal cancer, nasopharyngeal cancer, gallbladder cancer, pancreatic cancer, kidney cancer, tongue cancer, bladder cancer, melanoma, lipoma, thyroid cancer, thymus cancer or bone cancer.

20. Use according to claim 17, characterized in that: the autoimmune disease is any one of lupus erythematosus, rheumatoid arthritis, ankylosing spondylitis, type I diabetes, nephrotic syndrome, sjogren's syndrome, multiple sclerosis, psoriasis, dermatomyositis, allergic rhinitis, intractable urticaria, autoimmune hemolytic anemia, idiopathic thrombocytopenic purpura, nonspecific enteritis, scleroderma and rejection reaction after organ transplantation.