Polymorph of dihydroartemisinin-ursodeoxycholic acid conjugate and preparation method and application thereof

Abstract

The present invention relates to a polymorph of a dihydroartemisinin-ursodeoxycholic acid conjugate (I) and a preparation method thereof, as well as its use as an anti-tumor and immunosuppressant drug. Multiple crystal forms of the conjugate (I) are characterized by means such as X-ray powder diffraction, differential scanning calorimetry, and thermogravimetric analysis. Among them, the preparation method of crystal form A is simple, reproducible, has good thermodynamic stability, is conducive to the preparation process of the drug and the improvement of the physicochemical properties, and improves oral bioavailability. The preparation method of crystal form A can not only be used for the purification of raw materials, but also has important application value in pharmaceutical preparations.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical chemistry, and specifically relates to crystal forms such as crystal form A of a dihydroartemisinin-ursodeoxycholic acid conjugate (I) and a preparation method and use thereof. Background Art

[0002] Drug polymorphism refers to the formation of different polymorphs of the same drug due to its different crystal structures. In the field of drug research, in addition to anhydrous polymorphs, it also includes multi-component crystal forms such as organic solvates and hydrates. Drug polymorphism is widely present in the process of drug development and is an inherent characteristic of organic small molecule compounds. Different crystal forms have different melting points, solubility, dissolution properties, chemical stability, fluidity, mechanical stability, etc. These physicochemical properties or processability sometimes directly affect the bioavailability or effective performance of the drug. Therefore, crystal form research and control have become an important research content in the process of drug development.

[0003] Patents CN201710294320.6 (US10881636B2, EP18790816.5) and CN201910705451.8 disclose the following conjugates (general formula II) obtained by condensation of dihydroartemisinin with the 3-hydroxyl group of a steroid:

[0004]

[0005] Among them, the 10-hydroxyl group of dihydroartemisinin condenses with the 3-hydroxyl group of various steroids to form new dihydroartemisinin derivatives;

[0006] The structure of dihydroartemisinin is (ⅡI):

[0007]

[0008] The hydroxyl group at the 10-position of dihydroartemisinin can be in S or R configuration;

[0009] The 10-hydroxyl group of dihydroartemisinin and the 3-hydroxyl group of the steroid compound are condensed to form an ether bond. The 10-hydroxyl group of dihydroartemisinin can be in S or R configuration; the 3-hydroxyl group of the steroid compound can be in α or β configuration;

[0010] In the general formula (II), n=0 to 5;

[0011] In the general formula (II), R 1 It can be an H atom at position 6 or 7, or a hydroxyl group, an alkoxy group, an amino group or an amine group, or a ketocarbonyl group, or a double bond formed by positions 6 and 7; if position 6 or 7 is a hydroxyl group, an amino group or an amine group, it can be in α or β configuration;

[0012] In the general formula (II), R 2It can be an H atom or a hydroxyl group, an alkoxy group, an amino group or an amine group, or a ketocarbonyl group; R 2 If it is a hydroxyl, alkoxy, amino or amine group, it can be in α or β configuration;

[0013] In the general formula (II), R 3 It can be hydrogen, carboxyl and its derivatives, hydroxyl and its derivatives, amino and its derivatives, halogen, alkyl and olefin or alkyne with 1-10 carbon atoms, alcohol or polyol with 1-10 carbon atoms and derivatives generated from alcohol hydroxyl group, carboxylic acid or polycarboxylic acid with 1-10 carbon atoms and derivatives generated from carboxyl group, alkylsulfonic acid and salts with 1-10 carbon atoms, amine with 1-10 carbon atoms and derivatives generated from amine group, alkylimine and alkoxyimine with 1-10 carbon atoms, hydroxylamine, hydroxylamine sulfonate and its salt, 3-8-membered cyclic hydrocarbon or heterocyclic hydrocarbon containing or not containing heteroatoms, also including R 3 A derivative formed by connecting to the 16-carbon ring.

[0014] In the general formula (II), the isomers of the conjugate obtained by condensing the 10-hydroxyl group of dihydroartemisinin and the 3-hydroxyl group of the steroid compound include all isomers thereof, such as positional isomers, stereoisomers and optical isomers.

[0015] Such compounds include the following preferred compounds:

[0016]

[0017] Among them, R 1 It can be an H atom at position 6 or 7, or a hydroxyl group, an alkoxy group, an amino group or an amine group, or a ketocarbonyl group, or a double bond formed by positions 6 and 7; if position 6 or 7 is a hydroxyl group, an amino group or an amine group, it can be an α or β configuration.

[0018] Patent CN201710294320.6 (US20200046679, EP18790816.5) discloses the structure of dihydroartemisinin-ursodeoxycholic acid conjugate (I) and its use for cancer treatment. In addition, patent CN201910705451.8 further discloses the conjugate as a new immunosuppressant, which can effectively inhibit hyperimmunity, and can be used alone or in combination with other drugs to treat a variety of autoimmune diseases.

[0019] The structural formula of dihydroartemisinin-ursodeoxycholic acid conjugate (I) is as follows:

[0020]

[0021] (I)

[0022] In in vitro experiments, the compound can inhibit the proliferation of CD4+T cells and CD8+T cells in a dose-dependent manner. 50 The compound can also strongly inhibit the secretion of IFN-γ by T cells in a dose-dependent manner, with IC 50 The compound not only strongly inhibited the proliferation of mouse spleen T lymphocytes induced by concanavalin A (IC 50 15nM), and also strongly inhibited the proliferation of normal mouse spleen B lymphocytes induced by bacterial lipopolysaccharide LPS (IC 50 The compound was tested for inhibiting the proliferation of human PBMC stimulated by PHA in vitro, and the results showed that the compound could inhibit the proliferation of PBMC in a dose-dependent manner. 50 It is 141nM.

[0023] The compound can significantly inhibit the ear swelling induced by DNFB (2,4-dinitrofluorobenzene) in a mouse delayed hypersensitivity model, and the effect is dose-dependent. The compound can significantly inhibit the plantar swelling induced by sheep red blood cells in a delayed hypersensitivity model, and the effect is dose-dependent.

[0024] In conclusion, the dihydroartemisinin-ursodeoxycholic acid conjugate (I) showed good immunosuppressive activity in both in vitro and in vivo experiments. Summary of the invention

[0025] The object of the present invention is to provide a crystal form A of a dihydroartemisinin-ursodeoxycholic acid conjugate (I), a preparation method thereof and an application thereof. Compared with other crystal forms, the preparation method of the crystal form A is simple, has good reproducibility, has good thermodynamic stability, is conducive to the process treatment of the drug and the improvement of the physicochemical properties, improves the drug properties, and the preparation of the crystal form A has high bioavailability and has extremely high practical value in the application of preparation prescriptions.

[0026] The polymorphic study of the compound shows that the compound not only has multiple amorphous forms, but also has multiple different types of polymorphic forms, which are of great significance for the study of the compound for use in pharmaceutical dosage forms.

[0027] The present invention provides a crystalline form A of a dihydroartemisinin-ursodeoxycholic acid conjugate (I), which comprises at least the following 11 diffraction angles based on X-ray powder diffraction using copper Kα1 radiation and measured at 2θ±0.2°:

[0028] 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°.

[0029] Differential scanning calorimetry analysis of Form A showed a characteristic exothermic peak at 166-174°C, and thermogravimetric analysis showed no obvious weight loss and no obvious solvent residue when heated to 145-165°C, proving that it is an anhydrous crystalline form.

[0030] The one-week stability study of Form A showed that after being placed under 25°C / 60% RH and 40°C / 75% RH conditions for one week, no change in the solid morphology of Form A, no change in the characteristic peaks of the XRPD pattern, or a significant decrease in HPLC purity was observed.

[0031] Furthermore, no changes in the solid morphology of Form A, changes in the characteristic peaks of the XRPD pattern, or significant decrease in HPLC purity were observed in the long-term accelerated 6-month experiment (40°C / 75% RH) and the long-term 12-month stability experiment (25°C / 60% RH) of Form A.

[0032] These results all indicate that Form A has good physical and chemical stability.

[0033] Form A can be prepared by one of the following two methods:

[0034] 1. Method 1: Recrystallize or slurry the conjugate (I) using an ester solvent;

[0035] 2. Method 2: Recrystallizing or slurrying the conjugate (I) with an alcohol solvent containing or not containing water;

[0036] 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.

[0037] In the above operation, the recrystallization temperature is in the range of 0 to 120°C, and the beating temperature is in the range of -20 to 80°C.

[0038] The present invention also provides other polymorphs of the crystal form, and provides their X-ray powder diffraction characteristic diffraction angle (2θ). Specifically, including crystal form B, crystal form C, crystal form D, crystal form E, crystal form F, crystal form G, crystal form H, crystal form I, crystal form J and crystal form K. Studies have found that crystal form C, crystal form E and crystal form H are hydrates, crystal form B and crystal form I are solvates, crystal form F, crystal form J and crystal form K are metastable crystal forms, and only crystal form A, crystal form D and crystal form G are anhydrous crystal forms. Metastable crystal forms are unstable at room temperature, and solvates and hydrates are prone to lose solvents and water when heated and undergo crystal transformation. Since crystal form A has good thermodynamic stability, it has better practical value in the development of raw material drug technology and pharmaceutical preparations. In addition to metastable crystal forms F, crystal form J and crystal form K, hard capsules prepared with other crystal forms were tested for pharmacokinetic properties on eluted dogs, and the results showed that capsules prepared with crystal form A have higher bioavailability than other crystal forms.

[0039] The present invention provides the use of a polymorph of a dihydroartemisinin-ursodeoxycholic acid conjugate (I) in preparing a drug for treating cancer or autoimmune diseases.

[0040] The present invention provides a pharmaceutical composition comprising a therapeutically effective amount of any crystalline form of any one of the above-mentioned conjugates (I) and a pharmaceutically acceptable carrier or excipient.

[0041] The above-mentioned acceptable carrier is nontoxic, can assist in administration and has no adverse effect on the therapeutic effect of the conjugate. Such carriers can be any solid excipient, liquid excipient, semisolid excipient or gas excipient in aerosol composition that can be obtained by those skilled in the art usually. Solid pharmaceutical excipients include starch, cellulose, talcum, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, magnesium stearate, sodium stearate, glyceryl stearate, sodium chloride, anhydrous skim milk, etc. Liquid and semisolid excipients can be selected from glycerol, propylene glycol, water, ethanol and various oils, including those derived from petroleum, animals, plants or synthetic oils, for example, peanut oil, soybean oil, mineral oil, sesame oil, etc., preferred liquid carriers, particularly for injectable solutions, including water, saline, glucose aqueous solution and glycol. Other adjuvants such as flavoring agents, sweeteners, etc. can also be added to the composition in addition.

[0042] The pharmaceutical composition of the present invention can be administered orally or by other administration methods, such as injection, transdermal administration, spray administration, rectal administration, vaginal administration, etc. The preferred administration method is oral administration, which can be adjusted according to the severity of the disease.

[0043] The 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 compound is mixed with one or more carriers and then made into the desired dosage form, such as tablets, pills, capsules, semisolids, powders, sustained-release dosage forms, solutions, suspensions, formulations, aerosols, etc.

[0044] The pharmaceutical composition of the present invention can be used in combination with other anticancer drugs including traditional Chinese medicine anticancer drugs, such as doxorubicin, bleomycin, vinca alkaloids, taxanes, etoposide, 5-fluorouracil, cyclophosphamide, methotrexate, cisplatin, retinoic acid, temozolomide, actinomycin and targeted drugs such as imatinib, gefitinib, sorafenib, erlotinib, sunitinib, rituximab, cetuximab, trastuzumab, etc.

[0045] The pharmaceutical composition of the present invention can also be used in combination with other cancer treatment methods, such as surgical treatment, radiotherapy, bone marrow transplantation, etc.

[0046] The pharmaceutical composition of the present invention can be used in combination with other immunosuppressants, including antimetabolites: azathioprine (Aza), methotrexate, mycophenolate mofetil (MMF), etc.; alkylating agents: cyclophosphamide, etc.; corticosteroids: prednisone, dexamethasone, etc.; antibiotics: CsA, FK506, rapamycin, etc.; antibodies: anti-lymphocyte globulin (ALG), monoclonal T lymphocyte antibody (OKT3), etc.; Chinese herbal medicines: tripterygium wilfordii polyglycosides, cordyceps sinensis preparations, etc.

[0047] The cancers include brain cancer, 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.

[0048] The autoimmune diseases include lupus erythematosus, rheumatoid arthritis, ankylosing spondylitis, type I diabetes, nephrotic syndrome, Sjögren's syndrome, multiple sclerosis, psoriasis, dermatomyositis, allergic rhinitis, intractable urticaria, autoimmune hemolytic anemia, idiopathic thrombocytopenic purpura, nonspecific enteritis, scleroderma, rejection reaction after human organ transplantation, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 XRPD pattern of Form A.

[0050] Figure 2 DSC graph of Form A.

[0051] Figure 3 TGA chart of Form A.

[0052] Figure 4 1HNMR spectrum of Form A.

[0053] Figure 5 PLM image of Form A.

[0054] Figure 6 XRPD pattern of Form D.

[0055] Figure 7 DSC graph of Form D.

[0056] Figure 8 TGA graph of Form D.

[0057] Fig. 9 1HNMR spectrum of Form D.

[0058] Fig.10 PLM image of Form D.

[0059] Fig.11 XRPD pattern of Form G.

[0060] Fig.12 DSC graph of Form G.

[0061] Fig.13 TGA graph of Form G.

[0062] Fig.14 1HNMR spectrum of Form G.

[0063] Fig.15 PLM image of Form G.

[0064] Fig.16 XRPD pattern of Form C.

[0065] Fig.17 XRPD pattern of Form E.

[0066] Fig.18 XRPD pattern of Form H.

[0067] Fig.19 XRPD pattern of Form B.

[0068] Fig. 20 XRPD pattern of Form I.

[0069] Fig.21 XRPD pattern of Form F.

[0070] Fig. 22 XRPD pattern of Form J.

[0071] Fig.23 XRPD pattern of Form K.

[0072] Fig.24 Dynamic solubility study XRPD pattern of Form A.

[0073] Fig.25 One-week stability XRPD pattern of Form A.

[0074] Fig.26 HPLC chart of the stability of Form A for one week.

[0075] Fig. 27 Long-term accelerated 6-month (40°C / 75% RH) and long-term 12-month (25°C / 60% RH) stability XRPD patterns of Form A.

[0076] Fig.28 HPLC charts of the long-term accelerated stability of Form A for 6 months (40°C / 75% RH) and long-term stability for 12 months (25°C / 60% RH). DETAILED DESCRIPTION

[0077] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0078] 1. Main instruments:

[0079] 1.1 X-ray powder diffractometer (XRPD)

[0080]

[0081] 1.2 Thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC)

[0082]

[0083]

[0084] 2. Preparation of dihydroartemisinin-ursodeoxycholic acid conjugate (I):

[0085] At -78°C, boron trifluoride etherate (1 ml) was slowly added dropwise to an ether solution (50 ml) of dihydroartemisinin (569 mg, 2.0 mmol) and ursodeoxycholic acid (785 mg, 2.0 mmol), and the reaction system was naturally warmed to room temperature and stirred overnight. After the reaction was completed by thin layer silica gel chromatography, the reaction was slowly quenched with saturated sodium bicarbonate aqueous solution (30 ml), extracted with ethyl acetate (30 ml × 3), the organic phases were combined and washed once with water (50 ml), washed once with saturated brine (50 ml), dried over anhydrous magnesium sulfate, filtered, and the filtrate was decompressed and dried to obtain a crude product. The crude product was purified by silica gel column (eluent: petroleum ether / ethyl acetate = 1 / 1) to obtain 749 mg of white solid dihydroartemisinin-ursodeoxycholic acid conjugate (I), with a yield of 56.8%.

[0086] 1 H NMR (400 MHz, CDCl 3 ): 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).

[0087] 13 C NMR (100 MHz, CDCl 3 ): 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.

[0088] MS(+ESI):(m / z)[M+H] + =659.9

[0089] In addition, according to the similar synthesis method of dihydroartemisinin-ursodeoxycholic acid conjugate (I), other derivatives with similar structures as shown in the following table can be obtained by referring to patents CN201710294320.6 (US20200046679, EP18790816.5) and CN201910705451.8:

[0090]

[0091]

[0092]

[0093]

[0094]

[0095] 3. Preparation of dihydroartemisinin-ursodeoxycholic acid conjugate (I) crystalline form:

[0096] 3.1 Crystalline Form A

[0097] Form A can be prepared by one of the following two methods.

[0098] Method 1: Add 1 g of dihydroartemisinin-ursodeoxycholic acid conjugate (I) and 30 ml of ethyl acetate into a round-bottom flask and heat to reflux. When the solid is basically dissolved, filter it while hot, and place the clarified filtrate at room temperature overnight. Filter and collect the precipitated white solid product and dry it in vacuo to obtain 750 mg of the target product.

[0099] Method 2: Add 1 g of dihydroartemisinin-ursodeoxycholic acid conjugate (I) and 10 ml of methanol into a 50 ml round-bottom flask, heat until the sample is completely dissolved, and then drop 2-8 ml of pure water. Filter while hot, place the clarified filtrate at room temperature overnight, filter and collect the precipitated white solid product and vacuum dry to obtain 898 mg of the target product.

[0100] The XRPD image of the product is as follows Figure 1 Its main peak data are shown in the following table:

[0101] 2-Theta Height Height % 3.72 2314 20.3 6.28 4002 35.1 7.71 11413 100.0 8.45 2409 21.1 10.60 2525 22.1 11.24 5523 48.4 12.64 4029 35.3 14.98 6771 59.3 15.57 5795 50.8 17.00 2566 22.5 18.35 2153 18.9

[0102] The DSC diagram of the product is as follows: Figure 2 As shown in Figure 1, it has a characteristic exothermic peak at 166-174°C (single decomposition exothermic peak, peak temperature at 172.37°C), and the thermogravimetric analysis TGA graph is as follows Figure 3 It is shown that there is no obvious weight loss when heated to 145-165°C. 1 HNMR Figure 4There is no obvious solvent residue, and it is an anhydrous crystal type.

[0103] Product polarization microscope PLM picture Figure 5 As shown, it is a thin rod-shaped crystal, and birefringence is observed.

[0104] 3.2 Crystal Form D

[0105] At room temperature, 20 mg of dihydroartemisinin-ursodeoxycholic acid conjugate (I) was dissolved in 0.2 ml of 1,4-dioxane, filtered, and the clear solution was placed in a 1 ml vial. The vial was then placed in a 20 ml vial containing 3 ml of n-hexane. The 20 ml vial was sealed with a 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 precipitated in the 1 ml vial, and the solid product was collected by filtration.

[0106] The XRPD image of the product is as follows Figure 6 Its main peak data are shown in the following table:

[0107]

[0108]

[0109] The DSC diagram of the product is as follows: Figure 7 As shown in Figure 1, it has a characteristic exothermic peak at 150-171°C (single decomposition exothermic peak, peak temperature at 164.86°C), and the thermogravimetric analysis TGA graph is as follows: Figure 8 As shown, when the temperature rises to 135-145°C, the sample loses 3.3% of its weight. 1 HNMR images Fig. 9 As shown, a small amount of solvent residue was observed. In HNMR, the peak area of ​​dihydroartemisinin-ursodeoxycholic acid conjugate (I) was used as the standard, and the content was calculated by quantitative integration. The results showed that the dioxane content was 2.48% (wt); if a solvate is formed, the dioxane content needs to reach 12%. Since the sample lost 3.3% of its weight, what was lost was water and dioxane, and the water content was only 0.82%, which did not reach the water content of more than 2.7% required to form a hydrate. Therefore, Form D is an anhydrous crystalline form.

[0110] Product polarization microscope PLM picture Fig.10 As shown, it is a fine needle-shaped crystal, and birefringence is observed.

[0111] 3.3 Form G

[0112] 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.

[0113] The XRPD image of the product is as follows Fig.11 Its main peak data are shown in the following table:

[0114]

[0115]

[0116] The DSC diagram of the product is as follows: Fig.12 As shown, it has a characteristic exothermic peak at 167-173°C (there is an exothermic decomposition peak with a peak temperature at 171.11°C), and the thermogravimetric analysis TGA graph is as follows Fig.13 As shown in the figure, when the temperature rises to 130-160℃, the sample has no obvious weight loss and 1 HNMR images Fig.14 A small amount of solvent residue was observed, and the product was in the form of anhydrous crystals.

[0117] Product polarization microscope PLM picture Fig.15 As shown, it is an irregular crystal and birefringence is observed.

[0118] 3.4 Form C

[0119] About 20 mg of dihydroartemisinin-ursodeoxycholic acid conjugate (I) was weighed and dissolved in 0.3 ml of a mixed solvent of ethanol / methyl ethyl ketone (v / v=1 / 1), and the clear solution obtained was filtered and placed in a 1 ml vial. The vial was then placed in a 20 ml vial containing 3 ml of n-hexane. The 20 ml vial was sealed with a bottle cap, and the organic vapor was 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.

[0120] The XRPD image of the product is as follows Fig.16 Its main peak data are shown in the following table:

[0121]

[0122]

[0123] The study found that the product had a weight loss of 3.08% before 150-160°C by TGA analysis. 1 No obvious residual solvent was observed by HNMR. After heating to 150°C for 3 minutes, Form C transformed into Form A. Form C was a hydrate.

[0124] 3.5 Form E

[0125] About 20 mg of dihydroartemisinin-ursodeoxycholic acid conjugate (I) was weighed 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 under stirring until the total amount of solvent reached 5.0 ml. After stirring at room temperature for 1 day, the system remained in a clear solution state, the lid was opened for evaporation, and thin rod-shaped crystals were precipitated after 3 days, and the solid product was collected by filtration.

[0126] The XRPD image of the product is as follows Fig.17 Its main peak data are shown in the following table:

[0127] 2-Theta Height Height % 5.30 2939 6.9 10.66 42558 100.0 13.85 11092 26.1 20.11 5243 12.3

[0128] The study found that there is an endothermic peak in the DSC graph, located at 47.48°C (peak temperature); an exothermic decomposition peak, located at 164.87°C (peak temperature). In the TGA graph, when the temperature rises to 140-150°C, the sample loses 2.73% of its weight, and 1 No obvious residual solvent was observed by HNMR. After heating to 120°C for 3 minutes, Form E transformed into Form F. Form E was a hydrate.

[0129] 3.6 Crystal form H

[0130] At room temperature, an excess of dihydroartemisinin-ursodeoxycholic acid conjugate (I) is suspended in methyl tert-butyl ether and stirred for 2 hours. Filter to obtain a saturated solution of dihydroartemisinin-ursodeoxycholic acid conjugate (I) at room temperature, and then add a solid mixture of crystal form A and crystal form D to the saturated solution to obtain a suspension. After the suspension is magnetically stirred at room temperature for about 2 days, the solid product is collected by filtration.

[0131] The XRPD image of the product is as follows Fig.18 Its main peak data are shown in the following table:

[0132] 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

[0133] The study found that there was an exothermic decomposition peak in the DSC graph, with a peak temperature of 166.52°C. When the temperature was raised to 150-160°C in the TGA graph, the sample lost 6.3% of its weight, and 1 HNMR observed a small amount of residual solvent. After heating to 100°C for 10 minutes, Form H transformed into Form J. Form H was a hydrate.

[0134] 3.7 Form B

[0135] About 20 mg of dihydroartemisinin-ursodeoxycholic acid conjugate (I) was weighed and dissolved in 1.5 ml of acetone. After filtering, the filtrate was placed in a 5 ml vial, sealed with a sealing film, and left at room temperature to evaporate slowly through the pinhole on the sealing film. After 11 days, a large amount of solid precipitated from the system, and the solid product was collected by filtration.

[0136] The XRPD image of the product is as follows Fig.19 Its main peak data are shown in the following table:

[0137] 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

[0138] The study found that there is a broad endothermic peak in the DSC graph, located at 143.20°C (peak temperature); an exothermic decomposition peak, located at 162.41°C (peak temperature). In the TGA graph, when the temperature rises from 130°C to 160°C, the sample loses 5.5% of its weight, and 1 HNMR detected that the sample contained 5.58% acetone. After heating to 140°C for 60 minutes, Form B transformed into an amorphous form, which was a solvate of acetone.

[0139] 3.8 Form I

[0140] 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 large bottle containing 15 ml of n-hexane. The large bottle was sealed with a lid and placed 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.

[0141] The XRPD image of the product is as follows Fig. 20 Its main peak data are shown in the following table:

[0142] 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

[0143] The study found that there is an endothermic peak in the DSC graph, located at 136.43°C (peak temperature); an exothermic peak is located at 161.85°C (peak temperature). The TGA graph shows that when the temperature is raised to 120-130°C, the sample has no obvious weight loss; when the temperature is raised from 130°C to 160°C, the sample loses 9.7% of its weight, and 1 HNMR found that the residual 1,4-dioxane content was 11.69%. When heated to 135-145° C. for 20 minutes, Form I transformed into an amorphous form. Form I was a solvate of 1,4-dioxane.

[0144] 3.9 Form F

[0145] Form F can be obtained by heating Form E to 110-120°C for 3 minutes.

[0146] The XRPD image of the product is as follows Fig.21 Its main peak data are shown in the following table:

[0147]

[0148]

[0149] The study found that when the sample was placed at room temperature for 1 day, Form F transformed into Form E. Form F is a metastable form.

[0150] 3.10 Crystal Form J

[0151] Form J can be obtained by heating Form H to 80-110°C for 10 minutes.

[0152] The XRPD image of the product is as follows Fig. 22 Its main peak data are shown in the following table:

[0153] 2-Theta Height Height % 3.06 910 100.0 5.90 593 65.2 6.18 326 35.8 7.36 591 64.9 8.77 200 22.0 10.79 349 38.4 11.49 275 30.2 13.24 620 68.1

[0154] The study found that when the sample was left open at room temperature for 1 day, the crystal form did not change but the crystallinity was significantly weakened; when the sample was left open at room temperature for 3 days, the crystal form did not change but the crystallinity was weakened again. After a longer period of time, crystal form J gradually turned into amorphous. Polymorph J is a metastable crystal form.

[0155] 3.11 Crystal Form K

[0156] 50 mg of dihydroartemisinin-ursodeoxycholic acid conjugate (I) was weighed and suspended in 2.0 ml of methyl tert-butyl ether. The suspension was magnetically stirred at room temperature for about 3 days, and then the solid product was collected by filtration to obtain Form K.

[0157] The XRPD image of the product is as follows Fig.23 Its main peak data are shown in the following table:

[0158]

[0159]

[0160] The study found that when the sample was left open at room temperature for one day, Form K transformed into Form H. Form K is a metastable form.

[0161] 4. Dynamic solubility study of Form A:

[0162] At 37°C, the crystal form A was prepared into a supersaturated suspension (about 10 mg / mL) in water. The suspension was sampled at 1 hour, 2 hours, 4 hours and 24 hours, the supernatant was filtered, the filtrate was tested for concentration, and the solid was tested for crystal form (see Fig.24XRPD diagram). The results showed that within 24 hours, the dynamic solubility of Form A in water was less than 0.01 mg / mL, and the X-ray powder diffraction test showed that the crystal form did not change.

[0163] 5. One-week stability study of Form A:

[0164] The crystal form A with an initial HPLC purity of 99.28% was selected and placed under 25° C. / 60% RH and 40° C. / 75% RH conditions for one week, and then the HPLC purity and crystal form changes were tested.

[0165] The stability evaluation results of Form A are shown in the following table:

[0166]

[0167] Fig.25 This is the XRPD diagram of the stability of Form A for one week. Fig.26 This is a one-week stability HPLC test chart of Form A. The results showed that no change in the solid morphology of Form A was observed, and no change in the characteristic peaks of the spectrum was observed in the X-ray powder diffraction test. The initial HPLC purity of Form A did not decrease significantly after one week. The test results show that Form A has good physical and chemical stability.

[0168] 6. Long-term accelerated 6-month (40°C / 75%RH) and long-term 12-month stability experiments (25°C / 60%RH) of Form A:

[0169] Form A with an initial HPLC purity of 98.31% was selected for a long-term accelerated 6-month stability experiment at 40°C / 75%RH, and a long-term 12-month stability experiment at 25°C / 60%RH.

[0170] The stability evaluation results of Form A are shown in the following table:

[0171]

[0172] Fig. 27 The XRPD diagram of long-term accelerated 6-month (40°C / 75%RH) and long-term 12-month (25°C / 60%RH) stability test of Form A is shown in Figure 1. Fig.28 The HPLC test chart of the stability of Form A for long-term accelerated 6 months (40°C / 75%) and long-term 12 months (25°C / 60%RH). The results show that no change in the solid form of Form A was observed in the experiments under the two conditions, no change in the characteristic peaks of the spectrum was observed in the X-ray powder diffraction test, and no significant decrease in the HPLC purity of Form A. The test results show that Form A has good physical and chemical stability.

[0173] 7. Melting point test of crystal forms A to K:

[0174] The melting point test was performed using the Chinese Pharmacopoeia method (Ch.P 2015Appendix 0612the first method B) with a melting point apparatus (manufacturer: METTLER TOLEDO, model MP50), with the starting temperature set to 130°C, the ending temperature set to 170°C, and the heating rate set to 1.0°C / min. The melting points of crystal forms A to K were tested (each crystal form was tested three times and the average value was taken):

[0175]

[0176]

[0177] The results show that the melting points of Form A, Form D and Form G are higher than those of other forms. Generally speaking, due to the difference in lattice energy of polymorphs, there will be differences in melting points between isomorphs, and generally the form with a higher melting point is more thermodynamically stable. Therefore, from the melting point data, Form A is more stable than other forms.

[0178] 8. Dissolution and bioavailability tests of hard capsule preparations prepared from each crystal form:

[0179] Since the crystal forms F, J and K are metastable crystal forms and are not stable enough at room temperature, they are not used in this experiment. 10 mg of one polymorph of dihydroartemisinin-ursodeoxycholic acid conjugate (I) was taken and mixed with other excipients to prepare a hard capsule preparation according to the following formula:

[0180]

[0181]

[0182] The capsule preparation process is as follows:

[0183] (1) Raw material pretreatment: Take an appropriate amount of dihydroartemisinin-ursodeoxycholic acid conjugate (I), pass it through a 30-mesh manual sieve, and set aside;

[0184] (2) Material distribution: Weigh the excipients according to the prescription ratio and place them on weighing paper for later use;

[0185] (3) Premixing: Take about half of the microcrystalline cellulose colloidal silicon dioxide co-processed product (SMCC) in the above excipients and pass it through a 30-mesh manual sieve to pre-saturate the sieve, and then transfer it to a ziplock bag to rinse the entire ziplock bag. Add cross-linked polyvinylpyrrolidone to the dihydroartemisinin-ursodeoxycholic acid conjugate (I), mix it briefly with a spoon, add mannitol and mix it, pass the mixture through a 30-mesh manual sieve, and transfer it to a ziplock bag. Finally, sieve the remaining SMCC and transfer it to a ziplock bag, and mix it manually for 5 minutes;

[0186] (4) Total mixing: Finally, magnesium stearate is added to the ziplock bag in step (3) and mixed for 3 minutes to obtain a total mixed material;

[0187] (5) Filling: Manually fill 0# gelatin hollow capsules with a filling amount of 190-210 mg (200 mg ± 5%).

[0188] The dissolution test of the capsules prepared from each crystal form in artificial gastric fluid was carried out for 60 minutes:

[0189]

[0190] The results showed that the cumulative average dissolution of the capsules prepared from each crystal form in artificial gastric juice was between 35% and 66%.

[0191] Bioavailability Testing:

[0192] The capsules prepared from each crystal form were treated with male Non- Bioavailability test was conducted on beagle dogs (weight between 10 and 11 kg). Five male eluted dogs were used for injection administration. The dihydroartemisinin-ursodeoxycholic acid conjugate (I) was dissolved in a solvent and then injected (solvent: 5% volume DMSO + 15% volume Solutol HS15 + 80% volume HP-β-CD aqueous solution (20%, w / v)); the injection dose for each dog was 1 mg / kg; capsules prepared from each crystal form were orally administered to five male eluted dogs (dose: 10 mg dihydroartemisinin-ursodeoxycholic acid conjugate (I) / dog, i.e., one capsule for each dog). The animals were fasted overnight before administration, and were given feed 4 hours after the end of administration. The animals were free to eat during the rest of the test period.

[0193] Blood collection time:

[0194] Oral gavage (po) group: 0.25h, 0.5h, 1h, 1.5h, 2h, 2.5h, 3h, 3.5h, 4h, 6h, and 8h after administration.

[0195] Intravenous (iv) group: 0.083h, 0.25h, 0.5h, 1h, 1.5h, 2h, 2.5h, 3h, 3.5h, 4h, and 8h after administration.

[0196] Sample collection and handling:

[0197] Blood was collected through the jugular vein, and about 1 mL of blood was collected for each sample. The blood was anticoagulated with sodium heparin and placed on ice after collection. The plasma was separated by centrifugation within 1 hour (centrifugation conditions: centrifugal force 2200g, 10 minutes, 2-8°C). The collected plasma samples were stored in a -70°C refrigerator before analysis. After analysis, the remaining plasma samples continued to be stored in a -70°C refrigerator with a shelf life of one month.

[0198] Bioanalysis and data processing:

[0199] The concentration of dihydroartemisinin-ursodeoxycholic acid conjugate (I) in plasma samples was detected by LC-MS / MS. The accuracy of quality control samples was evaluated while analyzing the samples, and it was required that the accuracy of more than 66% of the quality control samples was between 80-120%.

[0200] WinNonlin was used to calculate pharmacokinetic parameters using blood drug concentration data at different time points. When plotting the plasma drug concentration-time curve, BLQ was recorded as 0. When calculating pharmacokinetic parameters, the concentration before administration was calculated as 0; the BLQ before Cmax (including "No peak") was calculated as 0; and the BLQ after Cmax (including "No peak") was not included in the calculation.

[0201] The bioavailability F is calculated according to the following formula:

[0202] F = [AUC (0-t,po) ×Dose (iv) ] / [AUC (0-t,iv) ×Dose (po) ]×100%

[0203] Wherein, AUC is the area under the drug-time curve, po means oral administration, iv means intravenous injection, and Dose means the dosage.

[0204] The key test data of dihydroartemisinin-ursodeoxycholic acid conjugate (I) dissolved in a solvent and injected are as follows (dose: 1 mg / kg; data are average data):

[0205]

[0206] The key data obtained from the intragastric administration test of capsules prepared from each crystal form are as follows (the data are average data):

[0207]

[0208]

[0209] The test results show that the bioavailability F (%) of the capsules prepared from Form A is higher than that of the capsules prepared from other forms. Combined with the stability data of Form A, Form A is an advantageous form suitable for use in pharmaceutical preparations.

[0210] The above-mentioned embodiments only express the implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that for ordinary technicians in this field, several modifications and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be based on the attached claims.

Claims

1. Crystalline Form A of Dihydroartemisinin-Ursodeoxycholic Acid Conjugate (I), ； X-ray powder diffraction based on copper Kα1 radiation at 2θ ± 0.2 o The design includes at least the following 11 diffraction angles: 3.72±0.2 o , 6.28±0.2 o ,7.71±0.2 o , 8.45±0.2 o , 10.60±0.2 o , 11.24±0.2 o ,12.64±0.2 o , 14.98±0.2 o , 15.57±0.2 o , 17.00±0.2 o , 18.35±0.2 o 。 2. The crystalline form A of the dihydroartemisinin-ursodeoxycholic acid conjugate (I) according to claim 1, characterized in that: It has an X-ray powder diffraction pattern as shown in FIG1 .

3. The crystalline form A of the dihydroartemisinin-ursodeoxycholic acid conjugate (I) according to claim 1, characterized in that: It has a differential scanning calorimetry analysis diagram as shown in FIG2, with a range of 166 to 174 o C has a characteristic exothermic peak.

4. The crystalline form A of the dihydroartemisinin-ursodeoxycholic acid conjugate (I) according to claim 1, characterized in that: It has a thermogravimetric analysis diagram as shown in FIG3 , and has no obvious weight loss when heated to 145-165°C.

5. The method for preparing the dihydroartemisinin-ursodeoxycholic acid conjugate (I) crystal form A according to claim 1, characterized in that It can be prepared by one of the following two methods: Recrystallizing or slurrying the conjugate (I) with an ester solvent or an aqueous alcohol solvent; 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, and the alcohol solvent is any one of methanol, ethanol, n-propanol, isopropanol, n-butanol, isobutanol, sec-butanol or tert-butanol.

6. Use of the crystalline form A of the dihydroartemisinin-ursodeoxycholic acid conjugate (I) according to claims 1 to 5 in the preparation of a drug for treating cancer or autoimmune diseases.

7. The use according to claim 6, characterized in that: The cancer is any one of brain cancer, 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, thymic cancer or bone cancer.

8. The use according to claim 6, characterized in that: The autoimmune disease is any one of lupus erythematosus, rheumatoid arthritis, ankylosing spondylitis, type I diabetes, nephrotic syndrome, Sjögren's syndrome, multiple sclerosis, psoriasis, dermatomyositis, allergic rhinitis, intractable urticaria, autoimmune hemolytic anemia, idiopathic thrombocytopenic purpura, nonspecific enteritis, scleroderma, and rejection reaction after human organ transplantation.

9. A pharmaceutical composition, characterized in that Comprising a therapeutically effective amount of the crystalline form A of the conjugate (I) described in claims 1 to 5 and a pharmaceutically acceptable carrier, diluent or excipient.

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