CN111825689A

CN111825689A - Novel crystal form of dihydropyrazolone compound and preparation method thereof

Info

Publication number: CN111825689A
Application number: CN201910308629.5A
Authority: CN
Inventors: 殷惠军; 褚国彪; 王斌; 屈磊磊; 王秀娟; 赵新; 闫旭; 王卓
Original assignee: National Institutes of Pharmaceutical R&D Co Ltd
Current assignee: National Institutes of Pharmaceutical R&D Co Ltd
Priority date: 2019-04-17
Filing date: 2019-04-17
Publication date: 2020-10-27
Anticipated expiration: 2039-04-17
Also published as: CN111825689B

Abstract

The invention relates to a novel crystal form of a dihydropyrazolone compound and a preparation method thereof. Specifically, the invention relates to crystal forms A and B of 1- (6- (2-oxa-8-azaspiro [4.5] decane-8-yl) pyrimidine-4-yl) -4- (1H-1,2, 3-triazole-1-yl) -1, 2-dihydro-1H-pyrazole-3-sodium phenolate. The compound is obtained by preparing 2- (6- (2-oxa-8-azaspiro [4.5] decane-8-yl) pyrimidine-4-yl) -4- (1H-1,2, 3-triazole-1-yl) -1, 2-dihydro-3H-pyrazol-3-ketone into a sodium salt in an organic solvent and crystallizing. It can be used for preventing and/or treating diseases related to PHD activity.

Description

Novel crystal form of dihydropyrazolone compound and preparation method thereof

Technical Field

The invention relates to crystal forms A and B of 1- (6- (2-oxa-8-azaspiro [4.5] decane-8-yl) pyrimidine-4-yl) -4- (1H-1,2, 3-triazole-1-yl) -1, 2-dihydro-1H-pyrazole-3-sodium phenolate, a preparation method thereof and application thereof in preparing a medicament for preventing and/or treating diseases related to PHD activity.

Background

In hypoxic environments, the body is able to spontaneously develop hypoxic reactions to maintain the body's oxygen-acquisition capacity. In 1992, Semenza et al discovered that a protein, termed Hypoxia Inducible Factor (HIF) (Semenza GL et al, mol. cell biol.,1992,12, 5447-. HIF has a wide range of target genes that can affect the body's hematopoietic function, angiogenesis, iron ion transport, glucose utilization, resistance to oxidative stress, cell differentiation, cell survival and apoptosis, extracellular matrix homeostasis, and tumorigenesis. HIF is a heterodimer composed of alpha and beta subunits, the alpha subunit belongs to a functional subunit, is very sensitive to the change of oxygen concentration in cells, is highly regulated and controlled, and has the function of regulating HIF activity; the beta subunit is a structural subunit, also known as an aryl hydrocarbon receptor nuclear transport protein (ARNT), which is stably expressed in cells, and whose mRNA transcription and protein expression levels are not affected by changes in oxygen concentration. The alpha and beta subunits of HIF belong to members of the basic helix-loop-helix transcription factor superfamily. Human HIF α has three subtypes HIF-1 α, HIF-2 α, and HIF-3 α. HIF-1 alpha is generally distributed in vivo and plays an important role in the angiogenesis process induced by ischemia or hypoxia of local tissues, but has little influence on the iron metabolic process; HIF-2 alpha presents localized distribution, have important effects in EPO (erythropoietin) gene expression of renal tissue and synthetic process, in addition, also through regulating the cytochrome of duodenum and expression of divalent metal transporter-1 and improving iron in the absorption of the enteric canal, and have effects of reducing the expression of the liver bactericidal peptide, play a leading role in the metabolic process of iron; HIF-3. alpha. has a structure different from other subtypes, and does not affect gene expression without a DNA binding domain. Studies have shown that HIF-3 α may have a negative regulatory role in HIF-mediated gene expression. Thus, HIF-1 α, HIF-2 α play a role in the hypoxia response. In a mouse experiment in which HIF-1 alpha and HIF-2 alpha genes are deleted, the necessity of HIF-1 alpha and HIF-2 alpha in the hypoxia response process is confirmed. In the development of compounds for treating chronic renal anemia, HIF-2 α changes are more important than HIF-1 α.

2001 research shows that HIF-PHD can convert O₂And 2-OG as a substrate, specifically hydroxylates HIF alpha proline residues, thereby regulating HIF bioactivity. Catalytic cycling of HIF-PHD in Fe²⁺And 2-OG to the PHD active site. PHD then binds to HIF alpha proline residues using O₂The hydroxylation is completed by replacing the water molecule. The ascorbic acid in the whole process isEssential catalyst, Fe²⁺、O₂2-OG are indispensable factors.

HIF-PHD possesses three distinct subtypes, PHD1, PHD2, and PHD3, exhibits distinct tissue distribution, and selectively hydroxylates HIF α subtypes. PHD1 has stable expression, is not induced by hypoxia, and has certain effect in maintaining oxygen balance in vivo; PHD2 plays an important role in the response to oxygen-dependent regulation of HIF α activity; under the normoxic environment, PHD2 and PHD3 respectively selectively act on HIF-1 alpha and HIF-2 alpha, respectively hydroxylate proline residues at 402 th and 564 th positions of the HIF-1 alpha and 405 th and 531 th positions of the HIF-2 alpha, and the hydroxylated HIF-1 alpha and HIF-2 alpha can be combined with a VHL E3 ubiquitin ligase complex to be ubiquitinated and then enter a protease body to be degraded. When the cell is in a hypoxia environment, the activity factors of PHD2 and PHD3 are O₂Scarcely inhibited, undegraded HIF-1 alpha and HIF-2 alpha enter nucleus to combine with HIF-beta, and act on Hypoxia Response Element (HRE) under the cooperation of p300/CBP, promote the expression of related gene (such as EPO), increase protein level, thus promote erythropoiesis and correct anemia symptoms.

In the study of the gene defects related to the human HIF-PHD system, pVHL (Von Hippel-Lindau tumor suppressor gene product, which is involved in mediating the degradation of ubiquitinated HIF) mutation, PHD deletion and body change under the condition of HIF deletion are involved. The pVHL mutation can induce human bodies to generate pVHL related diseases to cause vascular tumors; when the mutation occurs from the C end 598 to the T end of the pVHL, congenital polycythemia can be caused. In a renal cancer patient caused by pVHL deletion, HIF-1 alpha is also mutated, which indicates that the occurrence and development of renal cancer may be related to the mutation of HIF-1 alpha. Secondary erythrocytosis is easy to occur in the population with HIF-2 alpha missense mutation and PHD2 mutation, thereby increasing the incidence of thrombosis.

In view of the starting action of HIF-PHD on the degradation process of HIF, the HIF-PHD inhibitor can increase the HIF level, thereby promoting the expression of target genes such as EPO, VEGF, iNOS, GLUT-1 and the like, and achieving the effect of treating diseases related to the PHD activity. Thus, therapeutic uses of PHD (proline hydroxylase) inhibitors include, but are not limited to: treating and/or preventing cardiovascular diseases, especially cardiac insufficiency, coronary heart disease, angina pectoris, myocardial infarction, stroke, arteriosclerosis, primary, pulmonary and malignant hypertension and peripheral arterial occlusive disease; treatment and/or prevention of hematopoietic disorders, such as primary anemia, renal anemia and anemia associated with neoplastic disease (especially chemotherapy-induced anemia), infection (especially HIV infection), or other inflammatory diseases, such as rheumatoid arthritis; for the supportive treatment of anemia due to blood loss, iron deficiency anemia, vitamin deficiency anemia (e.g. due to vitamin B12 deficiency or due to folate deficiency), aplastic anemia and aplastic anemia or hemolytic anemia, or for the supportive treatment of anemia due to iron utilization disorders (iron-utilizing anemia) or due to other endocrine disorders (e.g. hypothyroidism); treatment and/or prevention of surgically-related ischemic conditions and their continuous symptoms following surgery, in particular cardiac interventions using a heart-lung machine (e.g. bypass surgery, heart valve transplantation), carotid interventions, aortic interventions and interventions using an instrument opening or penetration of the calvaria; general treatment and/or prevention with the aim of accelerating wound healing and shortening recovery time in surgery; treatment and/or prevention of cancer and for the treatment and/or prevention of damage to the health status that occurs during cancer treatment, in particular after treatment with cytostatics, antibiotics and radiation; the treatment and/or prevention of a range of diseases in the rheumatic form and other disease forms which are considered autoimmune diseases, in particular for the treatment and/or prevention of impairment of the health status which occurs during pharmacotherapy of such diseases; treatment and prevention of the continuous symptoms of acute and prolonged cerebral ischemic conditions (e.g. stroke, childbirth asphyxia).

The present inventors have described in patent application No. 201811237690.7 the compound 2- (6- (2-oxa-8-azaspiro [4.5] decan-8-yl) pyrimidin-4-yl) -4- (1H-1,2, 3-triazol-1-yl) -1, 2-dihydro-3H-pyrazol-3-one, which is useful as a PHD inhibitor for the prevention and/or treatment of diseases associated with PHD activity, the structure of which is shown in the following formula (I):

the inventor conducts repeated experiments on the compound of the formula (I) and the hydrochloride thereof, and finds that the compound of the formula (I) and the hydrochloride thereof have strong hygroscopicity, and the moisture absorption and the weight increase are respectively 1.81 percent and 1.86 percent under the relative humidity of 42 percent to 65 percent RH, so that the production and the application of a solid preparation are not facilitated. In addition, the compound of formula (I) and its hydrochloride have poor solubility in water and various lower alcohols, and are not suitable for the preparation of oral solid preparations.

Disclosure of Invention

The present inventors examined various crystallization conditions of the sodium salt of the compound of formula (I) and obtained a series of crystalline products, and carried out X-ray powder diffraction and DSC detection on the obtained crystalline products, and found novel crystals having improved properties such as solubility and hygroscopicity and good stability.

Accordingly, an object of the present invention is to provide a type a crystal of sodium 1- (6- (2-oxa-8-azaspiro [4.5] decan-8-yl) pyrimidin-4-yl) -4- (1H-1,2, 3-triazol-1-yl) -1, 2-dihydro-1H-pyrazole-3-olate characterized by an X-ray powder diffraction pattern comprising characteristic peaks at 2 θ diffraction angles of 4.398 ° ± 0.2 °, 8.740 ° ± 0.2 °, 12.949 ° ± 0.2 °, 13.352 ° ± 0.2 °, 16.353 ° ± 0.2 °, 18.761 ° ± 0.2 °, 22.542 ° ± 0.2 °, 23.564 ° ± 0.2 °, 26.564 ° ± 0.2 °.

In one embodiment, the form a crystal of sodium 1- (6- (2-oxa-8-azaspiro [4.5] decan-8-yl) pyrimidin-4-yl) -4- (1H-1,2, 3-triazol-1-yl) -1, 2-dihydro-1H-pyrazol-3-olate according to the invention is characterized by its X-ray powder diffraction pattern as shown in figure 1.

It is another object of the present invention to provide a type-B crystal of sodium 1- (6- (2-oxa-8-azaspiro [4.5] decan-8-yl) pyrimidin-4-yl) -4- (1H-1,2, 3-triazol-1-yl) -1, 2-dihydro-1H-pyrazole-3-olate characterized by an X-ray powder diffraction pattern comprising characteristic peaks at 2-theta diffraction angles of 7.460 DEG + -0.2 DEG, 16.188 DEG + -0.2 DEG, 16.423 DEG + -0.2 DEG, 17.072 DEG + -0.2 DEG, 21.158 DEG + -0.2 DEG, 22.554 DEG + -0.2 DEG, 23.677 DEG + -0.2 deg.

In one embodiment, 1- (6- (2-oxa-8-azaspiro [4.5] decan-8-yl) pyrimidin-4-yl) -4- (1H-1,2, 3-triazol-1-yl) -1, 2-dihydro-1H-pyrazole-3-phenol sodium crystal form B according to the present invention is characterized by an X-ray powder diffraction pattern comprising characteristic peaks at 2 θ diffraction angles of 7.460 ° ± 0.2 °, 9.368 ° ± 0.2 °, 9.636 ° ± 0.2 °, 16.188 ° ± 0.2 °, 16.423 ° ± 0.2 °, 17.072 ° ± 0.2 °, 17.473 ° ± 0.2 °, 21.158 ° ± 0.2 °, 22.554 ° ± 0.2 °, 23.677 ° ± 0.2 °, 29.368 ° ± 0.2 °.

In another embodiment, the form B crystal of sodium 1- (6- (2-oxa-8-azaspiro [4.5] decan-8-yl) pyrimidin-4-yl) -4- (1H-1,2, 3-triazol-1-yl) -1, 2-dihydro-1H-pyrazol-3-olate according to the invention is characterized by its X-ray powder diffraction pattern as shown in figure 4.

The present invention further provides a process for preparing form a crystals of sodium 1- (6- (2-oxa-8-azaspiro [4.5] decan-8-yl) pyrimidin-4-yl) -4- (1H-1,2, 3-triazol-1-yl) -1, 2-dihydro-1H-pyrazol-3-olate according to the invention, said process comprising the steps of:

1) dispersing 2- (6- (2-oxa-8-azaspiro [4.5] decan-8-yl) pyrimidin-4-yl) -4- (1H-1,2, 3-triazol-1-yl) -1, 2-dihydro-3H-pyrazol-3-one in an organic solvent under basic conditions; adding dropwise sodium salt solution under heating, preferably at 55-60 deg.C to obtain 1- (6- (2-oxa-8-azaspiro [4.5] decan-8-yl) pyrimidin-4-yl) -4- (1H-1,2, 3-triazol-1-yl) -1, 2-dihydro-1H-pyrazol-3-ol sodium solution;

2) stirring the 1- (6- (2-oxa-8-azaspiro [4.5] decan-8-yl) pyrimidin-4-yl) -4- (1H-1,2, 3-triazol-1-yl) -1, 2-dihydro-1H-pyrazol-3-phenol sodium solution obtained in the step 1) at room temperature for crystallization preferably for 1 to 2 hours, then continuing stirring at 5 ℃ to 10 ℃ for crystallization preferably for 2 hours, and filtering the crystals;

3) drying the crystals obtained in the step 2) to obtain the A-type crystals of 1- (6- (2-oxa-8-azaspiro [4.5] decan-8-yl) pyrimidin-4-yl) -4- (1H-1,2, 3-triazol-1-yl) -1, 2-dihydro-1H-pyrazol-3-ol sodium.

In a preferred embodiment, the process for the preparation of form a crystals according to the present invention, wherein in step 1), 2- (6- (2-oxa-8-azaspiro [4.5] decan-8-yl) pyrimidin-4-yl) -4- (1H-1,2, 3-triazol-1-yl) -1, 2-dihydro-3H-pyrazol-3-one is further dissolved in an organic solvent by heating, preferably at 55 ℃ to 60 ℃, hot-filtered, and the filtrate is added dropwise with a sodium salt solution.

In another preferred embodiment, the process for the preparation of form A crystals according to the present invention, wherein in step 1), the solution of sodium 1- (6- (2-oxa-8-azaspiro [4.5] decan-8-yl) pyrimidin-4-yl) -4- (1H-1,2, 3-triazol-1-yl) -1, 2-dihydro-1H-pyrazol-3-olate obtained is further incubated preferably for 15 to 30 minutes.

In another preferred embodiment, the method for preparing form a crystals according to the present invention comprises the steps of:

1) dispersing 2- (6- (2-oxa-8-azaspiro [4.5] decan-8-yl) pyrimidin-4-yl) -4- (1H-1,2, 3-triazol-1-yl) -1, 2-dihydro-3H-pyrazol-3-one in an organic solvent under alkaline conditions, heating preferably to 55-60 ℃ to clarify the solution, and hot filtering; adding the sodium salt solution dropwise to the filtrate under heating, preferably at 55 ℃ to 60 ℃, to obtain a solution of sodium 1- (6- (2-oxa-8-azaspiro [4.5] decan-8-yl) pyrimidin-4-yl) -4- (1H-1,2, 3-triazol-1-yl) -1, 2-dihydro-1H-pyrazol-3-olate, which is kept warm, preferably for 15 to 30 minutes;

In a preferred embodiment, the preparation method of form a crystal according to the present invention, wherein the organic solvent is selected from tetrahydrofuran, 1, 4-dioxane, acetone, acetonitrile or a mixture thereof, or a mixture of the above solvent and water. Mixtures of tetrahydrofuran and water are preferred. Particular preference is given to mixtures of tetrahydrofuran and water in a volume ratio of from 10:1 to 50:1, more particular mixtures of tetrahydrofuran and water in a volume ratio of from 20:1 to 40: 1.

In another preferred embodiment, the method for preparing form a crystals according to the present invention, wherein the basic conditions are preferably triethylamine, N-diisopropylethylamine, pyridine, dimethylamine, aqueous ammonia, preferably triethylamine; the sodium salt is selected from sodium bicarbonate, sodium hydrogen, sodium carbonate, sodium methoxide, sodium ethoxide and sodium hydroxide, preferably sodium hydroxide, and the concentration of the sodium hydroxide is preferably 20-45%.

The present invention further provides a process for preparing form B crystals of sodium 1- (6- (2-oxa-8-azaspiro [4.5] decan-8-yl) pyrimidin-4-yl) -4- (1H-1,2, 3-triazol-1-yl) -1, 2-dihydro-1H-pyrazol-3-olate according to the invention, said process comprising the steps of:

2) stirring the solution of 1- (6- (2-oxa-8-azaspiro [4.5] decan-8-yl) pyrimidin-4-yl) -4- (1H-1,2, 3-triazol-1-yl) -1, 2-dihydro-1H-pyrazol-3-olate sodium obtained in the step 1) at 0 ℃ to 10 ℃ for crystallization preferably for 2 to 20 hours, more preferably for 20 hours, and filtering the crystals;

3) drying the crystals obtained in the step 2) to obtain B-type crystals of 1- (6- (2-oxa-8-azaspiro [4.5] decan-8-yl) pyrimidin-4-yl) -4- (1H-1,2, 3-triazol-1-yl) -1, 2-dihydro-1H-pyrazol-3-ol sodium.

In a preferred embodiment, the preparation method of form B crystal according to the present invention, wherein, in step 1), 2- (6- (2-oxa-8-azaspiro [4.5] decan-8-yl) pyrimidin-4-yl) -4- (1H-1,2, 3-triazol-1-yl) -1, 2-dihydro-3H-pyrazol-3-one is further dissolved in an organic solvent by heating, preferably at 55 ℃ to 60 ℃, followed by hot filtration, and a sodium salt solution is added dropwise to the filtrate.

In a preferred embodiment, the process for the preparation of form B crystals according to the present invention, wherein, in step 1), the solution to obtain sodium 1- (6- (2-oxa-8-azaspiro [4.5] decan-8-yl) pyrimidin-4-yl) -4- (1H-1,2, 3-triazol-1-yl) -1, 2-dihydro-1H-pyrazol-3-olate is further incubated for preferably 15 to 30 minutes.

In a preferred embodiment, the preparation method of the B-type crystal according to the present invention, wherein, in the step 2), the solution of sodium 1- (6- (2-oxa-8-azaspiro [4.5] decan-8-yl) pyrimidin-4-yl) -4- (1H-1,2, 3-triazol-1-yl) -1, 2-dihydro-1H-pyrazol-3-olate obtained in the step 1) is first crystallized by stirring at room temperature for preferably 1 to 2 hours, and then at 0 ℃ to 10 ℃ for preferably 2 hours, and the crystal is filtered.

In another preferred embodiment, the method for preparing form B crystals according to the present invention comprises the steps of:

2) stirring the solution of 1- (6- (2-oxa-8-azaspiro [4.5] decan-8-yl) pyrimidin-4-yl) -4- (1H-1,2, 3-triazol-1-yl) -1, 2-dihydro-1H-pyrazol-3-olate sodium obtained in the step 1) at room temperature for crystallization preferably for 1 to 2 hours, then stirring at 0 ℃ to 10 ℃ for crystallization preferably for 2 hours, and filtering the crystals;

In a preferred embodiment, the preparation method of form B crystals according to the present invention, wherein the organic solvent is selected from C1-C4 alcohol, 1, 4-dioxane, acetonitrile, acetone or a mixture thereof, or a mixture of the above solvent and water; preferably a C1-C4 alcohol or a mixture of a C1-C4 alcohol and water; the C1-C4 alcohol is preferably methanol or ethanol.

In another preferred embodiment, the preparation method of the form B crystal according to the present invention, wherein the organic solvent is a mixture of C1-C4 alcohol and water, and the volume ratio of the C1-C4 alcohol to water is 1:2 to 40:1, preferably 1:2 to 15: 1.

In another preferred embodiment, the method for preparing form B crystals according to the present invention, wherein the organic solvent is a mixture of methanol and water, and the volume ratio of methanol to water is 1:2 to 15: 1.

In another preferred embodiment, the method for preparing form B crystals according to the present invention, wherein the basic conditions are preferably triethylamine, N-diisopropylethylamine, pyridine, dimethylamine, aqueous ammonia, preferably triethylamine; the sodium salt is selected from sodium bicarbonate, sodium hydrogen, sodium carbonate, sodium methoxide, sodium ethoxide and sodium hydroxide, preferably sodium hydroxide, and the concentration of the sodium hydroxide is preferably 10-45%.

The 2- (6- (2-oxa-8-azaspiro [4.5] decan-8-yl) pyrimidin-4-yl) -4- (1H-1,2, 3-triazol-1-yl) -1, 2-dihydro-3H-pyrazol-3-one used in the process for producing the a-type crystal or the B-type crystal of the present invention is a compound of the formula (I), which can be synthesized by a conventional production method by a retro-synthesis method well known to those skilled in the art, and in particular, can be obtained by the method described in example 8 in patent application No. 201811237690.7, and is described in detail in examples of the present invention. In addition, 2- (6- (2-oxa-8-azaspiro [4.5] decan-8-yl) pyrimidin-4-yl) -4- (1H-1,2, 3-triazol-1-yl) -1, 2-dihydro-3H-pyrazol-3-one used in the present invention may be in any crystal form or amorphous form.

The C1-C4 alcohol used in the present invention may be any linear or branched alkane alcohol commonly used in the art, such as methanol, ethanol, propanol, isopropanol, n-butanol, sec-butanol, tert-butanol, propylene glycol, ethylene glycol, and the like, preferably methanol or ethanol.

The sodium salt used in the present invention to form 1- (6- (2-oxa-8-azaspiro [4.5] decan-8-yl) pyrimidin-4-yl) -4- (1H-1,2, 3-triazol-1-yl) -1, 2-dihydro-1H-pyrazol-3-ol sodium may be any sodium salt commonly used in the art that can be reacted with a compound of formula (I) to form a salt, for example sodium bicarbonate, sodium hydrogen, sodium carbonate, sodium methoxide, sodium ethoxide, sodium hydroxide, preferably sodium hydroxide. The concentration of the sodium hydroxide is preferably 10-45%.

The crystal forms of the A-type crystal and the B-type crystal are determined and researched by X-ray diffraction (XRD), differential scanning thermal analysis (DSC) and thermogravimetric analysis (TGA).

The crystals of form A and form B of 1- (6- (2-oxa-8-azaspiro [4.5] decan-8-yl) pyrimidin-4-yl) -4- (1H-1,2, 3-triazol-1-yl) -1, 2-dihydro-1H-pyrazol-3-olate sodium prepared by the method of the present invention have good stability and bioavailability and can be advantageously used as pharmaceutically active ingredients.

In another aspect, the present invention provides a pharmaceutical composition comprising the form a crystal or the form B crystal of sodium 1- (6- (2-oxa-8-azaspiro [4.5] decan-8-yl) pyrimidin-4-yl) -4- (1H-1,2, 3-triazol-1-yl) -1, 2-dihydro-1H-pyrazol-3-olate according to the invention as an active ingredient together with a pharmaceutically acceptable carrier.

The invention further provides the use of crystals type a or crystals type B of sodium 1- (6- (2-oxa-8-azaspiro [4.5] decan-8-yl) pyrimidin-4-yl) -4- (1H-1,2, 3-triazol-1-yl) -1, 2-dihydro-1H-pyrazol-3-olate according to the invention or of a pharmaceutical composition containing same for the preparation of a medicament for the prevention and/or treatment of a disease associated with PHD activity, preferably selected from cardiovascular diseases, in particular cardiac insufficiency, coronary heart disease, angina pectoris, myocardial infarction, stroke, arteriosclerosis, primary, pulmonary and malignant hypertension and peripheral arterial occlusive diseases; chronic kidney disease; hematopoietic disorders, such as primary anemia, renal anemia, and anemia associated with neoplastic disease (particularly chemotherapy-induced anemia); infection (particularly HIV infection) or other inflammatory diseases, such as rheumatoid arthritis; anemia due to blood loss, iron deficiency anemia, vitamin deficiency anemia (e.g. due to vitamin B12 deficiency or due to folate deficiency), aplastic anemia and aplastic anemia or hemolytic anemia, anemia due to iron utilization disorders (iron-loss anemia) or due to other endocrine disorders (e.g. hypothyroidism); post-surgical procedures associated with ischemic conditions and their subsequent symptoms, particularly cardiac interventions using heart-lung machines (e.g. bypass surgery, heart valve transplantation), carotid interventions, aortic interventions and interventions using instrument openings or penetrating calvarial; surgical wound healing; cancer and the damage of the health state that occurs during the treatment of cancer, in particular after treatment with cytostatics, antibiotics and radiation, diseases ranging from the rheumatic forms and other diseases considered as autoimmune diseases, in particular the damage of the health state that occurs during the pharmacological treatment of such diseases; continuous symptoms of acute and prolonged cerebral ischemic conditions (e.g. stroke, childbirth asphyxia).

As used herein, "pharmaceutically acceptable" is useful in preparing pharmaceutical compositions that are generally safe, neither biologically nor otherwise undesirable, and that are acceptable for veterinary use and human pharmaceutical use.

As used herein, "carrier" refers to a diluent, adjuvant, or excipient with which the compound is administered. Pharmaceutically acceptable carriers can be liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, rapeseed oil and the like. The pharmaceutically acceptable carrier may also be physiological saline, gum arabic, gelatin, starch paste, talc, keratin, silica gel, urea, etc. In addition, auxiliary agents, stabilizers, thickeners, lubricants, colorants, and the like can also be used.

It will be appreciated by those skilled in the art that the pharmaceutical composition of the present invention may be formulated into various formulations well known in the art, such as oral dosage forms (powder, tablet, capsule, soft capsule, liquid drug, syrup, elixir pill, powder, sachet, granule), or topical formulations (cream, ointment, lotion, gel, balm, plaster, paste, spray, aerosol, etc.), or injectable formulations (solution, suspension, emulsion), depending on the particular mode of administration. Among the pharmaceutical compositions of the invention, mention may in particular be made of those suitable for oral, parenteral (intravenous or subcutaneous) or nasal administration, for example, tablets or dragees, sublingual tablets, gelatin capsules, lozenges, suppositories, creams, ointments, dermal gels, injectable preparations, drinkable suspensions and the like.

The pharmaceutical composition according to the invention may comprise a pharmaceutically acceptable carrier, adjuvant or diluent, for example: fillers, disintegrants, lubricants, suspending agents, binders, sweeteners, flavoring agents, preservatives, bases, and the like. Fillers such as: starch, pregelatinized starch, lactose, mannitol, chitin, microcrystalline cellulose, sucrose, etc.; disintegrants for example: starch, pregelatinized starch, microcrystalline cellulose, sodium carboxymethyl starch, cross-linked polyvinyl pyrrole, low-substituted hydroxypropyl cellulose, cross-linked sodium carboxymethyl cellulose, etc.; lubricants such as: magnesium stearate, sodium lauryl sulfate, talc, silica, and the like; suspending agents such as: polyvinylpyrrolidone, microcrystalline cellulose, sucrose, agar, hydroxypropyl methylcellulose, and the like; examples of the binder include starch slurry, polyvinylpyrrolidone, hydroxypropylmethylcellulose, and the like. The compositions of the present invention may be formulated by any method known in the art to provide rapid, sustained or slow release of the active ingredient after administration to a patient.

The pharmaceutical composition of the present invention is administered to an individual animal such as a mammal (rat, mouse, domesticated animal or human) by various routes, all of which are contemplated, for example, administration may be oral, topical, rectal or intravenous, intramuscular, transdermal, intrathecal, epidural or intracerebroventricular injection.

The dosage of the active ingredient of the present invention to be administered may vary depending on the individual condition and weight, the nature and severity of the condition, the form of the drug, the route of administration and the period of administration, and may be selected by those skilled in the art. The dosage may vary from 1 to 100 mg/day and may be administered in a single dose per day or in divided doses per day.

The invention will be further elucidated with reference to the drawings and specific examples, but it will be understood that these are by way of illustration only and do not in any way limit the scope of the invention.

Drawings

Figure 1 shows the X-ray powder diffraction pattern of form a of the present invention.

Figure 2 shows a DSC profile of form a of the present invention.

Figure 3 shows a TGA profile of crystalline form a of the present invention.

Figure 4 shows an X-ray powder diffraction pattern of form B of the present invention.

Figure 5 shows a DSC profile of form B of the invention.

Figure 6 shows a TGA profile of crystalline form B of the present invention.

Detailed Description

The present invention will be explained in more detail with reference to examples, which are provided only for illustrating the technical solutions of the present invention and are not intended to limit the spirit and scope of the present invention.

An experimental instrument:

1. DSC spectrum

The instrument model is as follows: TA Instruments Q200MDSC

And (3) purging gas: nitrogen gas

The heating rate is as follows: 10 ℃/min

Temperature range: 0 ℃ to 400 DEG C

2. TGA Spectrum

The instrument model is as follows: TA Instruments Q500TGA

And (3) purging gas: nitrogen gas

The heating rate is as follows: 10 ℃/min

Temperature range: 0 ℃ to 500 DEG C

3. X-ray diffraction spectrum

The instrument model is as follows: bruker D8advance diffracometer

Ray: 1.54nm Kalpha X-ray

The scanning mode is as follows: 0.2 sec/step, scan range: 3 to 40 degrees.

The structure of the compounds is determined by Nuclear Magnetic Resonance (NMR) or/and Mass Spectrometry (MS). NMR shift at 10^-6The units in (ppm) are given. NMR was measured using a Brukerdps model 300 nuclear magnetic spectrometer using deuterated dimethyl sulfoxide (DMSO-d)₆) Deuterated chloroform (CDCl)₃) Deuterated AAlcohol (CD)₃OD), internal standard Tetramethylsilane (TMS).

MS was measured using a 1100Series LC/MSD Trap (ESI) mass spectrometer (manufacturer: Agilent).

Known starting materials of the present invention can be synthesized by or according to methods known in the art, or can be purchased from the companies such as cyber-mart, beijing coup, Sigma, carbofuran, yishiming, shanghai kaya, enokay, nanjing yashi, ann naiji chemical, and the like.

Example 1: preparation of 2- (6- (2-oxa-8-azaspiro [4.5] decan-8-yl) pyrimidin-4-yl) -4- (1H-1,2, 3-triazol-1-yl) -1, 2-dihydro-3H-pyrazol-3-one (I)

The compound of formula (I) was prepared according to example 8 of patent application 201811237690.7, specifically as follows:

step 1: synthesis of ethyl 2- (1H-1,2, 3-triazol-1-yl) acetate (intermediate 1A)

1H-1,2, 3-triazole (100g, 1.45mol) was added to a 1L reaction flask, to which ethyl acetate (300mL), DIPEA (252mL, 1.45mol) were added, and stirred for 3 minutes under ice bath. Ethyl bromoacetate (152mL, 1.38mol) was added to 200mL of ethyl acetate, which was then slowly added dropwise to the reaction flask, after which it was stirred at room temperature overnight. After completion of the reaction, filtration was carried out, and the filtrate was washed once with water and saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain 100g of the title product as a yellow oil in yield: 44.3 percent.

Step 2: synthesis of ethyl 3- (dimethylamino) -2- (1H-1,2, 3-triazol-1-yl) acrylate (intermediate 1B)

Ethyl 2- (1H-1,2, 3-triazol-1-yl) acetate (100g, 0.64mol) and DMA (100mL) were added to a single-necked flask, and the reaction was stirred at 60 ℃ for 1 hour. After the reaction was completed, the reaction solution was cooled to room temperature and concentrated to dryness under reduced pressure, 300mL of water and 300mL of dichloromethane were added for extraction, the organic phase was concentrated until dichloromethane was substantially not remained, 400mL of methyl t-butyl ether was added with stirring, stirred at room temperature for 1 hour, and filtered to obtain 62.2g of the title product as a pale yellow solid in yield: 45.9 percent.

And step 3: synthesis of 4-chloro-6-hydrazinopyrimidine (intermediate 1C)

4, 6-dichloropyrimidine (10g, 68mmol) and ethanol (350mL) were charged into a 1L reaction flask, hydrazine hydrate (6.05g, 122mmol) was added dropwise at room temperature, while turbidity occurred, ethanol (200mL) was added thereto, and after the addition, the mixture was stirred at room temperature for 12 hours. After completion of the reaction, filtration was carried out, and the filter cake was washed twice with water and petroleum ether, respectively, and dried to obtain 6.3g of the title product as a yellow solid in a yield of 65.6%.

And 4, step 4: synthesis of 2- (4-chloropyrimidin-2-yl) -4- (1H-1,2, 3-triazol-1-yl) -1, 2-dihydro-3H-pyrazol-3-one (intermediate 1D)

4-chloro-6-hydrazinopyrimidine (5.19g, 36.3mmol), ethyl 3- (dimethylamino) -2- (1H-1,2, 3-triazol-1-yl) acrylate (6.25g, 29.7mmol), ethanol (63mL), TFA (1.37g, 8.8mmol) were sequentially added to a 250mL reaction flask, heated to reflux and stirred for 12 hours. The reaction solution was cooled to room temperature, 40mL of 1M dioxane hydrochloric acid gas was added dropwise, and after the addition was completed, the mixture was stirred for 1 hour and filtered. The filter cake was added to 90mL of ethanol, and 32mL of a 25% sodium methoxide solution in methanol was added thereto, followed by stirring at room temperature for 2 hours, and then the pH was adjusted to 4-5 with 1N hydrochloric acid, followed by stirring at room temperature for 2 hours. After the reaction was complete, filtration was carried out and the filter cake was washed once with ethanol and dried to give 6.9g of the title product as a yellow solid in yield: 87.8 percent.

And 5: 2- (6- (2-oxa-8-azaspiro [4.5] decan-8-yl) pyrimidin-4-yl) -4- (1H-1,2, 3-triazol-1-yl) -1, 2-dihydro-3H-pyrazol-3-one (Compound I)

2- (4-Chloropyrimidin-2-yl) -4- (1H-1,2, 3-triazol-1-yl) -1, 2-dihydro-3H-pyrazol-3-one (6.9g, 0.026mol), 2-oxa-8-azaspiro [4.5] decane oxalate (Nanjing Yashi science Co., Ltd.) (5.4g, 0.014mol), DMSO 50mL, and potassium carbonate (6.9g, 0.50mol) were sequentially added to a 250mL reaction flask, heated at 100 ℃ to 110 ℃ and stirred for 8 hours. After completion of the reaction, cooled to room temperature, concentrated to dryness, adjusted to PH 4-5 with hydrochloric acid, filtered and dried to give 3.3g of the title product as a white solid in yield: 54%, purity: 98.5 percent.

MS：m/z＝369.9[M+H]⁺。

¹H NMR(300MHz,DMSO):ppm 1.60(m,4H),1.79(m,2H),3.77(m,8H),7.42(s,1H),7.85(s,1H),8.21(s,1H),8.37(s,1H),8.51(s,1H)。

Example 2: preparation of crystal form A of 1- (6- (2-oxa-8-azaspiro [4.5] decan-8-yl) pyrimidin-4-yl) -4- (1H-1,2, 3-triazol-1-yl) -1, 2-dihydro-1H-pyrazol-3-olate sodium (II)

2- (6- (2-oxa-8-azaspiro [4.5] decan-8-yl) pyrimidin-4-yl) -5- (1H-1,2, 3-triazol-1-yl) -1, 2-dihydro-3H-pyrazol-3-one (I) (3g) obtained in example 1 was added to a mixed solution of 30ml of tetrahydrofuran and 1ml of water, triethylamine (0.83g) was added, and the temperature was raised to 60 ℃ until the solution was clear, and hot filtration was carried out. The filtrate was warmed to 60 ℃ and 45% aqueous NaOH solution (0.86g) was added dropwise. After the dripping is finished, the temperature is kept for 30 minutes, the heating is closed, the temperature is naturally reduced to the room temperature, the mixture is stirred and crystallized for 2 hours at the room temperature, the temperature is reduced to 10 ℃ and crystallized for 2 hours, and the mixture is filtered. The filter cake was dried at 55 ℃ under reduced pressure for 5 hours to give the product as a yellowish solid (1.8g) with a yield of 56.3%.

The X-ray diffraction pattern of the crystalline sample is shown in fig. 1, and the X-ray diffraction data are shown in table 1 below. The DSC spectrum is shown in figure 2, and the TGA spectrum is shown in figure 3. This form is defined as form a.

TABLE 1 values of 2theta values and corresponding values of strength for form A

Strength%	2-Theta
		100	4.398
5.1	8.740
		8.2	12.949
5.5	13.352
		18.9	16.353
4.5	18.761
		7.3	22.542
7.2	23.564
		22.1	26.564
10.7	29.563

Example 3: preparation of crystal form A of 1- (6- (2-oxa-8-azaspiro [4.5] decan-8-yl) pyrimidin-4-yl) -4- (1H-1,2, 3-triazol-1-yl) -1, 2-dihydro-1H-pyrazol-3-olate sodium (II)

2- (6- (2-oxa-8-azaspiro [4.5] decan-8-yl) pyrimidin-4-yl) -5- (1H-1,2, 3-triazol-1-yl) -1, 2-dihydro-3H-pyrazol-3-one (I) (30g) obtained in example 1 was added to a mixed solution of 200ml of tetrahydrofuran and 10ml of water, triethylamine (8.3g) was added thereto, the temperature was raised to 55 ℃ and a 20% aqueous NaOH solution (19.5g) was added dropwise. Keeping the temperature for 15 minutes after the dripping is finished, closing the heating and naturally cooling to room temperature, stirring and crystallizing for 2 hours at room temperature, cooling and crystallizing for 2 hours at 10 ℃, and filtering. The filter cake was dried at 55 ℃ under reduced pressure for 5 hours to give the product as a yellowish solid (28g) with a yield of 87.6%.

The XRD pattern of the product is determined to be A crystal form through research and comparison.

Example 4: preparation of crystal form A of 1- (6- (2-oxa-8-azaspiro [4.5] decan-8-yl) pyrimidin-4-yl) -4- (1H-1,2, 3-triazol-1-yl) -1, 2-dihydro-1H-pyrazol-3-olate sodium (II)

2- (6- (2-oxa-8-azaspiro [4.5] decan-8-yl) pyrimidin-4-yl) -5- (1H-1,2, 3-triazol-1-yl) -1, 2-dihydro-3H-pyrazol-3-one (I) (6g) obtained in example 1 was added to a mixed solution of 40ml of tetrahydrofuran and 1ml of water, triethylamine (1.66g) was added, the temperature was raised to 60 ℃ and a 25% aqueous NaOH solution (3.1g) was added dropwise. After the dripping is finished, the temperature is kept for 30 minutes, the heating is closed, the temperature is naturally reduced to the room temperature, the mixture is stirred and crystallized for 1 hour at the room temperature, and is cooled and crystallized for 2 hours at the temperature of 5 ℃, and then the mixture is filtered. The filter cake was dried at 55 ℃ under reduced pressure for 5 hours to give the product as a yellowish solid (5.8g) with a yield of 90.6%.

Example 5: preparation of crystal form B of 1- (6- (2-oxa-8-azaspiro [4.5] decan-8-yl) pyrimidin-4-yl) -4- (1H-1,2, 3-triazol-1-yl) -1, 2-dihydro-1H-pyrazol-3-olate sodium (II)

2- (6- (2-oxa-8-azaspiro [4.5] decan-8-yl) pyrimidin-4-yl) -5- (1H-1,2, 3-triazol-1-yl) -1, 2-dihydro-3H-pyrazol-3-one (I) (6g) obtained in example 1 was added to 60ml of methanol and 12ml of water, triethylamine (1.6g) was added, and the temperature was raised to 60 ℃ until the solution was clear, and hot filtration was carried out. The filtrate was warmed to 60 ℃ and 30% NaOH solution (2.6g) was added dropwise. After the dripping is finished, the temperature is kept for 20 minutes, the heating is closed, the temperature is naturally reduced to the room temperature, the mixture is stirred and crystallized for 2 hours at the room temperature, the temperature is reduced to 0 ℃ and crystallized for 2 hours, and the mixture is filtered. The filter cake was dried at 50 ℃ under reduced pressure for 5 hours to give the product as a yellowish solid (5.2g) in 82.3% yield.

The X-ray diffraction pattern of the crystalline sample is shown in fig. 4, and the X-ray diffraction data are shown in table 2 below. The DSC spectrum is shown in figure 5, and the TGA spectrum is shown in figure 6. This form is defined as form B.

TABLE 2 values of 2theta values for form B corresponding to the strength

Strength%	2-Theta
		59.7	7.460
10.8	9.368
		16.1	9.636
50.1	16.188
		54.6	16.423
25.1	17.072
		13.5	17.473
24.2	21.158
		100	22.554
26.6	23.677
		12.1	29.368

Example 6: preparation of crystal form B of 1- (6- (2-oxa-8-azaspiro [4.5] decan-8-yl) pyrimidin-4-yl) -4- (1H-1,2, 3-triazol-1-yl) -1, 2-dihydro-1H-pyrazol-3-olate sodium (II)

2- (6- (2-oxa-8-azaspiro [4.5] decan-8-yl) pyrimidin-4-yl) -5- (1H-1,2, 3-triazol-1-yl) -1, 2-dihydro-3H-pyrazol-3-one (I) (3g) obtained in example 1 was added to a mixed solution of 24ml of methanol and 0.8ml of water, triethylamine (0.83g) was added thereto, the temperature was raised to 60 ℃ and a 33% aqueous NaOH solution (1.19g) was added dropwise. After the dripping is finished, the temperature is kept for 30 minutes, and the heating is closed and the temperature is naturally reduced to the room temperature. Stirring at room temperature for crystallization for 2 hours, cooling at 10 ℃ for crystallization for 2 hours, and filtering. The filter cake was dried at 55 ℃ under reduced pressure for 5 hours to give the product as a yellowish solid (2.3g) with a yield of 71.5%.

The XRD pattern of the product is determined to be B crystal form through research and comparison.

Example 7: preparation of crystal form B of 1- (6- (2-oxa-8-azaspiro [4.5] decan-8-yl) pyrimidin-4-yl) -4- (1H-1,2, 3-triazol-1-yl) -1, 2-dihydro-1H-pyrazol-3-olate sodium (II)

2- (6- (2-oxa-8-azaspiro [4.5] decan-8-yl) pyrimidin-4-yl) -5- (1H-1,2, 3-triazol-1-yl) -1, 2-dihydro-3H-pyrazol-3-one (I) (12g, 8.2mmol) obtained in example 1 was added to a mixed solution of 120ml of methanol and 8ml of water, triethylamine (3.2g) was added, the temperature was raised to 60 ℃ and a 16% aqueous NaOH solution (9.2g) was added dropwise. Keeping the temperature for 15 minutes after the dripping is finished, closing the heating and naturally cooling to room temperature, stirring and crystallizing for 2 hours at room temperature, cooling and crystallizing for 2 hours at 0 ℃, and filtering. The filter cake was dried at 50 ℃ under reduced pressure for 5 hours to give the product as a yellowish solid (9.6g) in 78.3% yield.

Example 8: preparation of crystal form B of 1- (6- (2-oxa-8-azaspiro [4.5] decan-8-yl) pyrimidin-4-yl) -4- (1H-1,2, 3-triazol-1-yl) -1, 2-dihydro-1H-pyrazol-3-olate sodium (II)

2- (6- (2-oxa-8-azaspiro [4.5] decan-8-yl) pyrimidin-4-yl) -5- (1H-1,2, 3-triazol-1-yl) -1, 2-dihydro-3H-pyrazol-3-one (I) (4g) obtained in example 1 was added to a mixed solution of 40ml of methanol and 1ml of water, triethylamine (1.1g) was added, the temperature was raised to 55 ℃, and a 45% aqueous NaOH solution (0.86g) was added dropwise. After the dripping is finished, the temperature is kept for 20 minutes, the heating is closed, the temperature is naturally reduced to the room temperature, the mixture is stirred and crystallized for 2 hours at the room temperature, the temperature is reduced to 10 ℃ and crystallized for 2 hours, and the mixture is filtered. The filter cake was dried at 55 ℃ under reduced pressure for 5 hours to give the product as a yellowish solid (3.1g) with a yield of 94.8%.

Example 9: preparation of crystal form B of 1- (6- (2-oxa-8-azaspiro [4.5] decan-8-yl) pyrimidin-4-yl) -4- (1H-1,2, 3-triazol-1-yl) -1, 2-dihydro-1H-pyrazol-3-olate sodium (II)

2- (6- (2-oxa-8-azaspiro [4.5] decan-8-yl) pyrimidin-4-yl) -5- (1H-1,2, 3-triazol-1-yl) -1, 2-dihydro-3H-pyrazol-3-one (I) (24g) obtained in example 1 was added to a mixed solution of 240ml of methanol and 8ml of water, triethylamine (6.6g) was added, the temperature was raised to 55 ℃ and a 25% aqueous NaOH solution (12.5g) was added dropwise. After the dripping is finished, the temperature is kept for 30 minutes, the heating is closed, the temperature is naturally reduced to the room temperature, the mixture is stirred and crystallized for 1 hour at the room temperature, and is cooled and crystallized for 2 hours at the temperature of 10 ℃, and then the mixture is filtered. The filter cake was dried at 50 ℃ under reduced pressure for 5 hours to give the product as a yellowish solid (18.4g) with a yield of 71.5%.

Example 10: preparation of crystal form B of 1- (6- (2-oxa-8-azaspiro [4.5] decan-8-yl) pyrimidin-4-yl) -4- (1H-1,2, 3-triazol-1-yl) -1, 2-dihydro-1H-pyrazol-3-olate sodium (II)

2- (6- (2-oxa-8-azaspiro [4.5] decan-8-yl) pyrimidin-4-yl) -5- (1H-1,2, 3-triazol-1-yl) -1, 2-dihydro-3H-pyrazol-3-one (I) (30g) obtained in example 1 was added to a mixed solution of 300ml of methanol and 20ml of water, triethylamine (8.3g) was added, the temperature was raised to 60 ℃ and a 10% aqueous NaOH solution (39g) was added dropwise. After the dripping is finished, the temperature is kept for 30 minutes, the heating is closed, the temperature is naturally reduced to the room temperature, the mixture is stirred and crystallized for 2 hours at the room temperature, the temperature is reduced to 10 ℃ and crystallized for 2 hours, and the mixture is filtered. The filter cake was dried at 50 ℃ under reduced pressure for 5 hours to give the product as a yellowish solid (24g) in 78.3% yield.

Example 11: preparation of crystal form B of 1- (6- (2-oxa-8-azaspiro [4.5] decan-8-yl) pyrimidin-4-yl) -4- (1H-1,2, 3-triazol-1-yl) -1, 2-dihydro-1H-pyrazol-3-olate sodium (II)

2- (6- (2-oxa-8-azaspiro [4.5] decan-8-yl) pyrimidin-4-yl) -5- (1H-1,2, 3-triazol-1-yl) -1, 2-dihydro-3H-pyrazol-3-one (I) (3g) obtained in example 1 was added to 15ml of methanol and 30ml of water, stirred to raise the temperature to 55 ℃, triethylamine (0.83g) was added dropwise, stirred to clarify, and filtered while hot. The filtrate was warmed to 60 ℃ and 45% NaOH saturated solution (0.9g) was slowly added dropwise with stirring. After the dropwise addition, the temperature is reduced to 0 ℃, and the mixture is stirred for 20 hours for crystallization. Filtering, washing a filter cake by using a small amount of methanol, and drying at 60 ℃ under reduced pressure to obtain 0.8g of yellowish solid, wherein the yield is as follows: 25.1 percent.

Example 12: preparation of crystal form B of 1- (6- (2-oxa-8-azaspiro [4.5] decan-8-yl) pyrimidin-4-yl) -4- (1H-1,2, 3-triazol-1-yl) -1, 2-dihydro-1H-pyrazol-3-olate sodium (II)

2- (6- (2-oxa-8-azaspiro [4.5] decan-8-yl) pyrimidin-4-yl) -5- (1H-1,2, 3-triazol-1-yl) -1, 2-dihydro-3H-pyrazol-3-one (I) (6g, 16mmol) obtained in example 1 was added to 60ml of methanol and 60ml of water, triethylamine (1.6g) was added, and the temperature was raised to 60 ℃ until the solution was clear, and hot filtration was carried out. The filtrate was warmed to 60 ℃ and a 45% NaOH saturated solution (0.86g) was added dropwise. After the dripping is finished, the temperature is kept for 30 minutes, the heating is closed, the temperature is naturally reduced to the room temperature, the mixture is stirred and crystallized for 2 hours at the room temperature, the temperature is reduced to 10 ℃ and crystallized for 2 hours, and the mixture is filtered. The filter cake was dried at 55 ℃ under reduced pressure for 5 hours to give the product as a yellowish solid (4.72g) in 74.2% yield.

Example 13: hygroscopicity test

The hygroscopicity of the crystalline form a obtained in example 3, the crystalline form B obtained in example 9 and the compound of formula (I) was examined according to appendix XIX J of the second part of the chinese pharmacopoeia, 2010 edition. The specific method comprises the following steps: placing a dried glass weighing bottle (with an outer diameter of 50mm and a height of 15mm) in a suitable constant temperature drier with a temperature of 25 +/-1 ℃ for the previous day, and precisely weighing (m)₀) Taking a proper amount of sample, precisely weighing (m)₁) The sample is flatly paved in the weighing bottle, and the thickness of the sample is about 1 mm; opening the weighing bottle, placing the weighing bottle and the bottle cap under the conditions of constant temperature and constant humidity for 24 hours, and covering the weighing bottle and the bottle capThe bottle cap is weighed well, and precision weighing (m)₂) And calculating the weight gain percentage. Percent (%) moisturization (m)₂-m₁-m₀/m₁)×100％。

According to the guiding principle of the medicament hygroscopicity test, the experimental conditions are as follows: the hygroscopicity was examined at 25 ℃. + -. 1 ℃ under various relative humidities. The hygroscopicity results of the respective samples are shown in the following table 3.

TABLE 3

And (4) conclusion: under the relative humidity of 80% +/-2%, the calculated moisture absorption weight gain of the crystal form A is 0.82%, the moisture absorption weight gain of the crystal form B is 0.33%, and the crystal form A is slightly hygroscopic according to the defined standard of hygroscopic weight gain specified by pharmacopoeia. The weight of the compound of formula (I) increased by 3.33%. Therefore, the hygroscopicity of the crystal form A and the hygroscopicity of the crystal form B of the invention are both obviously improved compared with the compound shown in the formula (I), thereby being beneficial to the stability of the compound.

Example 14: stability study

A proper amount of the samples of the crystal form A prepared in example 4 and the crystal form B prepared in example 5 are put into a culture dish, spread into a thin layer with the thickness less than 5mm, exposed to the high temperature of 105 ℃ plus or minus 2 ℃, the high humidity of 92.5 percent plus or minus 2 percent, or the visible light of 4500 plus or minus 500lx, and the ultraviolet light intensity is not lower than 0.7W.h/m². Samples were obtained after exposure or irradiation and the impurity content was determined by HPLC using a Waters Aquacty Arc liquid chromatograph. The test conditions are shown in table 4 below.

TABLE 4HPLC detection conditions

The test results are shown in table 5 below.

Table 5 stability results for the crystalline forms of the invention

And (4) conclusion: the impurity content of the crystal form A and the impurity content of the crystal form B are not obviously changed under the conditions of high temperature, high humidity and illumination for 10 days or 20 days. Compound (I) had a significant increase in total impurities under these conditions. The A crystal form and the B crystal form have excellent stability.

Example 15: pharmacokinetic evaluation of crystal form of the invention in SD rat

The compound of formula (I) prepared in example 1, the crystalline form a prepared in example 3 or the crystalline form B prepared in example 10 were administered to male 7-week-old SD rats (sbefu (beijing) biotechnology limited) intravenously (i.v.) or orally (P.O.), at an intravenous dose of 1mg/kg and an oral dose of 5 mg/kg. Intravenous administration (1 mg/kg; 0.2mg/ml) formulation method: 2.5mg of the compound was weighed, suspended in 12.5ml of physiological saline, and mixed well. The preparation method of the oral administration (5 mg/kg; 0.5mg/ml) comprises the following steps: 37.5mg of the compound was weighed, suspended in 25ml of 0.5% CMC-Na and mixed well.

Intravenous injection of orbital bleeds at 0, 0.083, 0.25, 0.50, 1.00, 2.00, 4.00, 6.00, 8.00, and 24.00 post-dose, respectively; orbital bleeds were performed at 0, 0.167, 0.333, 0.50, 1.00, 2.00, 4.00, 6.00, 8.00, and 24.00 post-dose for oral administration, respectively. Blood was anticoagulated with sodium heparin (Sigma), centrifuged at 3500rpm for 10 minutes at 4 ℃ and plasma was taken and stored at-20 ℃ until testing.

Plasma samples of 50 μ L were taken in 1.5mL EP tubes and vortexed for 1 min to mix well. After vortexing, 0.2mL acetonitrile was added, vortexed vigorously for 1 min, and centrifuged at 16000rpm for 10 min. 0.2mL of the supernatant was removed, filtered in a 0.22 μ M organic membrane (Cleman), added to a sample vial, analyzed by LC/MS (Waters, Waters UPLCI Class, TQ-S micro) to obtain the plasma concentration, and analyzed by DAS software 3.0 for pharmacokinetic parameters.

The pharmacokinetic parameters are shown in table 6 below.

TABLE 6 pharmacokinetic parameters of the crystalline forms of the invention

And (4) conclusion: the bioavailability of the crystal form A and the crystal form B of the invention is obviously higher than that of the compound shown in the formula (I).

Example 16: effect of Compounds of the invention on Balb/C mouse EPO levels

The drug activity was measured by measuring changes in plasma EPO (erythropoietin) levels 4 hours after a single oral administration in mice.

Animals: Balb/C mice, male, 18-20 g; purchased from experimental animal technology limited of Viton Lihua, Beijing, SPF grade; animal production license number: SCXK (Jing) 2016-; issuing a certificate unit: the scientific and technical committee of Beijing.

Grouping: normal control group (0.5% CMC-Na vehicle group); form a prepared in example 3 (0.88mg/kg) and form B prepared in example 10 (0.88 mg/kg); each group had 5 animals.

Sample preparation: the crystal form of the invention is suspended in 0.5 percent of CMC-Na serving as a solvent.

The animals are purchased and adaptively fed for 5-7 days, and then used for tests, after the animals are orally administrated for 4 hours once, the animals are anesthetized by isoflurane for 3 minutes, blood is collected by an orbital venous plexus blood collection method (0.5 ml/animal), and heparin sodium (sigma) is anticoagulated. Centrifuging at 3500 rpm/min for 10 min, separating plasma, and storing at-20 deg.C.

The detection indexes and the method are as follows: the plasma EPO levels of mice were determined using an EPO ELISA kit (P137645, R & D System) and a microplate reader (rotation 3, BioTek).

Table 7 shows the effect of form A prepared in example 3 and form B prepared in example 10 of the present invention on EPO secretion in mice at a dose of 0.88 mg/kg. Wherein A represents >1000pg/mL, B represents 500-100pg/mL, and C represents <500 pg/mL.

Table 7 improvement of EPO levels in mice by the crystalline forms of the invention

Group of	EPO level (pg/mL)
		Normal control group	215
Crystal form A	A
		B crystal form	A

And (4) conclusion: the crystal form of the invention obviously improves the EPO level of Balb/C mouse blood plasma, which shows that the crystal form of the invention can obviously promote the EPO expression of Balb/C mice.

Example 17: the crystal form of the invention has the effect of improving the hemoglobin level of rats

Animals: wistar rat, male, 180-; purchased from experimental animal technology limited of Viton Lihua, Beijing, SPF grade; animal production license number: SCXK (Jing) 2016-; issuing a certificate unit: the scientific and technical committee of Beijing.

Grouping: normal control group (0.5% CMC-Na vehicle); form a group prepared in example 3 (1.0, 2.5, 5.0mg/kg) and form B group prepared in example 10 (1.0, 2.5, 5.0mg/kg), 10 animals per group.

Administration: one week after acclimatization of rats, gavage was started once a day. Normal control group was given vehicle 0.5% CMC-Na daily; the group of crystalline forms of the invention was given daily doses of the suspension. The administration was continued for 14 days.

The main detection indexes are as follows: hemoglobin levels, a conventional indicator of blood, were measured using a fully automatic hematology analyzer (Mythic22, alfine, switzerland).

As a result: after 14 days of continuous administration, the A crystal form and the B crystal form of the invention significantly improve the hemoglobin level of rats (table 8), wherein A represents more than 190 g/L; b represents 175-190 g/L; c represents 160-175 g/L; d represents 150-160 g/L; e represents <150 g/L.

Table 8 elevation of hemoglobin levels in rats by the crystalline forms of the invention

	Hemoglobin (g/L)
		Normal control group	E
Crystal form A (1.0mg/kg)	C
		Crystal form A (2.5mg/kg)	B
Crystal form A (5.0mg/kg)	A
		Crystal form B (1.0mg/kg)	C
Crystal form B (2.5mg/kg)	B
		Crystal form B (5.0mg/kg)	A

And (4) conclusion: the crystal form of the invention has obvious promotion effect on hemoglobin of normal rats.

Example 18: drug effect research of 5/6 nephrectomy for establishing renal anemia rat model by crystal form pair

Molding: after one week of adaptive feeding, 10% chloral hydrate (0.3ml/kg) is used for intraperitoneal injection and anesthesia, prone position fixation, local skin preparation and conventional skin disinfection. 5/6 nephrectomy is carried out by two-step method, wherein a cut of 3-4cm is made on the right side obliquely and outwards (renal region), the right side kidney is fully exposed, the renal capsule is separated, the renal pedicle is ligated, when the kidney becomes dark due to ischemia, the right kidney is excised, sutured and disinfected. The 2 nd operation is performed 7-10 days later. The left side of the kidney is cut 3-4cm in a direction obliquely outward from the lower side (renal area) to fully expose the left side of the kidney, and the renal capsule is peeled off. Clamping the upper and lower poles of the kidney, rapidly cutting off the upper and lower poles and the outer edge of the left kidney when the clamped area becomes purple black due to ischemia, cutting off about 2/3 kidney tissues (the upper and lower poles cut off 1/3 of the kidney respectively), compressing the wound surface with gelatin sponge to stop bleeding, flushing with normal saline, injecting penicillin into the abdominal cavity, suturing the muscular layer and the skin, closing the abdominal cavity, and sterilizing. The two procedures were performed to remove about 5/6 kidney. After the rat revives, the rat is put into a single cage for feeding, and the respiratory tract is kept unobstructed. After the operation, the patient is fasted for 24 hours, and the skin incision, the mental state and the food and water intake conditions are closely observed after the operation without water prohibition. The sham group did not excise kidney tissue, and only given 2 anaesthesia and isolated double renal fat capsules.

Blood samples were collected by orbital bleeding every 2 weeks after nephrectomy and tested for hematology (EDTA-2K anticoagulation) and renal function index creatinine and urea nitrogen (anticoagulation). The rats conforming to the characteristics of renal anemia are considered to be successfully modeled and are included in the subsequent intragastric administration test. Rats successfully molded were included in the compound in vivo pharmacodynamic evaluation study.

Grouping: sham group (0.5% CMC-Na vehicle); model group (0.5% CMC-Na vehicle); form a obtained in example 3 (0.5, 1.0 and 2.5mg/kg) and form B obtained in example 10 (0.5, 1.0 and 2.5mg/kg), 10 animals per group.

Administration: the preparation is administered by intragastric administration once a day. Sham and model groups were given daily vehicle 0.5% CMC-Na; the group of crystalline forms of the invention was given daily doses of the suspension.

Molding detection indexes: (1) detecting the blood routine index (EDTA anticoagulation), the erythrocyte count and the hemoglobin level by a full-automatic blood cell analyzer (Mythic22, Osofield, Switzerland); (2) renal function indexes: the levels of serum urea nitrogen (BUN) and creatinine (Scr) were measured using a colorimetric assay kit (Beijing Keliyida medical technology, Inc.).

The main detection indexes of the drug effect evaluation are as follows: the blood routine index and the hemoglobin level are detected by a Mythic22 full-automatic blood cell analyzer.

As a result: as shown in table 9, the crystalline form of the present invention significantly increased hemoglobin levels in the nephrectomized rat model after 4 weeks of administration. Wherein A represents more than 190 g/L; b represents 170-190 g/L; c represents 150-170 g/L; d represents 140-150 g/L; e represents <130 g/L.

TABLE 9 elevation of hemoglobin level in renal anemia rat by the crystalline form of the present invention

	Hemoglobin (g/L)
		Model set	E
Artificial operation group	D
		Crystal form A (0.5mg/kg)	D
Crystal form A (1.0mg/kg)	B
		Crystal form A (2.5mg/kg)	A
Crystal form B (0.5mg/kg)	D
		Crystal form B (1.0mg/kg)	B
Crystal form B (2.5mg/kg)	A

And (4) conclusion: the crystal form can obviously improve the hemoglobin level of a nephrectomized rat model, and has obvious improvement effect on anemia of the nephrectomized rat model.

Claims

1. A type-A crystal of 1- (6- (2-oxa-8-azaspiro [4.5] decan-8-yl) pyrimidin-4-yl) -4- (1H-1,2, 3-triazol-1-yl) -1, 2-dihydro-1H-pyrazole-3-phenol sodium characterized in that its X-ray powder diffraction pattern comprises characteristic peaks at 2-theta diffraction angles of 4.398 DEG + -0.2 DEG, 8.740 DEG + -0.2 DEG, 12.949 DEG + -0.2 DEG, 13.352 DEG + -0.2 DEG, 16.353 DEG + -0.2 DEG, 18.761 + -0.2 DEG, 22.542 DEG + -0.2 DEG, 23.564 DEG + -0.2 DEG, 26.564 DEG + -0.2 DEG, 29.563 DEG + -0.2 deg.

2. The form A crystal of sodium 1- (6- (2-oxa-8-azaspiro [4.5] decan-8-yl) pyrimidin-4-yl) -4- (1H-1,2, 3-triazol-1-yl) -1, 2-dihydro-1H-pyrazol-3-olate according to claim 1, characterized by the X-ray powder diffraction pattern shown in FIG. 1.

3. A B-type crystal of sodium 1- (6- (2-oxa-8-azaspiro [4.5] decan-8-yl) pyrimidin-4-yl) -4- (1H-1,2, 3-triazol-1-yl) -1, 2-dihydro-1H-pyrazole-3-olate characterized by an X-ray powder diffraction pattern comprising characteristic peaks at 2-theta diffraction angles of 7.460 DEG + -0.2 DEG, 16.188 DEG + -0.2 DEG, 16.423 DEG + -0.2 DEG, 17.072 DEG + -0.2 DEG, 21.158 DEG + -0.2 DEG, 22.554 DEG + -0.2 DEG, 23.677 DEG + -0.2 deg.

4. Type B crystals of sodium 1- (6- (2-oxa-8-azaspiro [4.5] decan-8-yl) pyrimidin-4-yl) -4- (1H-1,2, 3-triazol-1-yl) -1, 2-dihydro-1H-pyrazole-3-olate according to claim 3 characterized in that their X-ray powder diffraction pattern comprises characteristic peaks at the 2 θ diffraction angles of 7.460 ° ± 0.2 °, 9.368 ° ± 0.2 °, 9.636 ° ± 0.2 °, 16.188 ° ± 0.2 °, 16.423 ° ± 0.2 °, 17.072 ° ± 0.2 °, 17.473 ° ± 0.2 °, 21.158 ° ± 0.2 °, 22.554 ° ± 0.2 °, 23.677 ° ± 0.2 °, 29.368 ° ± 0.2 °.

5. The form B crystal of sodium 1- (6- (2-oxa-8-azaspiro [4.5] decan-8-yl) pyrimidin-4-yl) -4- (1H-1,2, 3-triazol-1-yl) -1, 2-dihydro-1H-pyrazol-3-olate according to claim 3 or 4, characterized by the X-ray powder diffraction pattern shown in FIG. 4.

6. A process for preparing form a crystals of sodium 1- (6- (2-oxa-8-azaspiro [4.5] decan-8-yl) pyrimidin-4-yl) -4- (1H-1,2, 3-triazol-1-yl) -1, 2-dihydro-1H-pyrazol-3-olate according to claim 1 or 2, comprising the steps of:

7. The production method according to claim 6, wherein, in step 1), 2- (6- (2-oxa-8-azaspiro [4.5] decan-8-yl) pyrimidin-4-yl) -4- (1H-1,2, 3-triazol-1-yl) -1, 2-dihydro-3H-pyrazol-3-one is further dissolved in an organic solvent by heating preferably at 55 ℃ to 60 ℃, followed by hot filtration, and a sodium salt solution is added dropwise to the filtrate.

8. The production method according to claim 6 or 7, wherein, in step 1), the solution obtained as sodium 1- (6- (2-oxa-8-azaspiro [4.5] decan-8-yl) pyrimidin-4-yl) -4- (1H-1,2, 3-triazol-1-yl) -1, 2-dihydro-1H-pyrazol-3-olate is further incubated for preferably 15 to 30 minutes.

9. The preparation method according to any one of claims 6 to 8, wherein the organic solvent is selected from 1, 4-dioxane, tetrahydrofuran, acetonitrile, acetone or a mixture thereof, or a mixture of the above solvent and water.

10. The production method according to any one of claims 6 to 9, wherein the organic solvent is a mixture of tetrahydrofuran and water, and the volume ratio of tetrahydrofuran to water is 10:1 to 50:1, preferably 20:1 to 40: 1.

11. The production method according to any one of claims 6 to 10, wherein the basic condition is selected from triethylamine, N-diisopropylethylamine, pyridine, dimethylamine, aqueous ammonia, preferably triethylamine; the sodium salt is selected from sodium bicarbonate, sodium hydrogen, sodium carbonate, sodium methoxide, sodium ethoxide and sodium hydroxide, preferably sodium hydroxide, and the concentration of the sodium hydroxide is preferably 20-45%.

12. A process for preparing form B crystals of sodium 1- (6- (2-oxa-8-azaspiro [4.5] decan-8-yl) pyrimidin-4-yl) -4- (1H-1,2, 3-triazol-1-yl) -1, 2-dihydro-1H-pyrazol-3-olate according to any one of claims 3 to 5, comprising the steps of:

2) stirring the solution of 1- (6- (2-oxa-8-azaspiro [4.5] decan-8-yl) pyrimidin-4-yl) -4- (1H-1,2, 3-triazol-1-yl) -1, 2-dihydro-1H-pyrazol-3-olate sodium obtained in the step 1) at 0 ℃ to 10 ℃ for crystallization, preferably for 2 to 20 hours, and filtering the crystals;

13. The production method according to claim 12, wherein, in step 1), 2- (6- (2-oxa-8-azaspiro [4.5] decan-8-yl) pyrimidin-4-yl) -4- (1H-1,2, 3-triazol-1-yl) -1, 2-dihydro-3H-pyrazol-3-one is further dissolved in an organic solvent by heating preferably at 55 ℃ to 60 ℃, followed by hot filtration, and a sodium salt solution is added dropwise to the filtrate.

14. The production method according to claim 12 or 13, wherein, in step 1), the solution obtained to obtain 1- (6- (2-oxa-8-azaspiro [4.5] decan-8-yl) pyrimidin-4-yl) -4- (1H-1,2, 3-triazol-1-yl) -1, 2-dihydro-1H-pyrazol-3-ol sodium is further incubated for preferably 15 to 30 minutes.

15. The production method according to any one of claims 12 to 14, wherein, in step 2), the sodium 1- (6- (2-oxa-8-azaspiro [4.5] decan-8-yl) pyrimidin-4-yl) -4- (1H-1,2, 3-triazol-1-yl) -1, 2-dihydro-1H-pyrazol-3-olate solution obtained in step 1) is first crystallized with stirring at room temperature, preferably for 1 to 2 hours, and then at 0 ℃ to 10 ℃ for preferably 2 hours, and the crystals are filtered.

16. The production method according to any one of claims 12 to 15, wherein the organic solvent is selected from a C1-C4 alcohol, 1, 4-dioxane, acetonitrile, acetone, or a mixture thereof, or a mixture of the above solvent and water; preferably a C1-C4 alcohol or a mixture of a C1-C4 alcohol and water; the C1-C4 alcohol is preferably methanol or ethanol.

17. The preparation method according to any one of claims 12 to 16, wherein the organic solvent is a mixture of a C1-C4 alcohol and water, and the volume ratio of the C1-C4 alcohol to water is from 1:2 to 40:1, preferably from 1:2 to 15: 1.

18. The production method according to any one of claims 12 to 17, wherein the organic solvent is a mixture of methanol and water, and the volume ratio of methanol to water is from 1:2 to 15: 1.

19. The production method according to any one of claims 12 to 18, wherein the basic condition is selected from triethylamine, N-diisopropylethylamine, pyridine, dimethylamine, aqueous ammonia, preferably triethylamine; the sodium salt is selected from sodium bicarbonate, sodium hydrogen, sodium carbonate, sodium methoxide, sodium ethoxide and sodium hydroxide, preferably sodium hydroxide, and the concentration of the sodium hydroxide is preferably 10-45%.

20. A pharmaceutical composition comprising the form a crystal of sodium 1- (6- (2-oxa-8-azaspiro [4.5] decan-8-yl) pyrimidin-4-yl) -4- (1H-1,2, 3-triazol-1-yl) -1, 2-dihydro-1H-pyrazol-3-olate according to any one of claims 1 or 2 as an active ingredient together with a pharmaceutically acceptable carrier.

21. A pharmaceutical composition comprising the crystal of 1- (6- (2-oxa-8-azaspiro [4.5] decan-8-yl) pyrimidin-4-yl) -4- (1H-1,2, 3-triazol-1-yl) -1, 2-dihydro-1H-pyrazol-3-ol sodium form B according to any one of claims 3 to 5 as an active ingredient together with a pharmaceutically acceptable carrier.

22. Use of a form a crystal of sodium 1- (6- (2-oxa-8-azaspiro [4.5] decan-8-yl) pyrimidin-4-yl) -4- (1H-1,2, 3-triazol-1-yl) -1, 2-dihydro-1H-pyrazol-3-olate according to any one of claims 1 or 2 or a pharmaceutical composition according to claim 20 for the preparation of a medicament for the prevention and/or treatment of a disease associated with PHD activity, preferably selected from cardiovascular diseases, in particular cardiac insufficiency, coronary heart disease, angina pectoris, myocardial infarction, stroke, arteriosclerosis, primary, pulmonary and malignant hypertension and peripheral arterial occlusive diseases; chronic kidney disease; hematopoietic disorders, such as primary anemia, renal anemia, and anemia associated with neoplastic disease (particularly chemotherapy-induced anemia); infection (particularly HIV infection) or other inflammatory diseases, such as rheumatoid arthritis; anemia due to blood loss, iron deficiency anemia, vitamin deficiency anemia (e.g. due to vitamin B12 deficiency or due to folate deficiency), aplastic anemia and aplastic anemia or hemolytic anemia, anemia due to iron utilization disorders (iron-loss anemia) or due to other endocrine disorders (e.g. hypothyroidism); post-surgical procedures associated with ischemic conditions and their subsequent symptoms, particularly cardiac interventions using heart-lung machines (e.g. bypass surgery, heart valve transplantation), carotid interventions, aortic interventions and interventions using instrument openings or penetrating calvarial; surgical wound healing; cancer and the damage of the health state that occurs during the treatment of cancer, in particular after treatment with cytostatics, antibiotics and radiation, diseases ranging from the rheumatic forms and other diseases considered as autoimmune diseases, in particular the damage of the health state that occurs during the pharmacological treatment of such diseases; continuous symptoms of acute and prolonged cerebral ischemic conditions (e.g. stroke, childbirth asphyxia).

23. Use of crystals of sodium 1- (6- (2-oxa-8-azaspiro [4.5] decan-8-yl) pyrimidin-4-yl) -4- (1H-1,2, 3-triazol-1-yl) -1, 2-dihydro-1H-pyrazol-3-olate according to any one of claims 3 to 5 or of a pharmaceutical composition according to claim 21 for the preparation of a medicament for the prevention and/or treatment of a disease associated with PHD activity, preferably selected from cardiovascular diseases, in particular cardiac insufficiency, coronary heart disease, angina pectoris, myocardial infarction, stroke, arteriosclerosis, primary, pulmonary and malignant hypertension and peripheral arterial occlusive diseases; chronic kidney disease; hematopoietic disorders, such as primary anemia, renal anemia, and anemia associated with neoplastic disease (particularly chemotherapy-induced anemia); infection (particularly HIV infection) or other inflammatory diseases, such as rheumatoid arthritis; anemia due to blood loss, iron deficiency anemia, vitamin deficiency anemia (e.g. due to vitamin B12 deficiency or due to folate deficiency), aplastic anemia and aplastic anemia or hemolytic anemia, anemia due to iron utilization disorders (iron-loss anemia) or due to other endocrine disorders (e.g. hypothyroidism); post-surgical procedures associated with ischemic conditions and their subsequent symptoms, particularly cardiac interventions using heart-lung machines (e.g. bypass surgery, heart valve transplantation), carotid interventions, aortic interventions and interventions using instrument openings or penetrating calvarial; surgical wound healing; cancer and the damage of the health state that occurs during the treatment of cancer, in particular after treatment with cytostatics, antibiotics and radiation, diseases ranging from the rheumatic forms and other diseases considered as autoimmune diseases, in particular the damage of the health state that occurs during the pharmacological treatment of such diseases; continuous symptoms of acute and prolonged cerebral ischemic conditions (e.g. stroke, childbirth asphyxia).