CN117257810B

CN117257810B - Application of imidazopyridine derivative in preparation of medicines for treating or preventing myeloproliferative neoplasms

Info

Publication number: CN117257810B
Application number: CN202311228111.3A
Authority: CN
Inventors: 姜德建; 刘学武; 信红亚; 梁广; 伍文奇; 江芝
Original assignee: Wenzhou Guangcheng Biotechnology Co ltd; Hunan Prima Pharmaceutical Research Center Co ltd
Current assignee: Wenzhou Guangcheng Biotechnology Co ltd; Hunan Prima Pharmaceutical Research Center Co ltd
Priority date: 2023-09-22
Filing date: 2023-09-22
Publication date: 2024-06-18
Anticipated expiration: 2043-09-22
Also published as: CN117257810A

Abstract

The invention belongs to the technical field of medicines, and provides an imidazole pyridine derivative and pharmaceutically acceptable salts thereof, a pharmaceutical composition containing the compound, and application of the compound in preparing medicines for treating and preventing myeloproliferative neoplasms, wherein the myeloproliferative neoplasms comprise polycythemia vera, thrombocythemia and myelofibrosis. The imidazole pyridine derivative is shown as a formula (I).

Description

Application of imidazopyridine derivative in preparation of medicines for treating or preventing myeloproliferative neoplasms

Technical Field

The invention relates to the technical field of medicines, in particular to application of a compound (code number X22) of a formula (I) or pharmaceutically acceptable salt thereof in preparing a medicament for treating or preventing myeloproliferative neoplasms.

Background

It has now been found that molecular markers associated with MPN are mainly JAK2, CARL, MPL, and the like. In 2005, the JAK2 gene acquired mutation-JAK 2 ^V617F point mutation in the disease is found, and the mutation is proved to have important diagnostic and prognostic significance in MPN. Over 90% of PV patients and about 60% of ET and PMF patients all have the V617F site mutation. In addition, in recent years, studies have revealed that erythropoietin receptor (MPL) mutations and somatic Calreticulin (CALR) mutations, such as MPL515 site mutations, and frame shift mutations of CALR exon 9, are found in MPN patients negative for JAK2V617F mutation.

JAK2, MPL and CALR mutations were all functionally verified and sufficient to generate MPN phenotypes in mice. All of these mutations have a functional effect on JAK2 signaling and the downstream transcription factor STAT. Studies of gene expression profiles clearly show that these mutations can see an activated JAK2 signal in all MPN patients. The JAK-STAT signaling pathway plays an important role in the primary signaling cascade of MPNs. The JAK2 gene encodes a tyrosine kinase receptor in the JAK-STAT pathway. The V617F mutation of the JAK2 gene in a human body can cause continuous activation and enhancement of JAK2 kinase and downstream signal transduction pathways, so that malignant proliferation and apoptosis inhibition of cells can be caused, and finally, polycythemia Vera (PV) and the like can be caused. JAK2 mutations have been identified as a potential molecular mechanism for primary polycythemia (ET), many PMF and PT cases.

After these mutations were found, pharmaceutical companies and researchers have obtained small molecule inhibitors of JAK2 signaling (JAKi) for treatment of MPNs ^[7]. JAKi inhibit JAK2 and STAT phosphorylation thereby reducing tumor cell proliferation and inducing apoptosis. The U.S. Food and Drug Administration (FDA) approves Ruxolitinib (RUX) for treatment of moderate and high risk Myelofibrosis (MF), including PMF and post-PV/post-ET MF, 11-16 2011. Subsequently, on month 4 of 2014, the FDA approved RUX treatment for patients with inadequate or intolerance to Hydroxyurea (HU).

JAKs inhibitors in clinical trials revealed different toxicity profiles that might be relevant to them, with enhanced specific inhibition being our aim of investigation. Specific JAK2 inhibitors, particularly against mutation sites, have received increasing attention in recent years.

The small molecule compound 4- {5' - [3' -propyl-2 ' - [3' - (1 ' -methyl) indolyl ] -imidazo [4,5-b ] pyridine ] yl } morpholin, the compound of formula (I), code X22, is a reported existing compound, and is known by search, application number 202010059361.9 discloses the application of X22 in preparing a medicament for treating or preventing diseases related to kidneys, application number 2012104847041 discloses the application of X22 in preparing a medicament for treating inflammation (such as acute inflammation or chronic inflammation) or diseases related to inflammation, and the application of X22 in preparing a medicament for treating or preventing myeloproliferative neoplasms is not discovered.

Disclosure of Invention

In view of the deficiencies of the prior art, it is an object of the present invention to provide a novel use of 4- {5' - [3' -propyl-2 ' - [3"- (1" -methyl) indolyl ] -imidazo [4,5-b ] naphthyridinyl } morpholines, compounds of formula (I), code X22. The invention is verified on various MPNs kinds of cells, in particular to evaluation on erythroleukemia cell line HEL of a human with natural mutation of JAK2 ^V617F, the selectivity of the compound to a JAK2 ^V617F mutation site is examined, the compound with better inhibition effect on the enhancement of JAK2 kinase activity caused by the mutation of the site is sought, and pharmacodynamics evaluation is carried out through an erythrocytosis model established by the cells.

The technical scheme of the invention is as follows:

the invention discloses an application of an imidazole pyridine derivative or a pharmaceutically acceptable salt thereof in preparing a medicament for treating or preventing myeloproliferative neoplasms, wherein the imidazole pyridine derivative is shown as a formula (I):

The invention also discloses application of the pharmaceutical composition containing the compound shown in the formula (I) in preparing medicines for treating or preventing myeloproliferative neoplasms.

Preferably, the pharmaceutical composition containing the compound represented by the formula (I) is formulated into tablets, capsules, oral liquids, injections, powders, ointments or topical medicinal liquids.

Preferably, the myeloproliferative neoplasm (MPN) includes Polycythemia Vera (PV), thrombocythemia (ET) and myelofibrosis (PMF). Over 90% of patients have JAKV617F gene mutation. The marketed myeloproliferative neoplasms include Lu Ke tinib, phenanthrene Zhuo Tini, which are inhibitors of JAK1/2/3, only with selective differences. JAK1/3 is mainly associated with inflammation and JAK2 is associated with the hematopoietic system.

Preferably, in the use of the present invention, the clinical manifestations of myeloproliferative neoplasms are splenomegaly, erythrocytosis, thrombocytosis and myelofibrosis.

The medicament for use in the present invention will generally also contain a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier used refers to nontoxic fillers, stabilizers, diluents, adjuvants or other formulation adjuvants. In addition, the pharmaceutical composition of the present invention may further contain other auxiliary materials such as flavoring agents, sweeteners, etc. The pharmaceutical composition may be formulated into various dosage forms according to the therapeutic purpose and the need of the administration route, preferably the composition is in the form of unit administration dosage such as lyophilized preparation, tablet, capsule, powder, emulsion, water injection or spray, more preferably the pharmaceutical composition is in the form of injection (such as lyophilized powder injection) or oral dosage form (such as tablet, capsule). The medicament may be administered by conventional routes, in particular enterally, e.g. orally, e.g. in the form of tablets or capsules, or parenterally, e.g. in the form of injectable solutions or suspensions, topically, e.g. in the form of lotions or gels, or in the form of nasal or test agents.

Pharmaceutically acceptable salts are those salts including those with mineral acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid and the like; salts with organic acids such as acetic acid, oxalic acid, tartaric acid, maleic acid, citric acid, ascorbic acid, and the like; and salts formed from elemental anions such as chlorine, bromine, and iodine; "pharmaceutical excipients" refers to pharmaceutical carriers conventional in the pharmaceutical arts and includes, but is not limited to, any adjuvant, carrier, excipient, glidant, sweetener, diluent, preservative, dye/colorant, flavoring agent, surfactant, wetting agent, dispersing agent, suspending agent, stabilizer, isotonic agent, solvent or emulsifying agent approved by the U.S. food and drug administration as acceptable for use in humans or livestock.

Compared with the prior art, the invention has the advantages that:

1. Compared with the similar medicines Lu Ke and Lu Ke, the compound X22 has lower effective dose, stronger medicinal effect and less toxic effect.

2. In terms of effect: (1) X22 can significantly inhibit JAK1, JAK2, JAK3 activity, and has strongest inhibitory activity on JAK2 and stronger selectivity; the inhibition activity of X22 on JAK2 is obviously stronger than Lu Ke tenib and phenanthroline tenib. (2) The X22 can obviously reduce the erythrocyte number and the hemoglobin concentration of a mouse model with polycythemia vera, obviously reduce the gene load of JAK2 ^V617F, obviously reduce the weight of spleen, obviously reduce the increase of a bone marrow erythrocyte system and obviously improve the fibrosis degree of bone marrow. (3) X22 can be absorbed into blood of rats, the bioavailability is 6.38-48.14%, and the drug can support the patent drug of X22 in terms of drug generation; (4) The in vitro Ames test result of X22 is negative, which indicates that the risk of mutagenicity is low; the toxicity test of repeated administration of the rat for 1 month does not show obvious toxic reaction, which indicates that the general toxicity is lower; two safety evaluation test results can support the patent medicine of X22 in terms of safety.

The detailed structure of the present invention is further described below with reference to the accompanying drawings and detailed description.

Drawings

FIG. 1 is a graph of the change in blood cells and hemoglobin.

FIG. 2 is a bar graph of TNF- α content;

FIG. 3 is a bar graph of IL-6 content;

FIG. 4 is a spleen factor histogram;

FIG. 5 is a map of spleen JAK2 ^V617F gene load

FIG. 6 is a bar graph of spleen p-JAK2/JAK2 protein expression ratio;

FIG. 7 is a bar graph of spleen p-STAT3/STAT3 protein expression ratio;

FIG. 8 is a bar graph of spleen p-STAT5/STAT55 protein expression ratio;

FIG. 9 is a diagram of spleen pathology in a JAK2 ^V617F mutant mouse model; and (3) injection: a to G are a normal control group, a model control group, a ruxotinib phosphate group, a phenanthroline tinib group and an X22 low, medium and high dose group in sequence;

FIG. 10 is a diagram of bone marrow pathology in a JAK2 ^V617F mutant mouse model; and (3) injection: a to G are a normal control group, a model control group, a ruxotinib phosphate group, a phenanthroline tinib group and an X22 low, medium and high dose group in sequence;

Fig. 11 is a photograph of brain HE staining of a blank (animal No. 1F 03) administered for 4 weeks, x 200. The grey matter and white matter structures of the brain are clear, the neuron structure is normal, degeneration and loss are not seen, and inflammatory cell infiltration is not seen;

fig. 12 is a photograph of a control group (animal No. 1F 03) stained with cerebellum HE for 4 weeks, x 200. The cerebellar cortex and medulla are clear in structure, and cerebellar atrophy and inflammatory cell infiltration are not seen;

Fig. 13 is a photograph of liver HE staining of a blank (animal No. 1F 03) administered for 4 weeks, x 200. The hepatic cells are arranged orderly, the hepatic cells are not denatured or necrotic, and the hepatic sinus is not blood stasis;

Fig. 14 is a photograph of a blank (animal No. 1F 03) kidney HE staining, 4 weeks after administration, x 200. The kidney cortex and medulla have clear structure, the nephron has normal morphological structure, and the interstitium is not infiltrated by inflammatory cells;

Fig. 15 is a photograph of a blank (animal No. 1F 03) heart HE stained for 4 weeks of dosing, x 200. The myocardial fiber is uniformly dyed, the transverse lines are clear, denaturation and necrosis are not seen, and bleeding and inflammatory cell infiltration are not seen in the interstitium;

FIG. 16 is a photograph of HE staining of the lungs of a blank (animal No. 1F 03) given 4 weeks of HE staining, x 200. The alveolar wall has normal structure, the interstitium is not infiltrated by inflammatory cells, and obvious exudates are not found in the alveolar space;

fig. 17 is a photograph of a spleen HE staining of a blank (animal No. 1F 03) administered for 4 weeks, x 200. Spleen white marrow and red pith knot are clear in structure, and blood stasis and fibrous tissue hyperplasia are not seen;

Fig. 18 is a thymus HE staining photograph of a blank (animal No. 1F 03) administered for 4 weeks, x 200. The cortex and medulla structures are clear, inflammatory cell infiltration is not seen, and focal necrosis is not seen;

Fig. 19 is a photograph of ovaries HE staining of a blank (animal No. 1F 03) administered for 4 weeks, x 200. The morphological structure of each level of follicle is clear, and the degeneration necrosis and inflammatory cell infiltration are not seen. The stroma is not infiltrated by bleeding and inflammatory cells;

FIG. 20 is a photograph of a blank (animal No. 1F 03) stained for bone marrow HE at 4 weeks of administration, x 200. Bone marrow hematopoietic granulocytes were not abnormal;

Fig. 21 is a photograph of a blank (animal No. 1M 03) testis HE stained for 4 weeks of dosing, x 200. The arrangement of the sperm cells in different development stages in the testis seminiferous tubules is clear, the morphology is normal, and the interstitial tissue is not infiltrated by inflammatory cells;

Fig. 22 is a photograph of HE staining of the brain of the X22 high dose group (animal No. 4F 03) administered for 4 weeks, X200. The grey matter and white matter structures of the brain are clear, the neuron structure is normal, degeneration and loss are not seen, and inflammatory cell infiltration is not seen;

fig. 23 is a plot of cerebellum HE staining for the X22 high dose group (animal No. 4F 03) administered for 4 weeks, X200. The cerebellar cortex and medulla are clear in structure, and cerebellar atrophy and inflammatory cell infiltration are not seen;

FIG. 24 is a photograph of HE staining of the liver of the X22 high dose group (animal No. 4F 03) given 4 weeks of HE staining, X200. The hepatic cells are arranged orderly, the hepatic cells are not denatured or necrotic, and the hepatic sinus is not blood stasis;

fig. 25 is a photograph of the HE staining of the kidneys of the X22 high dose group (animals No. 4F 03) administered for 4 weeks X200. The kidney cortex and medulla have clear structure, the nephron has normal morphological structure, and the interstitium is not infiltrated by inflammatory cells;

Fig. 26 is a photograph of HE staining of the heart of the X22 high dose group (animal No. 4F 03) administered for 4 weeks, X200. The myocardial fiber is uniformly dyed, the transverse lines are clear, denaturation and necrosis are not seen, and bleeding and inflammatory cell infiltration are not seen in the interstitium;

Fig. 27 is a photograph of HE staining of the lungs of the X22 high dose group (animal No. 4F 03) administered for 4 weeks X200. The alveolar wall has normal structure, the interstitium is not infiltrated by inflammatory cells, and obvious exudates are not found in the alveolar space;

Fig. 28 is a photograph of spleen HE staining for the X22 high dose group (animal No. 4F 03) administered for 4 weeks, X200. Spleen white marrow and red pith knot are clear in structure, and blood stasis and fibrous tissue hyperplasia are not seen;

Fig. 29 is a graphic image of thymus HE staining of the X22 high dose group (animal No. 4F 03) administered for 4 weeks, ×200. The cortex and medulla structures are clear, inflammatory cell infiltration is not seen, and focal necrosis is not seen;

fig. 30 is a photograph of ovaries HE staining of the X22 high dose group (animal No. 4F 03) administered for 4 weeks X200. The morphological structure of each level of follicle is clear, and the degeneration necrosis and inflammatory cell infiltration are not seen. The stroma is not infiltrated by bleeding and inflammatory cells;

FIG. 31 is a photograph of a X22 high dose group (animal No. 4F 03) stained for sternum marrow HE, administered for 4 weeks, X200. Bone marrow hematopoietic granulocytes were not abnormal;

fig. 32 is a photograph of the HE staining of testis of the X22 high dose group (animal No. 4M 03) administered for 4 weeks, X200. The arrangement of the sperm cells in different development stages in the testis seminiferous tubules is clear, the morphology is normal, and the interstitial tissue is not infiltrated by inflammatory cells;

Fig. 33 is a photograph of brain HE staining of a blank (animal No. 1F 03) administered for 4 weeks, x 200. The grey matter and white matter structures of the brain are clear, the neuron structure is normal, degeneration and loss are not seen, and inflammatory cell infiltration is not seen;

Fig. 34 is a photograph of a blank (animal No. 1F 03) cerebellum HE staining, 4 weeks after administration, x 200. The cerebellar cortex and medulla are clear in structure, and cerebellar atrophy and inflammatory cell infiltration are not seen;

Fig. 35 is a photograph of liver HE staining of a blank (animal No. 1F 03) administered for 4 weeks, x 200. The hepatic cells are arranged orderly, the hepatic cells are not denatured or necrotic, and the hepatic sinus is not blood stasis;

Fig. 36 is a photograph of a blank (animal No. 1F 03) kidney HE staining, 4 weeks after administration, x 200. The kidney cortex and medulla have clear structure, the nephron has normal morphological structure, and the interstitium is not infiltrated by inflammatory cells;

FIG. 37 is a photograph of a blank (animal No. 1F 03) stained heart HE for 4 weeks at x 200. The myocardial fiber is uniformly dyed, the transverse lines are clear, denaturation and necrosis are not seen, and bleeding and inflammatory cell infiltration are not seen in the interstitium;

Fig. 38 is a photograph of a lung HE staining of a blank (animal No. 1F 03) administered for 4 weeks, x 200. The alveolar wall structure is normal, the interstitium is not infiltrated by inflammatory cells, and no obvious exudates are found in the alveolar space.

Detailed Description

The invention is further illustrated in the following examples. These examples are only for illustrating technical effects of the present invention and are not intended to limit the scope of the present invention.

Pharmacological experiments:

example 1: JAKs kinase Activity assay

(1) The experimental method is divided into a control group (control) and a sample group (sample), and the experimental system is provided with 4 control groups, namely kinase complete activity control (MAX), kinase-free negative control (NEG), pure detection system control and buffer control. The sample group contains TK Substrate-biotin, JAK kinase, ATP and a compound I (a compound shown as (I), X22), which can be obtained directly through purchase or prepared through the existing synthetic method.

In this experiment, 10. Mu.l of a kinase reaction system was selected, and each system contained 10ng of JAK1 kinase and 3.92. Mu.M ATP in the JAK1 inhibitor screening. In the screening of the JAK2 inhibitor, each system contains 0.04ng of JAK2 kinase and 3.96 mu M of ATP. In the screening of the JAK3 inhibitor, each system contains 0.12ng of JAK3 kinase and 1.43 mu M of ATP. The TK Substrate-biotin is 1. Mu.M, and the compound I is required to be prepared at the required concentration. Specifically comprising preparation of ① kinase reaction buffer: 1ml of 5 XKinase Buffer was diluted to 1X in 4ml of double distilled water, 5. Mu.l of 1M DTT and 25. Mu.l of 1M MgCl ₂ (JAK 1 reaction Buffer plus 5. Mu.l of 1M MnCl ₂) were added and stored at room temperature. ② Configuration of compounds to be screened: the sample compound was dissolved in DMSO to prepare a mother solution having a concentration of 40mM, and the compound was diluted and prepared with a kinase reaction buffer, and the final concentration of the reaction of the compound I was 10. Mu.M, 1. Mu.M, 0.1. Mu.M, 0.01. Mu.M, 0.001. Mu.M. ③ The above components were added sequentially to a black 384-well microplate to make the reaction system 10. Mu.l. The fluorescence detection uses 330nm excitation light to detect the emission intensities of 665nm and 620nm, and the enzyme label instrument automatically calculates ratio=665/620×10000. ④ Data analysis: inhibition Ratio% = (Ratio MAX-Ratio sample)/(Ratio MAX-Ratio NEG) 100.

(2) Experimental results

Compound I has a better inhibitory effect on the kinase activity of JAK2, with IC ₅₀ of 0.15 μm. The inhibitory activity on both JAK1 and JAK3 is significantly weaker than JAK 2. The selectivity of X22 to JAK2 was 400-fold and 480-fold that of JAK1 and JAK2, the selectivity of Lu Ke to JAK2 was 1.8-fold and 7.3-fold that of JAK1 and JAK2, respectively, and the selectivity of phenanthrene Zhuo Tini to JAK2 was 15.2-fold and 54-fold that of JAK1 and JAK2, respectively, suggesting that the selectivity of X22 to JAK2 (vs JAK1 and JAK 3) was both stronger than Lu Ke and phenanthrene Zhuo Tini, as shown in table 1.

TABLE 1 IC of Compound I inhibiting JAKs kinase Activity ₅₀

Example 2: effect of X22 on murine polycythemia vera model: 90C 57BL/6 mice are selected, males are irradiated by adopting X rays (9 Gy/mouse), and after irradiation, bone marrow cells mutated by JAK2 ^V617F are injected into tail vein for bone marrow transplantation so as to induce a model of bone marrow hyperplasia of the mice, and 10 male C57BL/6 mice are taken as a normal control group without treatment. The number of red blood cells and the concentration of hemoglobin of mice are detected 8 weeks after the molding, 72 mice of a model with obviously increased number of red blood cells and concentration of hemoglobin are selected, and the mice are randomly divided into 6 groups according to the layering of the number of red blood cells, namely a model control group, lu Ke tinib group (10 mg/kg), a phenanthroline tinib group (50 mg/kg), and X22 low, medium and high dose groups (2.5, 5.0 and 10.0 mg/kg) and 12 mice/group respectively. Each group of mice was orally and gastro-orally administered with a corresponding concentration of the drug solution at 20mL/kg for 1 time/day for 4 weeks, and the normal control group and the model control group were administered with an equal volume of vehicle (20% HP-beta-CD).

Blood was collected at 2, 3 and 4 weeks of administration, and the number of erythrocytes and the concentration of hemoglobin were measured. The results, as seen in FIG. 1, show that the low, medium and high doses of X22 (5, 10 mg/kg) significantly reduced the number of erythrocytes and the hemoglobin concentration 2, 3, 4 weeks after the administration of polycythemia vera mice.

Blood was collected at 2, 3 and 4 weeks of administration, and the amounts of serum cytokines TNF- α and IL-6 were measured using ELISA kits. The results are shown in figures 2 and 3, and the results show that the high dosage (5, 10 mg/kg) of X22 can obviously reduce the content of serum TNF-alpha and IL-6.

The next day after the last dose, animals of each group were euthanized under anesthesia, dissected, spleens were taken and weighed, and spleen coefficients were calculated. See FIG. 4, which shows that low, medium and high doses of X22 (5, 10 mg/kg) significantly reduce spleen factor in polycythemia vera mice.

The next day after the last dose, animals of each group were euthanized under anesthesia, spleen and bone marrow were taken and divided into three parts, and one part was used to detect JAK 2V 617F gene load by PCR. See FIG. 5, which shows that medium, high doses of X22 (5, 10 mg/kg) significantly reduced spleen JAK 2V 617F gene load in polycythemia vera mice.

The next day after the last dose, animals of each group were euthanized, spleened and bone marrow were taken, divided into three, one was examined for expression of JAK2, STAT3, STAT5, p-JAK2, p-STAT3, p-STAT5 using Westernblot, and ratios of p-JAK2/JAK2, p-STAT3/STAT3 and p-STAT5/STAT5 were calculated and counted. The results, as shown in figures 6, 7 and 8, show that the medium and high doses (5, 10 mg/kg) of X22 can significantly reduce the ratio of p-JAK2/JAK2, p-STAT3/STAT3 and p-STAT5/STAT in a polycythemia vera mouse.

The next day after the last dose, animals of each group were euthanized under anesthesia, spleen and bone marrow were taken, divided into three parts, one part was fixed with formalin, HE stained, and observed for histopathological changes under a microscope. The results, as shown in figures 9 and 10, show that the high dose (5, 10 mg/kg) in X22 can significantly reduce the spleen and bone marrow lesion level of polycythemia vera mice, and improve the bone marrow fibrosis level.

Pharmacokinetics:

Example 3: rat plasma pharmacokinetics and bioavailability

The study used SD rats to be orally and gastrolavaged with different doses (2 mg/kg, 10mg/kg, 50 mg/kg) of X22, single administration; meanwhile, a venous administration group (1 mg/kg) is arranged, and the pharmacokinetic characteristics and the bioavailability of different doses of X22 after intragastric administration are examined by detecting the blood concentration of the X22 at different time points and calculating main pharmacokinetic parameters, so that a reference basis is provided for clinical experiments and clinical administration.

The concentration of X22 in SD rat plasma was determined by validated LC-MS/MS analysis. The plasma sample is deposited by methanol and then the supernatant liquid is taken for sample injection analysis. Chromatographic column Agela VenusilASB C (2.1X10 mm,5 μm) was used with 0.1% formic acid-water: gradient elution is carried out by taking 0.1% formic acid-acetonitrile solution as a mobile phase, the flow rate is 0.6mL/min, an electrospray ion source (ESI) is adopted, multiple Reaction Monitoring (MRM) scanning mode is adopted for monitoring, and a positive ion mode is adopted. The linear range of X22 is 1.000-1000 ng/mL, and the lower limit of quantification is 0.1000ng/mL.

The parallel drug administration experimental design is adopted, 32 SD rats are selected, and the weight is as follows: 210.9-242.9 g, and the male and female halves are orally irrigated with X22 with low, medium and high doses (2 mg/kg, 10mg/kg, 50 mg/kg) and the administration volume is 10mL/kg; a intravenous administration group (1 mg/kg) was set at the same time, and the administration volume was 2mL/kg. The intravenous administration group is collected before administration (0 h), 15min, 30min, 1h, 2h, 4h, 6h, 8h and 10h after administration; the low, medium and high dose groups were collected before administration (0 h), 15min, 30min, 1h, 2h, 4h, 6h, 8h and 10h blood, and the blood plasma was separated by centrifugation at 12000rpm for 2min after blood sampling from the jugular vein, and the blood plasma concentration was measured by the LC-MS/MS method. The concentration values measured were 80% below the lower limit of analyte quantification, i.e., as undetected or below the lower limit of quantification, zero before C _max reached the peak, and ND (Not detectable) after reaching the peak. Pharmacokinetic parameter calculations were performed using Phoenix winnonlin 8.0.0 software.

Test results: after the SD rat is orally and gastrolavaged, low, medium and high doses (2 mg/kg, 10mg/kg, 50 mg/kg) of X22 and 1mg/kg of X22 are intravenously administrated, the main pharmacokinetic parameters AUC _0-t、C_max、T_max、t_1/2 are shown in the following table, and the medicine that the X22 can be absorbed into blood of the rat and has the bioavailability of 6.38-48.14% and can support the X22 in terms of drug generation is shown in the following table.

TABLE 2 pharmacokinetic parameters of X22 in rats

Safety evaluation:

example 4: genotoxicity Ames test

The test used histidine auxotroph salmonella typhimurium strains TA97a, TA98, TA100, TA102 and TA1535. In the pre-test, TA100 strains were divided into vehicle control group (DMSO), X22 group (8, 40, 200, 1000, 5000 μg/dish); in the formal experiments, the 5 strains were divided into vehicle control group (DMSO), positive control group, and X22 group (100, 50, 25, 12.5, 6.25 μg/dish), respectively. Each group was given a different concentration of test/control solution, after incubation at 37 ℃ for 48h bacterial colony counts were performed and colony background was observed. The test was performed on two series of X22,3 parallel plates with S9 (+S9) and without S9 (-S9). And determining whether to repeat the test according to the result. The results showed that X22 was not mutated in the concentration range of 6.25-100. Mu.g/dish for all 5 strains of histidine-auxotrophic Salmonella typhimurium (TA 97a, TA98, TA100, TA102, TA 1535) and that Ames test results were negative.

Example 5: toxicity test of repeated administration for one month

The experiment observed long-term toxic response of SD rats following oral gavage administration of different doses of X22 for 1 month (4 weeks). SD rats were randomly divided into 4 groups of 30 animals each, male and female halves, according to sex body weight. The test was divided into a blank group and X22 low, medium and high dose groups (50, 100, 200 mg/kg), which are respectively 10, 20 and 40 times (in terms of body surface area) of the clinical intended dose of human, and the administration was performed by gastric lavage in a volume of 10mL/kg, 1 time a day, 7 days a week, and 1 month (4 weeks) in total. At the end of the administration (weekend 4) and at the end of the recovery (weekend 8), 80 and 40 SD rats were dissected as planned, each half of the male and female. The inspection items include: general clinical observations; body weight and food intake were measured; urine routine, hematology, blood biochemistry, coagulation, electrolyte examination, ophthalmic examination, bone marrow smear; measuring organ coefficients; histopathological examination, pharmacokinetic detection, and the like.

Referring to fig. 11-38, the results showed that SD rats were orally administered with the stomach for 1 month (4 weeks), during which no significant abnormalities were observed in the appearance signs, behavioral activities, general conditions of animals, etc. at each period of the animals in each of the administration dose groups compared with the contemporaneous blank control group. The low, medium and high doses (50, 100, 200 mg/kg) of X22 had no significant effect on rat body weight, feeding amount, hematology, coagulation, hematochemistry, electrolytes, urine convention, bone marrow cells, and no abnormal changes in organ weight (brain, heart, liver, spleen, thymus, kidney, adrenal gland, ovary, uterus, testis, epididymis) and organ tissues (including brain, spinal cord, pituitary gland, thymus, esophagus, trachea, thyroid/parathyroid gland, salivary gland, stomach, small intestine, large intestine, pancreas, liver, kidney, adrenal gland, spleen, lymph node, lung, aorta, heart, bladder, sternum, sciatic nerve and skeletal muscle, male animal testis, epididymis, prostate, female ovary and fallopian tube, uterus and cervix, vagina, ha gland, eye, seminal vesicle, skin and breast femur) were seen with respect to the subject.

Conclusion: SD rats were given X22 by continuous 1 menstrual lavage with no apparent toxic response dose (NOAEL) of 200mg/kg, approximately 40 times the pharmacodynamically equivalent dose.

The foregoing is a specific embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be able to apply equivalents and modifications to the technical solution and the concept thereof within the scope of the present invention as defined in the appended claims.

Claims

1. Use of an imidazopyridine derivative or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for the treatment or prophylaxis of myeloproliferative neoplasms, such as polycythemia vera, thrombocythemia and myelofibrosis; the imidazole pyridine derivative is 4- {5' - [3' -propyl-2 ' - [3' - (1 ' -methyl) indolyl ] -imidazo [4,5-b ] pyridine ] yl } morpholinine, and is shown as a formula (I):

（I）。

2. The use according to claim 1, characterized in that the pharmaceutical composition comprising the compound of formula (I) is used for the preparation of a medicament for the treatment or prevention of myeloproliferative neoplasms.

3. The use according to claim 2, wherein the pharmaceutical composition comprising the compound of formula (I) is formulated as a tablet, capsule, oral liquid, injection or powder.