Application of pyrimido pyrazole derivative in treatment of postmenopausal osteoporosis

Abstract

The invention relates to the technical field of biological medicines, in particular to application of pyrimido pyrazole derivatives in treatment of postmenopausal osteoporosis. The pyrimido pyrazole derivative is a derivative as shown in a formula I or a pharmaceutically acceptable salt thereof. Wherein R1 is as described in the claims and the specification. The pyridinopyrazole derivative is a derivative shown in a formula I or a pharmaceutically acceptable salt thereof, and can be used for preparing a medicine for treating and / or preventing osteoporosis, especially postmenopausal osteoporosis. # imgabs0 #

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to application of pyrimidopyrazole derivatives in treating postmenopausal osteoporosis. Background Art

[0002] In the prior art, osteoporosis is a common subclinical chronic bone metabolic disease, which is mainly characterized by low bone density, progressive bone loss and bone microstructure destruction, and is more common in middle-aged and elderly people.

[0003] Currently, the commonly used anti-osteoporosis drugs in clinical practice mainly include anti-bone resorption drugs (such as bisphosphonates, RANKL monoclonal antibodies) and bone formation-promoting drugs (such as parathyroid hormone analogs). However, current drug treatments face many limitations, including safety risks of long-term medication, many contraindications, easy rebound after discontinuation of medication, and high treatment costs. An ideal drug that is safe, reliable, precisely targeted and economical has not yet been obtained.

[0004] In the preclinical studies on the treatment of osteoporosis with allopurinol and febuxostat in the prior art, the impact of muscle-bone interaction is unclear, the efficacy is limited, and the long-term safety is uncertain.

[0005] People are eager to obtain a safe, reliable and precisely targeted drug for the treatment of osteoporosis, especially postmenopausal osteoporosis.

[0006] In the prior art, pyrimidopyrazole derivatives are used to prepare drugs for treating hyperuricemia or gout, but no related reports on their use for treating osteoporosis have been found. Summary of the invention

[0007] The technical problem solved by the present invention is to provide application of pyrimidopyrazole derivatives in preparing drugs for treating and / or preventing osteoporosis.

[0008] The pyrimidopyrazole derivative is a pyrimidopyrazole derivative having a general formula I or a pharmaceutically acceptable salt thereof:

[0009]

[0010] Among them, R 1 is a C1-C6 alkyl group;

[0011] Furthermore, R 1 is a branched C3-C6 alkyl group;

[0012] Furthermore, the pyrimidopyrazole derivative or a pharmaceutically acceptable salt thereof may be one or more of the following compounds:

[0013]

[0014] Preferably:

[0015]

[0016] 2-Isopropoxy-5-(3-oxo-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-yl)benzonitrile (XOI-501)

[0017] The pyrimido[1,2-a]pyrazole derivative or a pharmaceutically acceptable salt thereof according to the present invention can be used to prepare a medicament for treating osteoporosis.

[0018] Furthermore, the osteoporosis is osteoporosis with reduced bone mass or increased bone fragility caused by estrogen deficiency.

[0019] Even further, the osteoporosis is postmenopausal osteoporosis.

[0020] Even further, the pyrimido[1,2-a]pyrazole derivative or a pharmaceutically acceptable salt thereof exerts a therapeutic effect on osteoporosis by regulating "muscle-bone interaction".

[0021] The pyrimido[1,2-a]pyrazole derivative or a pharmaceutically acceptable salt thereof according to the present invention is prepared by the following method:

[0022] Step 1: Using the corresponding 3-cyano-4-alkoxy-benzamidine as a starting material, reacting with ethoxymethylene at a molar ratio of 1:(0.1-10) under the condition of 20-80 °C for cyclization. After the product is filtered and dried, at the condition of 45-70 °C, and then reacting with SOC1 2 to obtain the corresponding intermediate compound 1;

[0023] Step 2: The intermediate compound 1 in Step 1 reacts with hydrazine hydrate at a molar ratio of 1:(1-100) at room temperature to obtain the corresponding intermediate compound 2;

[0024] Step 3: The intermediate compound 2 is cyclized under the catalytic action of sodium methoxide under the condition of refluxing in methanol to obtain the pyrimido[1,2-a]pyrazole derivative of formula I or a pharmaceutically acceptable salt thereof.

[0025]

[0026] wherein, R 1 is a C1-C6 alkyl group.

[0027] Furthermore, the pyrimido[1,2-a]pyrazole derivative or a pharmaceutically acceptable salt thereof according to the present invention can be prepared into a pharmaceutical composition with a pharmaceutically acceptable carrier for preparing a medicament for treating and / or preventing osteoporosis.

[0028] In the above composition, the weight percentage of the pyrimidopyrazole derivative or its pharmaceutically acceptable salt is 0.5%-20%.

[0029] Furthermore, the weight percentage of the pyrimidopyrazole derivative or the pharmaceutically acceptable salt thereof is 0.5% to 10%.

[0030] The pyrimidopyrazole derivatives or pharmaceutically acceptable salts thereof or pharmaceutical compositions containing the derivatives or pharmaceutically acceptable salts thereof of the present invention can be further prepared into one of the following product forms: medical drug preparations, kits, health products, and medical devices.

[0031] The medical drug preparation also includes pharmaceutically acceptable excipients;

[0032] The auxiliary materials include at least one of pharmaceutically acceptable carriers, diluents, fillers, binders, preservatives, lubricants, dispersants, flavoring agents, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, antioxidants, colorants, and stabilizers;

[0033] The dosage form of the medical drug preparation includes at least one of decoction, powder, pill, wine, lozenge, gel, tea, koji, cake, dew, stick, thread, strip, nail, moxibustion, ointment, pill, liposome preparation, aerosol, injection, mixture, oral ampoule, tablet, capsule, pill, emulsion, film, and sponge.

[0034] After in-depth research, the present invention analyzed the differentially expressed proteins in the muscle tissue of ovariectomized osteoporosis model (OVX) mice and normal mice by non-target metabolomics sequencing and tissue protein Western blot, revealing that osteoporosis model mice have purine metabolism disorders, screening XO as a key protein target, and inducing osteogenic differentiation experiments by transfecting primary BMSCs of OVX osteoporosis model mice with XO siRNA, revealing for the first time that inhibiting XO protein activity can promote BMSCs osteogenic differentiation; inducing osteogenic differentiation experiments by transfecting primary myoblasts of OVX osteoporosis model mice with XO siRNA and treating the obtained cell supernatant with primary BMSCs, and injecting adenovirus AAV9 XO into OVX model mice. shRNA targeted knockdown of muscle / bone tissue XO gene expression revealed for the first time that muscle tissue XO protein affects osteogenesis and / or osteoclast activation through "muscle-bone interaction", providing a theoretical basis and technical foundation for the development of a new generation of tissue XO protein small molecule inhibitor drugs that are effective for the treatment of osteoporosis, and further research and development of therapeutic drugs for osteoporosis, which is of great significance for promoting the development of new osteoporosis drugs and improving the quality of life of osteoporosis patients.

[0035] The pyrimidopyrazole derivatives or pharmaceutically acceptable salts thereof of the present invention can inhibit the activity of muscle / bone tissue xanthine oxidase (XO) protein, reduce ROS generation by inhibiting XO activity, thereby improving tissue oxidative stress and inflammatory state, reducing osteoblast differentiation inhibition and inhibiting osteoclast activation, thereby benefiting osteoporosis; the present invention provides a theoretical basis for XO protein as a target for the development of a new generation of small molecule inhibitor drugs against osteoporosis. The targeted small molecule drug inhibits muscle / bone tissue XO protein within a larger safety window, has good safety, has great clinical transformation prospects in the treatment of osteoporosis, and is of great significance for improving the quality of life of postmenopausal osteoporosis patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 The non-targeted metabolomics results of muscle tissue of OVX osteoporosis model mice were compared with those of normal mice, and KEGG enrichment analysis was performed on the differential metabolites obtained by the metabolomics;

[0037] A: Volcano plot of differential metabolites;

[0038] B: KEGG bubble diagram of differential metabolite enrichment pathways;

[0039] Figure 2 It is part of the KEGG purine metabolism pathway and the target of XOI-501 pathway;

[0040] Figure 3 This is the protein immunoblotting of muscle tissue of OVX osteoporosis model mice and normal mice;

[0041] Figure 4 Simulate molecular docking of XOI-501 and XO;

[0042] Figure 5 is the maximum inhibitory concentration (IC) of pyrimidopyrazole derivatives for XO 50 );

[0043] A: Maximum inhibitory concentration curve of XOI-501 on myoblast XO;

[0044] B: Maximum inhibitory concentration curve of XOI-501 on osteoblast XO;

[0045] Figure 6 Western blot of muscle / bone tissue proteins in OVX model mice treated with XOI-501 or XOI-502 by oral gavage and OVX model mice;

[0046] A: Changes in the content of XO protein in muscle tissue of OVX model mice treated with XOI-501 and XOI-502;

[0047] B: Changes in the content of XO protein in the bone tissues of OVX model mice treated with XOI-501 and XOI-502 and OVX model mice;

[0048] C: Content of myogenic differentiation marker protein (MyoG) in OVX model mice treated with XOI-501 and XOI-502 and OVX model mice;

[0049] D: Changes in the content of bone differentiation marker proteins (COL1, RUNX2, ALP) in OVX model mice treated with XOI-501 and XOI-502 and OVX model mice;

[0050] Figure 7 Histological staining of muscle / bone tissues in OVX model mice and normal mice gavaged with XOI-501, XOI-502, and Alendronate;

[0051] A: Immunohistochemistry of XO protein in muscle tissues of OVX model mice treated with XOI-501 and untreated OVX model mice;

[0052] B: Goldner staining of bone tissues in OVX model mice gavaged with XOI-501 and Alendronate and untreated OVX model mice;

[0053] C: Calcein staining of bone tissues in OVX model mice, untreated OVX model mice, and normal mice gavaged with XOI-501, XOI-502, and Alendronate;

[0054] D: OCN immunofluorescence of bone tissues in OVX model mice, untreated OVX model mice, and normal mice gavaged with XOI-501, XOI-502, and Alendronate;

[0055] Figure 8 Micro-CT and bone mass detection of the femurs of OVX model mice and normal mice gavaged with XOI-501, XOI-502, Alendronate, and TMC-5;

[0056] A: Micro-CT imaging of the femurs of OVX model mice, untreated OVX mice, and normal mice gavaged with XOI-501, XOI-502, Alendronate, and TMC-5;

[0057] B: Comparison of the average bone density of the femurs of six groups of mice by micro-CT;

[0058] C: Comparison of the average bone density of the bone cortex of the femurs of six groups of mice by micro-CT;

[0059] D: Comparison of average bone density of femoral micro-CT trabeculae in six groups of mice. DETAILED DESCRIPTION

[0060] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0061] Example 1 Preparation of pyrimidopyrazole derivatives

[0062] Pyrimidopyrazole derivatives were prepared by referring to the method of Example 1 in Chinese patent CN 112778318B to obtain compounds XOI-500, XOI-501, XOI-502, and XOI-503, which correspond to compounds b-1, b-2, b-3, and b-4 in the above patent, respectively.

[0063] Example 2 Non-targeted metabolomics analysis of OVX osteoporosis model mice

[0064] The OVX osteoporosis model mice were established by surgical castration. The specific steps are as follows: 8-week-old female mice were selected, and the abdominal position was fixed after anesthesia. An incision of about 1 cm was made on both sides of the lumbar vertebrae on the back, the muscle tissue was separated, the ovaries were exposed and the fallopian tubes were ligated, the bilateral ovaries were completely removed, and the incisions were sutured. The sham operation group (control group) only exposed the ovaries without removal. After surgery, the mice were fed routinely for 3 months, and the OVX model mice were obtained when the estrogen level decreased and bone loss was induced.

[0065] Ten OVX osteoporosis model mice and ten control mice of the same age and sex were selected. After sacrifice, the lower limb muscles were extracted and labeled and classified in sequence. After quick freezing in liquid nitrogen, the muscle tissues of each group were ground into powder, and 500 μl of 70% methanol / water (-20 °C pre-cooled) extraction solution was added. The samples were oscillated at 2500 r / min for 5 min, left standing for 5 min, and the operation was repeated twice. After centrifugation at 12000 r / min for 5 min at 4 °C, 400 μl of the supernatant was aspirated into a new centrifuge tube. It was left standing in a -20 °C refrigerator for 30 min. After centrifugation at 15000 r / min for 20 min at 4 °C, 200 μl of the supernatant was taken for injection into LC-MS for analysis. Compound Discoverer (CD) software was used to analyze the mass spectrometry data, and the peaks of each sample were aligned according to a retention time deviation of 0.1 min and a mass deviation of 5 ppm. Signal peaks were extracted by setting information such as a mass deviation of 5 ppm, a signal intensity deviation of 30%, a signal-to-noise ratio of 3, a minimum signal intensity of 100000, and sum ions. The peak areas were quantified, and metabolites were identified by combining databases (such as HMDB, KEGG).

[0066] Partial Least Squares Discrimination Analysis (PLS-DA) was used to screen for differential metabolites between different groups and distinguish samples between groups. Then, the significantly differential metabolites were subjected to KEGG pathway enrichment to generate volcano plots and KEGG bubble plots respectively.

[0067] Result analysis:

[0068] As Figure 1 shown in A of [reference], a total of 375 differential metabolites were screened out between the OVX model group and the normal mouse group. There were 195 differential metabolites with significant significance, 122 significantly up-regulated differential metabolites, and 73 significantly down-regulated differential metabolites. A volcano plot was used to show the overall distribution of the differential metabolites. The X-axis and Y-axis represent the fold change in metabolite alteration and the significance of the P-value respectively. Red and blue represent up-regulated and down-regulated metabolites respectively. Among the down-regulated metabolites, xanthine and hypoxanthine are included.

[0069] As Figure 1 shown in B of [reference], a bubble plot was generated by performing KEGG pathway enrichment analysis on the differential metabolites. The X-axis represents the ratio of the number of significantly differential genes in a certain pathway to the total number of differential genes, the Y-axis represents the specific KEGG pathway name, the bubble size represents the number of significantly differential genes in a certain pathway, and the bubble color usually represents the significance level of the enrichment analysis.

[0070] We selected the top 20 pathways as significantly changed pathways, and further selected the pathway with both high enrichment fold and significance - Caffeine metabolism pathway as the research direction. As Figure 2 shown, the Purine metabolism pathway is part of the Caffeine metabolism pathway, which includes the processes of xanthine and hypoxanthine generation. Since the differential metabolites in the metabolome are mainly enriched in purine metabolism, and the results suggest that xanthine and hypoxanthine are significant differential metabolites between the skeletal muscle samples of the OVX group and the normal group, and the contents of xanthine and hypoxanthine in the muscle tissue of the OVX group are lower than those of the normal group, indicating that there is a purine disorder in the OVX group, and it may be related to the increased activity of xanthine oxidase (XO) in the purine pathway.

[0071] Example 3

[0072] Western blot detection of XO protein immunoblot in muscle tissues of OVX osteoporosis model mice and normal mice

[0073] Muscle tissues of OVX osteoporosis model mice and normal mice were respectively taken and quickly placed on ice, rinsed with pre - cooled PBS to remove blood and impurities. The tissues were minced and lysate buffer (containing protease inhibitor) was added, and then homogenized thoroughly with an ultrasonic crusher. Centrifuge at 12000 rpm for 15 minutes at 4°C, and the supernatant was the total protein extract. The protein concentration was measured using the BCA method. The protein samples were mixed with loading buffer, loaded after boiling and denaturation, and separated by SDS - PAGE electrophoresis.

[0074] The separated proteins were transferred onto PVDF or NC membranes. The membranes were blocked with 5% skim milk or BSA to reduce non - specific binding. Specific primary antibodies were added and incubated overnight at 4°C. The next day, HRP - labeled secondary antibodies were added and incubated at room temperature for 1 hour. ECL chemiluminescence reagent was used for color development, and protein bands were detected through an imaging system.

[0075] Result analysis: The Western blot results of XO protein in muscle tissues of OVX osteoporosis model mice (OVX) and normal mice (Ctrl) are shown in Figure 3 , and the results show that the content of XO protein in the muscle tissue of the OVX group is significantly higher than that of the normal group, suggesting that the activity of XO protein in the OVX group is increased, which may be related to purine disorder.

[0076] Example 4 Docking of XOI - 501 with XO mimic

[0077] Use the chemical drawing software (ChemDraw) to draw the two - dimensional structure of XOI - 501 and use the molecular modeling software ( Suite) to convert the two - dimensional structure into a three - dimensional structure and perform initial geometric optimization.

[0078] The three-dimensional structure was energy-minimized using the molecular mechanics force field (OPLS-2005) to optimize bond lengths, bond angles, and dihedral angles, ensuring the rationality of the molecular conformation. The molecular structure was further optimized using quantum chemical methods (DFT) to calculate electron distributions and energies, obtaining a more accurate geometric configuration and charge distribution. The charge assignment method (AM1-BCC) was used to assign atomic charges to XOI-501 to ensure accurate charge distribution in subsequent docking or molecular dynamics simulations. The crystal structure of xanthine oxidase (XO) (PDB: 1N5X) was obtained from the RCSB Protein Data Bank, and the protein was optimized and energy-minimized using Suite. The docking active site was defined as a grid box centered on the centroid of the known inhibitor (such as febuxostat) in the crystal structure, with a size of . The GLIDE module was used to dock XOI-501 into the active site of xanthine oxidase, and the docking calculations were performed in standard precision (SP) or extra precision (XP) mode. The docking results were visually analyzed using PyMOL to evaluate the binding mode of the ligand to the protein and the key interactions (such as hydrogen bonds, hydrophobic interactions, etc.). Finally, the docking method was verified by redocking the known inhibitor to ensure the reliability of the results.

[0079] Result analysis:

[0080] Multiple hydrogen bonds were formed between the carbonyl group of XOI-501 and Arg 880 and Thr 1010 (red circles), and hydrogen bond interactions with Glu 802 were also observed. In addition, the cyano group of XOI-501 was able to retain hydrogen bonds with Asn768 and Lys771 (blue circles), and these residues were located in the sub-pocket of XO. These interactions explained the significant XO inhibitory activity of 6-(4-alkoxy-3-cyanophenyl)-1,2-dihydro-3H-pyrazolo[3,4-d]pyrimidin-3-one compounds.

[0081] Example 5 Dose-protein activity curve of pyrimido-pyrazole derivative XOI-501 against XO

[0082] C2C12 myoblasts and MC3T3 osteoblasts were seeded at 8×10 cells per well 3Individuals were inoculated and cultured in a 96-well culture plate for 24 hours. The pyrimidine pyrazole derivative XOI-501 was fully dissolved in DMSO to the required concentrations (0, 0.9375 μM, 1.875 μM, 3.75 μM, 7.5 μM, 15 μM). After treatment with the pyrimidine pyrazole derivative XOI-501 at the corresponding concentrations, dead cells were removed with PBS buffer, and then the CCK-8 detection kit was used to detect the absorbance (OD value) of each well by an enzyme-linked immunosorbent assay (ELISA) reader to calculate cell viability. And the IC 50 curve was plotted using Prism5. Tables 1-2 show the survival rates of myoblasts / osteoblasts after treatment with XOI-501, respectively.

[0083] Table 1 Survival rate of myoblasts after treatment with XOI-501

[0084]

[0085]

[0086] Table 2 Survival rate of osteoblasts after treatment with XOI-501

[0087] XOI-501 (μM) Osteoblast Activity (%) 0 99.83±1.72 0.9375 87.67±3.72 1.875 58.00±7.48 3.75 28.17±1.17 7.5 17.00±1.26 15 16.50±4.59

[0088] Result analysis:

[0089] As Figure 5 shown in A, 5B, XOI-501 had a significant inhibitory effect on the activity of XO protein in C2C12 myoblasts in the concentration range of 1.893 μM - 2.229 μM (IC 50 = 2.057 μM), and had a significant inhibitory effect on the activity of XO protein in MC3T3 osteoblasts in the concentration range of 2.192 μM - 2.717 μM (IC 50 = 2.439 μM).

[0090] Example 6

[0091] Protein immunoblotting of muscle / bone tissues of OVX model mice treated by gavage with pyrimidine pyrazole derivatives (XOI-501, XOI-502) and untreated OVX model mice

[0092] XOI-501 and XOI-502 were fully dissolved in PBS to 3 μM, and used as XOI-501 group and XOI-502 group, respectively. Use a 1 ml syringe to connect the gavage needle, and draw 0.2 ml XOI-501 solution, 0.2 ml XOI-502 solution, and 0.2 ml Alendronate solution (30 μM) respectively. Normal mice were used as the Ctrl group. The mice were placed with their heads facing up, and the gavage needle was gently inserted into the mouse mouth and slowly pushed into the esophagus to inject the solution. The above operation was repeated once a day. After continuous gavage for 4 weeks, the mice were killed, and the target muscles and bone tissues were quickly removed and rinsed with pre-cooled PBS to remove blood and impurities. The muscle tissue was cut into pieces, added to RIPA lysis buffer (containing protease inhibitors), homogenized on ice for 10 minutes, and allowed to stand at 4 ° C for 30 minutes. The bone tissue was cut into pieces, ground into powder with liquid nitrogen, added to RIPA lysis buffer (containing protease inhibitors), and lysed on ice for 1 hour, with vortex mixing during the period. Centrifuge at 4°C and 12,000 rpm for 15 min, collect the supernatant, and determine the protein concentration using the BCA method and unify the quantification to 3ug / uL.

[0093] The protein samples were subjected to SDS-PAGE electrophoresis, transferred to PVDF membrane, blocked with 5% skim milk, incubated with primary antibody, and HRP-labeled secondary antibody was added the next day for incubation at room temperature for 1 hour. ECL chemiluminescent reagent was used for color development, and protein bands were detected by imaging system.

[0094] Result analysis:

[0095] Figure 6 A and B show that the XO content in muscle / bone tissue of OVX model mice treated with 3 μM pyrimidopyrazole derivatives was lower than that in untreated OVX model mice, indicating that 3 μM pyrimidopyrazole derivatives can effectively inhibit the XO activity in muscle / bone tissue of OVX model mice.

[0096] Figure 6 C shows that the content of myogenic differentiation marker protein (MyoG) in OVX model mice treated with XOI-501 was significantly higher than that in untreated OVX model mice, and was better than that in the XOI-502 treated group. Compared with the untreated OVX model mice, the XOI-502 treated group did not show significant changes.

[0097] Figure 6 D shows that the levels of osteogenic differentiation marker proteins (COL1, RUNX2, ALP) in OVX model mice treated with XOI-501 were significantly higher than those in untreated OVX model mice, and better than those in the XOI-502 treatment group.

[0098] The results showed that at a concentration of 3 μM in vitro, pyrimidine pyrazole derivatives XOI-501 and XOI-502 could effectively inhibit the activity of muscle / bone XO protein while promoting the differentiation function of the corresponding tissues, with XOI-501 having the best effect.

[0099] Example 7

[0100] Histological staining of muscle tissues of OVX model mice treated by intragastric administration with pyrimidine-pyrazole derivatives (XOI-501), untreated OVX model mice, and normal mice

[0101] XOI-501 was fully dissolved in PBS to 3 μM to study the effect of intragastric administration of XOI-501 on myogenic differentiation of OVX model mice.

[0102] A 1-ml syringe was connected to a gavage needle, and 0.2 ml of XOI-501 was aspirated. The mouse was held with its head up, and the gavage needle was gently inserted into the mouse's mouth and slowly advanced into the esophagus to inject the drug solution. The above operation was repeated once a day. After continuous intragastric administration for 4 weeks, the mice were sacrificed. Muscle tissues of two groups of mice were taken and fixed in 4% paraformaldehyde and embedded in paraffin for sectioning. The sections were dewaxed to water, antigen retrieval was performed, endogenous peroxidase was blocked, incubated with the primary antibody against XO protein, washed, and then incubated with the secondary antibody labeled with HRP. DAB was used for color development, hematoxylin was used for counterstaining, dehydrated, cleared, and sealed. Immunohistochemical staining was used to evaluate the XO protein activity in the muscle tissues of mice.

[0103] Result analysis: As Figure 7 shown in A, the content of XO in the muscle tissues of OVX model mice treated with XOI-501 was significantly reduced compared with that of untreated OVX model mice. And compared with the XOI-501 treatment group, the arrangement of muscle fibers in the untreated group was disordered, the diameters of muscle fibers were uneven, some fibers were significantly thinner, showing an atrophic state. The width of the muscle fiber gap increased, and the cross-striation structure of the muscle fibers became blurred, indicating that the muscle tissues might be affected by damage, degeneration, etc.

[0104] The results showed that at a concentration of 3 μM in vivo, treatment with the pyrimidine-pyrazole derivative XOI-501 could effectively inhibit the XO protein activity in the muscle tissues of OVX model mice and improve the differentiation function of muscle tissues to a certain extent.

[0105] Example 8

[0106] Bone tissue staining results of OVX model mice treated by intragastric administration with pyrimidine-pyrazole derivatives (XOI-501, XOI-502) and Alendronate, and untreated OVX mice

[0107] The pyrimidine-pyrazole derivatives (XOI-501, XOI-502) were fully dissolved in PBS to 3 μM.

[0108] (1) Connect a 1 ml syringe to a gavage needle, and separately aspirate 0.2 ml of XOI-501 and 0.2 ml of Alendronate (30 μM) solution. Hold the mouse with its head up, gently insert the gavage needle into the mouse's oral cavity and slowly push it into the esophagus, then inject the drug solution. Do this twice a week for 4 consecutive weeks, and then sacrifice the mouse. Take the femurs of the three groups of mice: those treated with XOI-501, those treated with Alendronate (30 μM), and untreated OVX mice. Fix them in 4% paraformaldehyde, decalcify, embed in paraffin, and section. Stain them successively with Weigert iron hematoxylin, acid fuchsin - orange G solution, and aniline blue solution. Finally, dehydrate, clear, and mount the slides. Evaluate the histological condition of the femur after XOI-501 gavage treatment using Goldner staining. The results are shown in Figure 7 B.

[0109] Figure 7 B shows that compared with untreated OVX model mice, in OVX model mice treated with XOI-501 and Alendronate, mineralized bone (green) accounts for the main part, and osteoid (red) is widened. Osteoblasts are arranged neatly, osteoclasts are rare, and the Goldner staining of the femurs in the two groups of XOI-501 and Alendronate is similar.

[0110] (2) Connect a 1 ml syringe to a gavage needle, and separately aspirate 0.2 ml of XOI-501, 0.2 ml of XOI-502, and 0.2 ml of Alendronate (30 μM) solution. Gavage the mice using the same method as above. Dissolve calcein in PBS to prepare a 10 mg / mL solution, filter and sterilize it for later use. Inject intraperitoneally at a dose of 10 mg / kg in the 3rd week of gavage, and inject again at the same dose 7 days later. After sacrificing the animals, take the femurs of the five groups of mice: those treated with XOI-501 + OVX, those treated with XOI-502 + OVX, those treated with Alendronate (30 μM), OVX, and normal mice (Ctrl). Fix them in 4% paraformaldehyde. Prepare undecalcified bone sections (5 - 7 μm thick) after dehydration with gradient ethanol and embedding in resin. Immerse the sections in calcein solution (2 mg / mL) and incubate in the dark for 5 minutes. Wash the sections with PBS and mount them with an anti-fluorescence quenching mounting medium. Observe the mineralization front marked by green fluorescence using a fluorescence microscope (excitation wavelength 494 nm, emission wavelength 517 nm). The results are shown in Figure 7 C.

[0111] Figure 7 C shows that the bone formation thickness (red arrow) in the mice treated with XOI-501 and Alendronate is close within the same time interval, higher than that of untreated OVX mice, and XOI-501 is better than the XOI-502 group.

[0112] (3) Treat the mice by gavage in the same method as above. Sacrifice the mice after continuous gavage for 4 weeks. Take the femurs of the five groups of mice: XOI-501 + OVX treatment, XOI-502 + OVX treatment, Alendronate (30 μM) + OVX treatment, OVX, and normal mice (Ctrl). Fix them in 4% paraformaldehyde, decalcify, dehydrate with gradient ethanol, and then embed them in paraffin for sectioning (5-7 μm thickness). After dewaxing the sections, perform antigen heat repair with sodium citrate buffer, and block them with 5% BSA for 30 minutes. Add the anti-OCN primary antibody and incubate overnight at 4°C. The next day, add the fluorescent secondary antibody (Alexa Fluor 647) and incubate for 1 hour at room temperature in the dark. After washing with PBS, stain the nuclei with DAPI for 5 minutes. Observe under a fluorescence microscope. The OCN positive signal is red fluorescence, and the cell nucleus is blue fluorescence. The results are shown in Figure 7 D.

[0113] Figure 7 D shows that the fluorescence intensities of the key osteogenic maturation proteins in the bone tissues of the mice in the XOI-501 and Alendronate treatment groups shown by OCN immunofluorescence are similar, higher than those of the untreated OVX mice, and better than those of the XOI-502 group.

[0114] The results show that at a concentration of 3 μM in vivo, the pyrimidine and pyrazole derivative XOI-501 can effectively promote the osteogenic differentiation function of the bone tissues of OVX model mice. The effect is close to that of the Alendronate treatment group for anti-osteoporosis, and better than that of the XOI-502 group. It is suggested that XOI-501 has a certain positive effect on improving the bone loss state of postmenopausal osteoporosis.

[0115] Example 9

[0116] Micro-CT and bone mass detection of the femurs of OVX model mice treated by gavage with pyrimidine and pyrazole derivatives (XOI-501, XOI-502), Alendronate, and TMC-5, untreated OVX model mice, and normal mice

[0117] The structure of TMC-5 is as follows:

[0118]

[0119] Pyrimidine and pyrazole derivatives were fully dissolved in PBS to 3 μM. 0.2 ml XOI-501, 0.2 ml XOI-502 solution, 0.2 ml Alendronate solution (30 μM), and 0.2 ml TMC-5 solution (30 μM) were taken respectively. The mouse head was facing up, and the gavage needle was gently inserted into the mouse mouth and slowly pushed into the esophagus to inject the solution. The above operation was repeated once a day. The mice were killed after continuous gavage for 4 weeks. Six groups of femurs were taken from XOI-501+OVX treated, Alendronate (30 μM)+OVX treated, OVX, XOI-502+OVX treated, TMC-5+OVX treated and normal mice (Ctrl), and fixed in 4% paraformaldehyde for 48 hours. Continuous sections of each bone growth plate were reconstructed and analyzed. The bone structure image was reconstructed into a three-dimensional image using CTVox software, and parameters such as bone density and bone volume fraction were reconstructed and analyzed by the software.

[0120] Result analysis: Figure 8 As shown in A, micro-CT three-dimensional reconstruction showed that compared with the OVX group, the femoral cortical bone of OVX mice in the XOI-501-treated and Alendronate (30 μM)-treated groups was thickened and the trabecular structure was dense, and the three-dimensional reconstruction of the femur of mice in the XOI-501-treated and Alendronate-treated groups was similar, which was significantly better than that in the XOI-502-treated and TMC-5-treated groups.

[0121] like Figure 8 As shown in B and 8C, compared with the OVX group, the bone cortical and trabecular densities of OVX mice in the XOI-501-treated and Alendronate (30 μM)-treated groups were significantly increased, which were significantly better than those in the TMC-5 and XOI-502-treated groups, with significant statistical significance.

[0122] The results showed that at a concentration of 3 μM in vivo, the pyrimidine pyrazole derivative XOI-501 can effectively promote the osteogenic differentiation of bone tissue in OVX model mice, with an effect close to that of the anti-osteoporosis Alendronate treatment group, and better than the XOI-502 group and TMC-5 group. This suggests that XOI-501 has a certain positive effect on improving the bone loss state of postmenopausal osteoporosis.

[0123] In summary, the present invention provides the use of pyrimidopyrazole derivatives in the preparation of drugs or foods for preventing or treating postmenopausal osteoporosis. Pyrimidopyrazole derivatives can play a role in treating osteoporosis by inhibiting XO protein. And through research, it was found that XO protein may become a new target for the development, research and screening of osteoporosis drugs. Drugs for treating osteoporosis can target and inhibit the activity of XO protein in muscle / bone tissues of postmenopausal osteoporosis purine metabolism disorders, while reducing side effects on non-muscle / bone tissues, having good safety, and having great clinical transformation prospects in the fight against osteoporosis.

[0124] The above disclosure is only the preferred embodiment of the present invention, which certainly cannot be used to limit the scope of the present invention. Therefore, equivalent changes made according to the claims of the present invention are still within the scope of the present invention.

Claims

1. Use of a pyrimidopyrazole derivative in the preparation of a drug or food for treating and / or preventing osteoporosis, wherein the pyrimidopyrazole derivative is a pyrimidopyrazole derivative represented by formula I or a pharmaceutically acceptable salt thereof; in, R1 is a C1-C6 alkyl group, preferably a C3-C6 branched alkyl group.

2. The use according to claim 1, characterized in that: The osteoporosis is osteoporosis with decreased bone mass or increased bone fragility caused by estrogen deficiency.

3. The use according to claim 1 or 2, characterized in that: The osteoporosis is postmenopausal osteoporosis.

4. Use of pyrimidopyrazole derivatives in the preparation of medicines or foods that help improve bone density, wherein the pyrimidopyrazole derivatives are pyrimidopyrazole derivatives represented by formula I or pharmaceutically acceptable salts thereof; in, R1 is a C1-C6 alkyl group, preferably a C3-C6 branched alkyl group.

5. The use according to any one of claims 1 to 4, characterized in that: The pyrimidopyrazole derivative or a pharmaceutically acceptable salt thereof is selected from the following compounds:

6. The use according to any one of claims 1 to 3, characterized in that: The pyrimidopyrazole derivatives or pharmaceutically acceptable salts thereof play a role in treating osteoporosis by regulating "muscle-bone interaction".

7. The use according to any one of claims 1 to 6, characterized in that: The pyrimidopyrazole derivative or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier are prepared into a pharmaceutical composition.

8. The use according to any one of claims 1 to 7, characterized in that: The pyrimidopyrazole derivatives or their pharmaceutically acceptable salts or their pharmaceutical compositions are further prepared into medical preparations, test kits, health products and medical devices.

9. The use according to claim 8, characterized in that The dosage form of the medical drug preparation includes at least one of decoction, powder, pill, wine, lozenge, gel, tea, koji, cake, dew, stick, thread, strip, nail, moxibustion, ointment, pill, liposome preparation, aerosol, injection, mixture, oral ampoule, tablet, capsule, pill, emulsion, film, and sponge.

10. Application of xanthine oxidase as a target in the development, research and screening of drugs for the treatment and / or prevention of osteoporosis.

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