Heterocyclic compound, method for producing same, pharmaceutical composition comprising same, and use of same

Abstract

The invention relates to a heterocyclic compound with osteoporosis treatment activity, a preparation method thereof, a pharmaceutical composition containing the heterocyclic compound and application of the heterocyclic compound. According to the invention, the compound is synthesized, and it is verified that the compound has anti-osteoporosis pharmacological activity in vivo and in vitro, wherein the heterocyclic compound has important value in the aspects of treating and preventing osteoporosis and bone metabolism related diseases.

Description

Technical Field

[0001] The present invention relates to a heterocyclic compound and application thereof in preparing medicine for treating osteoporosis and bone metabolism-related diseases. Background Art

[0002] Osteoporosis is a systemic metabolic bone disease characterized by a gradual decrease in bone mass, microstructural destruction, increased bone brittleness, and an increased risk of fracture. The most common complication of osteoporosis is fracture, which has a high disability and mortality rate and seriously threatens human health and quality of life [Postmenopausalosteoporosis. Nat Rev Dis Primers. 2016 Sep 29: 2: 16069. doi: 10.1038 / nrdp.2016.69.; Wnt signaling in osteoporosis: mechanisms and novel therapeutic approaches, Nat Rev Endocrinol,. 2013 Oct; 9(10): 575-83. doi: 10.1038 / nrendo.2013.154]. Osteoporosis has a high incidence rate and is common in middle-aged and elderly people. Postmenopausal women are prone to postmenopausal osteoporosis (PMO) due to aging and decreased hormone levels [Clinical Practice. Postmenopausal Osteoporosis. N Engl J Med. 2016 Jan 21; 374(3): 254-62. doi: 10.1056 / NEJMcp1513724.]. According to statistics, approximately one-third of women and one-fifth of men over the age of 50 will suffer a fracture, seriously affecting their quality of life and accompanied by pain. With the extension of life expectancy and the acceleration of population aging, osteoporosis has become a major public health issue of global concern.

[0003] Bone metabolism includes two processes: bone resorption and bone remodeling. Osteoclasts, which absorb bone matrix, and osteoblasts, which synthesize bone matrix, play an important role in bone metabolism. Imbalance in bone remodeling is a key factor in the etiology of osteoporosis [Hedgehog signaling regulates bone homeostasis through orchestrating osteoclast differentiation and osteoclast-osteoblast coupling. Cell Mol Life Sci. 2023 Jun 1; 80(6): 171. doi: 10.1007 / s00018-023-04821-9]. Under normal conditions, bone metabolism-related regulatory factors regulate bone anabolism (bone formation function of osteoblasts) and bone catabolism (bone resorption function of osteoclasts) in a dynamic balance, maintaining a constant bone mass. When bone metabolism is unbalanced, the function of osteoclasts is greater than that of osteoblasts, and bone resorption will be greater than bone formation, resulting in a decrease in bone mass and the occurrence of osteoporosis.

[0004] Currently, drugs for treating osteoporosis are mainly divided into drugs that inhibit bone resorption and drugs that promote bone formation. The toxic side effects of existing anti-osteoporosis drugs restrict their clinical application [Treatment of Low Bone Density or Osteoporosis to Prevent Fractures in Men and Women: A Clinical Practice Guideline Update From the American College of Physicians. Ann Intern Med. 2017 Jun 6; 166(11): 818-839. doi: 10.7326 / M15-1361]. For example, long-term use of bisphosphonates (BPs) can excessively inhibit bone turnover, affect bone strength, cause atypical fractures, increase the risk of acute myocardial infarction, jaw osteonecrosis, and rickets, and also have side effects such as toxicity to the gastrointestinal tract, kidneys, blood, and liver, and immunosuppression. Estrogen replacement therapy for osteoporosis can cause breast cancer, uterine bleeding, and cardiovascular disease, and is no longer used as a routine medication. Side effects of calcitonin include rhinitis and back pain. Parathyroid hormone is expensive, and its side effects primarily include hypercalcemia, renal side effects, and a potential risk of osteosarcoma. It is generally reserved for patients with severe osteoporosis or intolerance to other anti-osteoporotic medications. Fluoride can increase cancellous bone mass, but its effect on reducing fracture incidence is offset by increased cortical bone cavitation, leading to its seldom-used use. Supplementation with 25-hydroxyvitamin D (25(OH)D) levels >150 μg / L may lead to vitamin D toxicity, causing hypercalcemia and symptoms such as constipation, headache, and vomiting. In severe cases, cardiac arrhythmias and renal failure may occur. Denosumab is a fully human monoclonal antibody targeting RANKL (Receptor Activator for Nuclear Factor-κB Ligand). It has been approved by the European Commission (EMA) for the treatment of osteoporosis in postmenopausal women and hormone-induced bone loss in prostate cancer patients. However, its side effects include immune system impairment, eczema, and cellulitis, as well as high cost and complex dosing. Discontinuation of denosumab can lead to a further decrease in bone density, significantly increasing the risk of fractures. Strontium ranelate, due to its increased risk of myocardial infarction and thrombosis, is limited to patients with severe osteoporosis who have no other treatment options.In summary, currently only potent BPs (alendronate, risedronate, and zoledronic acid) and denosumab are effective in reducing fractures. Most osteoporosis medications have limitations and side effects, which affect long-term use and compliance. Furthermore, bone resorption inhibitors reduce the space available for bone formation and remodeling, and long-term treatment can only achieve a 10% increase in bone density at best. Bone formation promoters can increase bone density and strength, potentially overcoming some of the limitations of bone resorption inhibitors. Therefore, the development of dual regulators that can simultaneously regulate bone resorption and bone formation to achieve normal bone turnover and bone dynamic balance is a new strategy for the research and development of safe and effective new anti-osteoporosis drugs with independent intellectual property rights [WHI-131 Promotes Osteoblast Differentiation and Prevents Osteoclast Formation and Resorption in Mice.J Bone Miner Res.2016 Feb;31(2):403-15.doi:10.1002 / jbmr.2612;DOI:10.1002 / jbmr.2612;A Novel Rhein Derivative Modulates Bone Formation and Resorptionand Ameliorates Estrogen-Dependent Bone Loss.J Bone Miner Res.2019Feb;34(2):361-374.doi:10.1002 / jbmr.3604].

[0005] The OPG-RANKL-RANK pathway is an important component of the bone metabolism signal regulation network. Receptor activator of nuclear factor-κB ligand (RANKL) and osteoprotegerin (OPG) play an important role in regulating bone remodeling [Clinical implications of the osteoprotegerin / RANKL / RANK system for bone and vascular diseases.JAMA.2004 Jul 28;292(4):490-5.doi:10.1001 / jama.292.4.490;Recent advances in seafood bioactive peptides and their potential for managing osteoporosis.Crit Rev Food Sci Nutr.2022;62(5):1187-1203.doi:10.1080 / 10408398.2020.1836606.;Postmenopausal osteoporosis.Nat Rev Dis Primers.2016 Sep 29:2:16069.doi:10.1038 / nrdp.2016.69]. RANKL is expressed by osteoblasts and bone marrow stromal cells. It binds to its receptor (receptor activator of nuclear factor-κB, RANK) to stimulate downstream signaling, initiating the transcription of osteoclastogenic genes, inducing the formation of mature osteoclasts from osteoclast precursors, and leading to enhanced osteoclast differentiation. OPG, produced by bone marrow mesenchymal stem cells and osteoblasts, acts as a soluble decoy receptor for RANKL, competitively binding to RANKL and preventing RANKL from binding to RANK. This, in turn, blocks osteoblast-induced differentiation and fusion of osteoclast precursors, thereby inhibiting osteoclast differentiation and further inhibiting bone resorption. Therefore, the balance between OPG and RANKL in bone tissue is a key determinant of bone metabolism and an important target for the treatment of osteoporosis. The monoclonal antibody drug denosumab mimics the action of endogenous OPG protein, competitively binding to RANKL, inhibiting osteoclast activation and development, reducing bone resorption, and increasing bone density. Peroxisome proliferator-activated-β / δ (PPARβ / δ, or PPARβ, or PPARδ (hereinafter referred to as PPARδ in this patent)) plays a key regulatory role in bone turnover and musculoskeletal homeostasis.PPARδ agonists increase the expression of target proteins that promote osteogenesis by binding to and stimulating PPARδ, thereby promoting osteogenesis [PPARβ / δgoverns Wnt signaling and bone turnover.Nat Med.2013May;19(5):608-13.doi:10.1038 / nm.3146;Synthesis and Evaluation of PPARδAgonists That Promote Osteogenesis in a Human MesenchymalStem Cell Culture and in a Mouse Model of Human Osteoporosis.J MedChem.2021May 27;64(10):6996-7032.doi:10.1021 / acs.jmedchem.1c00560]. In addition, activation of PPARδ can induce the expression of OPG, thereby inhibiting the formation and differentiation of osteoclasts, and plays an important role in the regulation of bone metabolism. Bone morphogenetic proteins (BMPs), also known as bone morphogenetic proteins, are a group of highly conserved functional proteins with similar structures and belong to the TGF-β family. Human bone morphogenetic protein 2 (BMP2) is one of the most widely studied BMPs and has the strongest osteogenic activity. BMP2 can bind to receptors on the cell membrane surface, activate the Smad signaling pathway in cells, increase the expression of Runt-related transcription factor 2 (Runx2), and promote osteogenesis [TGFβ / BMPSignaling Pathway in Cartilage Homeostasis. Cells. 2019Aug 24; 8(9): 969. doi: 10.3390 / cells8090969.; Specification of BMP Signaling. Cells. 2019Dec 5; 8(12): 1579. doi: 10.3390 / cells8121579.]. Therefore, searching for small molecule drugs for the treatment of osteoporosis that can stimulate PPARδ, increase BMP2 expression, increase OPG expression, block the binding of RANKL to osteoclast RANK, and have the effect of regulating both bone formation and bone resorption has important research value and prospects. Summary of the Invention

[0006] Problems to be solved by the invention

[0007] In view of the above situation, the technical problem to be solved by the present invention is to provide a class of heterocyclic compounds that can simultaneously regulate both bone formation and bone resorption to achieve the effect of treating osteoporosis and bone metabolism-related diseases, while avoiding the adverse reactions of existing osteoporosis treatment drugs such as denosumab.

[0008] Means for solving problems

[0009] The present invention aims to solve the above-mentioned technical problems by designing and synthesizing novel small molecule drugs for treating osteoporosis, providing a heterocyclic compound represented by the above-mentioned general formula I or a pharmaceutically acceptable salt or solvate thereof, and providing the use of the heterocyclic compound or a pharmaceutically acceptable salt or solvate thereof in the preparation of a therapeutic agent for treating osteoporosis and bone metabolism-related diseases.

[0010] Technical Effects

[0011] The heterocyclic compound of the present invention can specifically excite PPARδ, upregulate the activity of BMP2 expression, upregulate the activity of OPG expression, promote osteoblast differentiation and bone formation, inhibit osteoclast differentiation and bone resorption, thereby achieving excellent effects in treating osteoporosis and bone metabolism-related diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] [ Figure 1 ] is a graph showing the agonistic activity of compound I-16 on PPARδ in MC3T3-E1 cells.

[0013] [ Figure 2 ] is a graph showing alkaline phosphatase (ALP) activity in MC3T3-E1 cells after treatment with compound I-16.

[0014] [ Figure 3 ] is a graph showing the effect of compound I-16 on the formation of calcified nodules in MC3T3-E1 cells. Figure 3 A in the figure represents the effect of the conditioned medium of MC3T3-E1 cells treated with compound I-16 on the formation of calcified nodules. Figure 3 B is a quantitative graph showing the effect of the conditioned medium of MC3T3-E1 cells treated with compound I-16 on calcified nodules.

[0015] [ Figure 4 ] Figure 4 A in FIG. 1 is a Trap staining diagram showing the effect of MC3T3-E1 cell conditioned medium treated with compound I-16 on osteoclast formation. Figure 4 B is a quantitative graph showing the effect of the conditioned medium of MC3T3-E1 cells treated with compound I-16 on osteoclast formation.

[0016] [ Figure 5 ] Figure 5 A in the figure represents the effect of the conditioned medium of MC3T3-E1 cells treated with compound I-16 on the formation of bone pits. Figure 5 B is a quantitative graph showing the effect of the conditioned medium of MC3T3-E1 cells treated with compound I-16 on bone pit formation.

[0017] [ Figure 6 ] Figure 6 A in FIG is a graph showing the effect of compound I-16 on the levels of OPG and RANKL proteins in MC3T3-E1 cells. Figure 6 B is a graph showing the effect of compound I-16 on OPG and RANKL protein levels in U-2OS cells.

[0018] [ Figure 7 ] is a graph showing the effect of compound I-16 on the OPG protein secretion level of MC3T3-E1 cells.

[0019] [ Figure 8 ] is a graph showing the effect of compound I-16 on the levels of genes related to osteoblast differentiation.

[0020] [ Figure 9 ] is a graph showing the femoral bone density of rats in each group after oral administration of compound I-16 for 90 days.

[0021] [ Figure 10 ] is a graph showing representative Micro-CT images of the femurs of rats in each group after oral administration of compound I-16 for 90 days.

[0022] [ Figure 11 ] represents the structural parameters of femoral trabecular bone in rats of each group after oral administration of compound I-16 for 90 days. Figure 11 A in the equation represents the bone volume fraction of rat trabecular bone (BV / TV). Figure 11 B in the equation represents trabecular thickness (Tb.Th). Figure 11 The C in the equation represents the number of trabecular bone (Tb.N). Figure 11 D in the equation represents the trabecular spacing (Tb.Sp). Figure 11 E in the equation represents the ratio of bone surface area to bone volume (BS / BV).

[0023] [ Figure 12 ] is a H&E staining image of the femur of rats in each group after oral administration of compound I-16 for 90 days.

[0024] [ Figure 13 ] is a toluidine blue staining image of the femur of rats in each group after oral administration of compound I-16 for 90 days. Figure 13A in the figure represents the representative images of each group stained with toluidine blue. Figure 13 B in the figure indicates the counting of the number of osteoblasts per millimeter of trabecular bone surface (left) and the area of ​​osteoblasts per unit bone surface (right).

[0025] [ Figure 14 ] is a TRAP staining image of the femur of each group of rats after oral administration of compound I-16 for 90 days, scale: 200 μm, Figure 14 A in the figure shows the representative TRAP staining images of femurs in each group. Figure 14 B in the figure represents the number of osteoclasts per bone surface (left) and the osteoclast area per unit bone surface (right).

[0026] [ Figure 15 ] Figure 15 A in the figure represents the fluorescence imaging of two calcein deposition lines generated at the two time points after two calcein injections before the rats were sacrificed. B represents MAR = width of two calcein deposition lines / time interval between the two injections in days.

[0027] [ Figure 16 ] represents the serum OPG / RANKL and osteocalcin indicators of rats in each group after oral administration of compound I-16 for 90 days. DETAILED DESCRIPTION

[0028] The present invention provides the following inventions.

[0029] <Heterocyclic compound represented by general formula I>

[0030] One object of the present invention is to provide a heterocyclic compound represented by general formula I or a pharmaceutically acceptable salt or solvate thereof.

[0031]

[0032] in,

[0033] A is an aryl group which may have a substituent or a heteroaryl group which may have a substituent, and is preferably a benzothiazolyl group, a benzoxazolyl group, a thiazolyl group, an oxazolyl group, a phenyl group or a pyridyl group;

[0034] B is absent or is an optionally substituted cycloalkyl group, an optionally substituted heterocycloalkyl group, an optionally substituted aryl group, or an optionally substituted heteroaryl group;

[0035] X is -(CR 4 R 5 ) p -、-(CR 4 R 5 ) p -O-、-(CR 4 R5 ) p -S-、-(CR 4 R 5 ) p -NR 6 -、-O-(CR 4 R 5 ) p -、-S-(CR 4 R 5 ) p -or-N(R 6 )-(CR 4 R 5 ) p -;

[0036] Y is -(CR 4 R 5 ) g -、-CONR 6 -、-(CR 4 R 5 ) g -CONR 6 -、-CONR 6 -(CR 4 R 5 ) g -、-CONR 6 SO2-、-CONR 6 SO2-(CR 4 R 5 ) g -or-(CR 4 R 5 ) g -CONR 6 SO2-;

[0037] J and Q are each independently selected from carbon and nitrogen;

[0038] W is selected from carbon and nitrogen;

[0039] Z is selected from carbon, oxygen, sulfur, and nitrogen;

[0040] R 1 、R 2 、R 3each independently selected from hydrogen, fluorine, chlorine, bromine, iodine, nitro, cyano, aldehyde, ester, an alkylcarbonyl group optionally having a substituent, an amino group, an aryl group optionally having a substituent, an aryloxy group optionally having a substituent, a linear or branched alkyl group having 1 to 6 carbon atoms optionally having a substituent, a linear or branched alkoxy group having 1 to 6 carbon atoms optionally having a substituent, a linear or branched oxaalkyl group having 1 to 6 carbon atoms optionally having a substituent, a linear or branched azaalkyl group having 1 to 6 carbon atoms optionally having a substituent, a linear or branched aliphatic carboxyl group having 1 to 6 carbon atoms optionally having a substituent, a cycloalkyl group having 3 to 6 carbon atoms optionally having a substituent, and an alicyclic carboxyl group having 4 to 6 carbon atoms optionally having a substituent;

[0041] R 4 、R 5 、R 6 Each is independently selected from hydrogen, fluorine, chlorine, bromine, iodine, and a linear or branched alkyl group having 1 to 6 carbon atoms which may have a substituent;

[0042] m is an integer selected from 0, 1, 2, 3, 4, 5;

[0043] n is an integer selected from 0, 1, 2, 3, 4;

[0044] k is an integer selected from 0, 1, 2, and 3;

[0045] p is an integer selected from 0, 1, 2, 3, 4, 5, 6;

[0046] g is an integer selected from 0, 1, 2, 3, 4, 5, and 6.

[0047] "Pharmaceutically acceptable salts or solvates" as used herein refer to salts formed by reacting the heterocyclic compound with an acid commonly used in the art by conventional methods, or solvates obtained by solvation with a solvent commonly used in the art. "Aryl" as used herein refers to phenyl and naphthyl, and the aryl group may be unsubstituted or substituted.

[0048] In the present invention, "aryloxy" refers to phenoxy, benzyl, and naphthyloxy, which may be unsubstituted or substituted. In the present invention, "alkylcarbonyl" refers to C1-10 alkylcarbonyl, such as methylcarbonyl, ethylcarbonyl, propylcarbonyl, butylcarbonyl, pentylcarbonyl, hexylcarbonyl, heptylcarbonyl, octylcarbonyl, nonylcarbonyl, and decylcarbonyl, which may be unsubstituted or substituted.

[0049] The "heteroaryl" in the present invention refers to pyridyl, pyrimidinyl, pyrrolyl, thiazolyl, and oxazolyl. The heteroaryl may be unsubstituted or have a substituent.

[0050] The "cycloalkyl group" in the present invention refers to a C3-12 cycloalkyl group, preferably a C3-9 cycloalkyl group, which is unsubstituted or has a substituent.

[0051] The "heterocycloalkyl" in the present invention refers to tetrahydrofuranyl, tetrahydropyranyl, dihydropyranyl, dioxanyl, pyrrolidinyl, piperidinyl, tetrahydrothiopyranyl, and tetrahydrothiophenyl. The heterocycloalkyl group may be unsubstituted or substituted.

[0052] The term "halogen" as used herein refers to fluorine, chlorine, bromine, and iodine.

[0053] The "substituent" in the present invention includes alkyl groups having 1 to 6 carbon atoms, alkoxy groups having 1 to 6 carbon atoms, chlorine, bromine, fluorine, C3 to 9 cycloalkyl groups, nitro, amino, carboxyl, and hydroxyl groups.

[0054] In the compound of the general formula I of the present invention, A is preferably benzoxazolyl, benzothiazolyl, or thiazolyl;

[0055] In the compound of the general formula I of the present invention, B is preferably a dioxane group;

[0056] The compound of the general formula I of the present invention preferably has B absent, R 2 is methoxy, n=2;

[0057] The compound of the general formula I of the present invention preferably has B absent, R 2 is methoxy, n=1;

[0058] The compound of the general formula I of the present invention is more preferably m=1, R 1 is fluorine, chlorine, methyl, methoxy, or nitro;

[0059] More preferably, X in the compound of formula I of the present invention is -S-CH2-, -O-CH2-, -S-CH(CH3)-, -SC(CH3)2-;

[0060] More preferably, Y of the compound of formula I of the present invention is -CONH-; more preferably, J and Q of the compound of formula I of the present invention are each a carbon atom;

[0061] The preferred compounds of formula I of the present invention are as follows:

[0062] N-(5,6-dimethoxybenzo[d]thiazol-2-yl)-2-((5-fluorobenzo[d]oxazol-2-yl)thio)acetamide;

[0063] 2-(Benzo[d]oxazole-2-thio)-N-(5,6-dimethoxybenzo[d]thiazol-2-yl)propionamide;

[0064] 2-(Benzo[d]oxazole-2-oxo)-N-(5,6-dimethoxybenzo[d]thiazol-2-yl)acetamide;

[0065] 2-(Benzo[d]oxazole-2-oxo)-N-(6,7-dihydro-[1,4]dioxanyl[2',3':4,5]benzo[1,2-d]thiazol-2-yl)acetamide;

[0066] N-(6,7-Dihydro-[1,4]dioxano[2',3':4,5]benzo[1,2-d]thiazol-2-yl)-2-((5-fluorobenzo[d]oxazol-2-yl)thio)acetamide;

[0067] 2-(Benzo[d]oxazole-2-thio)-N-(6,7-dihydro-[1,4]dioxane[2',3':4,5]benzo[1,2-d]thiazol-2-yl)propionamide;

[0068] N-(5,6-dimethoxybenzo[d]thiazol-2-yl)-2-((5-methylbenzo[d]oxazol-2-yl)thio)acetamide;

[0069] N-(6,7-Dihydro-[1,4]dioxanyl[2',3':4,5]benzo[1,2-d]thiazol-2-yl)-2-((5-methylbenzo[d]oxazol-2-yl)thio)acetamide;

[0070] 2-((5-fluorobenzo[d]oxazol-2-yl)thio)-N-(5-methoxybenzo[d]thiazol-2-yl)acetamide;

[0071] 2-(Benzo[d]oxazole-2-thio)-N-(5-methoxybenzo[d]thiazol-2-yl)propionamide;

[0072] 2-(Benzo[d]oxazole-2-oxo)-N-(5-methoxybenzo[d]thiazol-2-yl)acetamide;

[0073] N-(5-methoxybenzo[d]thiazol-2-yl)-2-((5-methylbenzo[d]oxazol-2-yl)thio)acetamide;

[0074] 2-((5-methoxybenzo[d]oxazol-2-yl)thio)-N-(5-methoxybenzo[d]thiazol-2-yl)acetamide;

[0075] N-(6,7-dihydro-[1,4]dioxano[2',3':4,5]benzo[1,2-d]thiazol-2-yl)-2-((5-methoxybenzo[d]oxazol-2-yl)thio)acetamide;

[0076] N-(5,6-dimethoxybenzo[d]thiazol-2-yl)-2-((5-methoxybenzo[d]oxazol-2-yl)thio)acetamide;

[0077] 2-(Benzo[d]oxazole-2-thio)-N-(6,7-dihydro-[1,4]dioxanyl[2',3':4,5]benzo[1,2-d]thiazol-2-yl)acetamide;

[0078] 2-(Benzo[d]oxazole-2-thio)-N-(5-methoxyoxybenzo[d]thiazol-2-yl)acetamide;

[0079] 2-(Benzo[d]oxazole-2-thio)-N-(5,6-dimethoxybenzo[d]thiazol-2-yl)acetamide;

[0080] N-(5,6-dimethoxybenzo[d]thiazol-2-yl)-2-((5-chlorobenzo[d]oxazol-2-yl)thio)acetamide;

[0081] 2-((5-chlorobenzo[d]oxazol-2-yl)thio)-N-(5-methoxybenzo[d]thiazol-2-yl)acetamide;

[0082] 2-((5-chlorobenzo[d]oxazol-2-yl)thio)-N-(6,7-dihydro-[1,4]dioxanyl[2',3':4,5]benzo[1,2-d]thiazol-2-yl)acetamide;

[0083] 2-((5-bromobenzo[d]oxazol-2-yl)thio)-N-(6,7-dihydro-[1,4]dioxanyl[2',3':4,5]benzo[1,2-d]thiazol-2-yl)acetamide;

[0084] 2-((5-bromobenzo[d]oxazol-2-yl)thio)-N-(5,6-dimethoxybenzo[d]thiazol-2-yl)acetamide;

[0085] 2-((5-bromobenzo[d]oxazol-2-yl)thio)-N-(5-methoxybenzo[d]thiazol-2-yl)acetamide;

[0086] 3-(Benzo[d]oxazole-2-thio)-N-(5-methoxybenzo[d]thiazol-2-yl)-2,2-dimethylpropionamide;

[0087] 2-((5-(tert-Butyl)benzo[d]oxazol-2-yl)thio)-N-(5-methoxybenzo[d]thiazol-2-yl)acetamide;

[0088] N-(5-methoxybenzo[d]thiazol-2-yl)-2-((5-methylbenzo[d]oxazol-2-yl)thio)propanamide;

[0089] N-(6,7-dihydro-[1,4]dioxane-[2',3':4,5]benzo[1,2-d]thiazol-2-yl)-2-((5-methylbenzo[d]oxazol-2-yl)thio)propionamide;

[0090] 3-(Benzo[d]oxazole-2-thio)-N-(6,7-dihydro-[1,4]dioxane[2',3':4,5]benzo[1,2-d]thiazol-2-yl)-2,2-dimethylpropionamide;

[0091] 2-((5-(tert-Butyl)benzo[d]oxazol-2-yl)thio)-N-(6,7-dihydro-[1,4]dioxanyl[2',3':4,5]benzo[1,2-d]thiazol-2-yl)acetamide.

[0092] Table 1. Preferred compound structures of the present invention

[0093]

[0094]

[0095]

[0096]

[0097]

[0098] <Method for preparing heterocyclic compounds represented by general formula I>

[0099] Another object of the present invention is to provide a method for preparing the heterocyclic compound represented by general formula I, which comprises the following reaction:

[0100]

[0101] The specific steps include:

[0102] (1) Compound 1 undergoes a nucleophilic substitution reaction with a halogen-substituted carboxylic acid compound in the presence of a base and a catalyst to obtain intermediate 2.

[0103] The solvent used in this step can be a mixed solvent selected from one or more of benzene, toluene, chloroform, n-hexane, cyclohexane, dichloromethane, 1,2-dichloroethane, methyl tert-butyl ether, ether, ethyl acetate, tetrahydrofuran, acetone, acetonitrile, ethanol, methanol, N,N-dimethylformamide or dimethyl sulfoxide, preferably N,N-dimethylformamide, tetrahydrofuran, acetonitrile or dichloromethane; the base used can be selected from triethylamine, diisopropylethylamine, pyridine, 4-dimethylaminopyridine, 1,8-diazacyclo[ A mixture of one or more of 5,4,0]undecene-7, sodium carbonate, potassium carbonate, cesium carbonate, sodium bicarbonate, potassium bicarbonate, potassium tert-butoxide, sodium hydride, methylmagnesium chloride, isopropylmagnesium chloride, tert-butylmagnesium chloride, lithium diisopropylamide, and lithium hexamethyldisilazide; the catalyst used can be a mixture of one or more selected from tetrabutylammonium bromide, tetraethylammonium bromide, benzyltriethylammonium bromide, and potassium iodide; the reaction temperature is -20°C to 100°C, and the preferred reaction temperature is 25°C to 80°C.

[0104] (2) Intermediate 2 and compound 3 are condensed in the presence of an activating agent and a base to produce a compound of formula 1.

[0105] The activation reagent used is selected from 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, oxalyl chloride, thionyl chloride, carbonyl diimidazole, chloroformate, 2-(7-benzotriazole oxide)-N,N,N',N'-tetramethyluronium hexafluorophosphate, 1-hydroxybenzotriazole, preferably carbonyl diimidazole; the base used can be selected from triethylamine, diisopropylethylamine, pyridine, 4-dimethylaminopyridine, 1,8-diazacyclo[5,4,0]undecene-7, sodium carbonate, potassium carbonate, bicarbonate A mixture of one or more of sodium, potassium bicarbonate, potassium tert-butoxide, sodium hydride, methylmagnesium chloride, isopropylmagnesium chloride, tert-butylmagnesium chloride, lithium diisopropylamide, and lithium hexamethyldisilazide; the solvent used can be a mixed solvent of one or more selected from dichloromethane, chloroform, tetrahydrofuran, toluene, 1,2-dichloroethane, acetonitrile, N,N-dimethylformamide, or dimethyl sulfoxide, preferably dichloromethane; the temperature used is selected from -20°C to 100°C, and the preferred reaction temperature is 30°C to 70°C;

[0106] In the above reaction formula, A, B, W, Z, J, Q, X, Y, R 1 、R 2 、R 3 The definitions of m, n, and k are the same as those of the heterocyclic compound represented by general formula I.

[0107] It should be noted that the pharmaceutically acceptable salt or solvate of the heterocyclic compound represented by general formula I can be obtained by forming a salt with an acid commonly used in the art, or by solvating with a solvent commonly used in the art by conventional methods. The acid used herein is an acid commonly used in the art, and can be an inorganic acid such as hydrochloric acid, hydrofluoric acid, or hydroiodic acid, or an organic acid such as formic acid, acetic acid, or propionic acid. The solvent used herein is a solvent commonly used in the art, and can be water, methanol, ethanol, N,N-dimethylformamide, dimethyl sulfoxide, ethyl acetate, dichloromethane, etc.

[0108] <Uses of the heterocyclic compound represented by Formula I>

[0109] Another object of the present invention is to provide a heterocyclic compound represented by general formula I or a pharmaceutically acceptable salt or solvate thereof for use in pharmaceutical preparation.

[0110] The heterocyclic compounds described in the present invention can specifically excite PPARδ, significantly upregulate the expression of OPG, upregulate the expression of BMP2, promote osteoblast differentiation, inhibit osteoclast differentiation in vitro, and effectively improve osteoporosis in ovariectomized rats. They provide drug lead compounds and drug candidates for the development of new anti-osteoporosis drugs, and are of great significance for the prevention and / or treatment of osteoporosis and bone metabolism-related diseases.

[0111] <Pharmaceutical Compositions and Preparations Containing Heterocyclic Compounds of Formula I>

[0112] The present invention also provides a pharmaceutical composition for preventing or treating bone metabolic diseases such as osteoporosis, comprising a therapeutically effective amount of a heterocyclic compound represented by Formula I or a pharmaceutically acceptable salt or solvate thereof. The pharmaceutical composition can be in the form of conventional pharmaceutical preparations such as ordinary tablets or capsules, sustained-release tablets or capsules, controlled-release tablets or capsules, granules, powders, syrups, oral solutions, and injections.

[0113] Example

[0114] The following examples are provided to illustrate the present invention in detail. The following examples are provided to better illustrate the present invention and are not intended to limit the scope of the present invention.

[0115] Example 1. Preparation of intermediates

[0116] (1,3-Benzoxazole-2-oxo)acetic acid: Under ice-bath conditions, 2-benzoxazolinone (500 mg, 3.70 mmol, sold by Bidex Pharmaceuticals), chloroacetic acid (350 mg, 3.70 mmol, sold by Bidex Pharmaceuticals), sodium hydride (296 mg, 7.40 mmol, sold by Beijing Coupling), and tetrabutylammonium bromide (36 mg, 0.11 mmol, sold by Bidex Pharmaceuticals) were dissolved in DMF and stirred at 70°C for 10 h. After the disappearance of the starting material by TLC, 1N HCl was added to adjust the pH to 4. The organic phase was separated, and the aqueous phase was extracted with ethyl acetate (25 mL × 3), washed with water and saturated brine in sequence. The organic phases were combined, dried over anhydrous sodium sulfate, the solvent was removed, and purified by column chromatography (petroleum ether:ethyl acetate = 2:1) to obtain a milky white solid with a yield of 62%.

[0117] (1,3-Benzoxazole-2-thio)acetic acid: Under ice bath conditions, 2-mercaptobenzoxazole (1 g, 6.61 mmol, sold by Bidex Pharmaceuticals), chloroacetic acid (625 mg, 6.61 mmol, sold by Bidex Pharmaceuticals), cesium carbonate (2.16 g, 6.61 mmol, sold by Bidex Pharmaceuticals), and tetrabutylammonium bromide (64 mg, 0.198 mmol, sold by Bidex Pharmaceuticals) were dissolved in DMF and stirred at room temperature for 12 h. After the disappearance of the starting material by TLC, 1N HCl was added to adjust the pH to 4. The organic phase was separated, and the aqueous phase was extracted with ethyl acetate (25 mL × 3), washed with water and saturated brine in sequence. The organic phases were combined, dried over anhydrous sodium sulfate, the solvent was removed, and purified by column chromatography (petroleum ether:ethyl acetate = 2:1) to obtain a milky white solid in a yield of 59%.

[0118] (5-Fluoro-[1,3]-benzoxazole-2-thio)acetic acid: Prepared according to the method of (1,3-benzoxazole-2-thio)acetic acid, except replacing 2-mercaptobenzoxazole with 5-fluorobenzo[d]oxazole-2-thiol (sold by Bidex Pharmaceuticals), other conditions were the same to obtain 880 mg of a white solid, with a yield of 43%.

[0119] 2-([1,3]-Benzoxazole-2-thio)propionic acid: Prepared by referring to the method for (1,3-benzoxazole-2-thio)acetic acid, except that chloroacetic acid was replaced with 2-bromopropionic acid (sold by Bidex Pharmaceuticals), other conditions were the same to obtain 961 mg of a white solid with a yield of 43%.

[0120] (5-Methyl-[1,3]-benzoxazole-2-thio)acetic acid: Prepared according to the method of (1,3-benzoxazole-2-thio)acetic acid, except that 2-mercaptobenzoxazole was replaced with 5-methylbenzo[d]oxazole-2(3H)-thione (sold by Bidex Pharmaceuticals). Other conditions were the same to obtain 614 mg of a white solid with a yield of 91%.

[0121] (5-Methoxy-[1,3]-benzoxazole-2-thio)acetic acid: Prepared according to the method of (1,3-benzoxazole-2-thio)acetic acid, except that 2-mercaptobenzoxazole was replaced with 5-methoxybenzo[d]oxazole-2(3H)-thione (sold by Bidex Pharmaceuticals). Other conditions were the same to obtain 345 mg of a light yellow solid with a yield of 87%.

[0122] 2-((5-Fluorobenzo[d]oxazol-2-yl)thio)acetic acid: Prepared according to the method of (1,3-benzoxazole-2-thio)acetic acid, except replacing 2-mercaptobenzoxazole with 5-chlorobenzo[d]oxazole-2(3H)-thione (sold by Bidex Pharmaceuticals), other conditions are the same to obtain 981 mg of an off-white solid with a yield of 85%.

[0123] (5-Bromo-[1,3]-benzoxazole-2-thio)acetic acid: Prepared according to the method of (1,3-benzoxazole-2-thio)acetic acid, except that 2-mercaptobenzoxazole was replaced with 5-bromobenzo[d]oxazole-2(3H)-thione (sold by Bidex Pharmaceuticals), to obtain 865 mg of off-white solid, with a yield of 88%.

[0124] Example 2, N-(5,6-dimethoxybenzo[d]thiazol-2-yl)-2-((5-fluorobenzo[d]oxazol-2-yl)thio)acetamide (Compound I-1)

[0125] 5,6-dimethoxy-1,3-benzothiazol-2-amine (117 mg, 0.55 mmol, sold by Bidex Pharmaceuticals) was dissolved in anhydrous dichloromethane (2 ml) at room temperature. Under ice bath conditions, 2-((5-fluorobenzo[d]oxazol-2-yl)thio)acetic acid (126 mg, 0.55 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (128 mg, 0.67 mmol, sold by Bidex Pharmaceuticals), 1-hydroxybenzotriazole (90 mg, 0.67 mmol, sold by Bidex Pharmaceuticals), and triethylamine (308 μl, 2.22 mmol, sold by Bidex Pharmaceuticals) prepared as described above were added sequentially and stirred at room temperature for 12 h. The reaction was monitored by TLC. After the starting material disappeared, water was added to the system, and dichloromethane was added and extracted repeatedly (10 mL × 3). The organic phases were combined and dried over anhydrous sodium sulfate. The solvent was removed and purified by column chromatography (DCM:EA=100:1) to obtain 75 mg of an off-white solid with a yield of 6-60%.

[0126] 1H NMR (400MHz, DMSO-d6) δ12.65(s,1H),7.69(m,1H),7.58–7.49(m,2H),7.34(s,1H),7.19(m,1H),4.52(s,2H),3.84(s,3H),3.80(s,3H). 13 C NMR(100MHz,DMSO-d6)δ166.25,166.19,161.13,158.76,156.50,149.45,148.40,147.55,142.97 ,142.59,142.45,112.21,111.95,111.53,111.43,105.69,105.43,104.16,104.08,56.40,56.20.

[0127] The obtained compound was identified as compound I-1 based on the spectrum results.

[0128] Example 3, 2-(Benzo[d]oxazole-2-thio)-N-(5,6-dimethoxybenzo[d]thiazole-2-

[0129] 1-[4-[4-[4-(2 ...methyl-1-oxo-2-yl)propionamide)] (Compound I-2)

[0130] Prepared by referring to the method of Example 2, except replacing 2-((5-fluorobenzo[d]oxazol-2-yl)thio)acetic acid with 2-([1,3]-benzoxazole-2-thio)propionic acid, other conditions were the same, to obtain 105 mg, with a yield of 6-60%.

[0131] 1 H NMR(400MHz,DMSO-d6)δ12.72(s,1H),7.72–7.62(m,2H),7.57(s,1H),7.39–7.2 9(m,3H),4.94(q,J=7.0Hz,1H),3.84(s,3H),3.81(s,3H),1.75(d,J=7.1Hz,3H). 13 CNMR(100MHz,DMSO-d6)δ169.78,162.94,156.36,151.71,149.47,147.61,142.96,141.5 6,125.26,125.14,123.49,118.95,110.83,104.13,104.07,56.40,56.20,46.13,19.20.

[0132] The obtained compound was determined to be compound I-2 based on the spectral results.

[0133] Example 4, 2-(Benzo[d]oxazole-2-oxo)-N-(5,6-dimethoxybenzo[d]thiazol-2-yl)acetamide (Compound I-3)

[0134] Prepared by referring to the method of Example 2, except replacing 2-((5-fluorobenzo[d]oxazol-2-yl)thio)acetic acid with (1,3-benzoxazole-2-oxo)acetic acid, other conditions were the same, to obtain 110 mg, with a yield of 6-60%.

[0135] 1 H NMR (400MHz, DMSO-d6) δ12.71(s,1H),7.56(s,1H),7.42-7.40(m,1H),7.34-7.33(m,2H),7.26-7.15(m,2H),4.91(s,2H),3.84-3.80(m,6H).

[0136] The obtained compound was determined to be compound I-3 based on the spectral results.

[0137] Example 5, 2-(Benzo[d]oxazole-2-oxo)-N-(6,7-dihydro-[1,4]dioxane[2',3':4,5]benzo[1,2-d]thiazol-2-yl)acetamide (Compound I-4)

[0138] Prepared by referring to the method of Example 2, except that 2-((5-fluorobenzo[d]oxazol-2-yl)thio)acetic acid was replaced by (1,3-benzoxazole-2-oxo)acetic acid, and 5,6-dimethoxy-1,3-benzothiazol-2-amine was replaced by 6,7-dihydro-[1,4]dioxane[2',3':4,5]benzo[1,2-d]thiazole-2-amine (seller: Bid Pharmaceuticals), other conditions were the same, and 15 mg was obtained with a yield of 6-60%.

[0139] 1 H NMR (400MHz, DMSO-d6) δ12.71(s,1H),7.46(s,1H),7.41-7.39(m,1H),7.33-7.31(m,1H),7.25-7.15(m,3H),4.90(s,2H),4.27(m,4H). 13 C NMR(100MHz,DMSO-d6)δ166.25,156.86,154.47,143.64,142.46,141.97,131 .73,130.13,124.52,123.05,110.25,110.02,109.24,108.45,64.48,44.61.

[0140] The obtained compound was determined to be compound I-4 based on the spectral results.

[0141] Example 6, N-(6,7-dihydro-[1,4]dioxane[2',3':4,5]benzo[1,2-d]thiazol-2-yl)-2-((5-fluorobenzo[d]oxazol-2-yl)thio)acetamide (Compound I-5)

[0142] Prepared by referring to the method of Example 2, except that 5,6-dimethoxy-1,3-benzothiazol-2-amine was replaced with 6,7-dihydro-[1,4]dioxane[2',3':4,5]benzo[1,2-d]thiazole-2-amine (sold by Bid Pharmaceuticals), other conditions were the same, and 95 mg was obtained with a yield of 6-60%.

[0143] 1 H NMR (400MHz, DMSO-d6) δ12.66(s,1H),7.69(dd,J=8.0,3.7Hz,1H),7.53(d,J=8.7Hz,1H),7 .46(d,J=2.0Hz,1H),7.24(s,1H),7.18(dd,J=12.7,5.9Hz,1H),4.51(s,2H),4.28(s,4H). 13 C NMR(100MHz,DMSO-d6)δ166.39,166.21,161.12,158.76,157.10,148.39,143.62,143.56,142 .58,142.44,141.91,112.20,111.94,111.52,111.42,109.20,108.45,105.69,105.43,64.48.

[0144] The obtained compound was determined to be compound I-5 based on the spectral results.

[0145] Example 7, 2-(Benzo[d]oxazole-2-thio)-N-(6,7-dihydro-[1,4]dioxane[2',3':4,5]benzo[1,2-d]thiazol-2-yl)propionamide (Compound I-6)

[0146] Prepared by referring to the method of Example 2, except that 2-((5-fluorobenzo[d]oxazol-2-yl)thio)acetic acid was replaced by 2-([1,3]-benzoxazol-2-thio)propionic acid, and 5,6-dimethoxy-1,3-benzothiazole-2-amine was replaced by 6,7-dihydro-[1,4]dioxane[2',3':4,5]benzo[1,2-d]thiazole-2-amine (seller: Bid Pharmaceuticals), other conditions were the same, and 86 mg was obtained with a yield of 6-60%.

[0147] 1 H NMR (400MHz, CDCl3) δ11.39(s,1H),7.80(d,J=7.6Hz,1H),7.47(d,J=8.1Hz,1H),7.34(dtd,J=25.7, 7.6,1.1Hz,2H),7.26(s,1H),7.24(s,1H),4.65(q,J=7.3Hz,1H),4.28(s,4H),1.76(d,J=7.4Hz,3H). 13 C NMR(100MHz,DMSO-d6)δ169.98,162.90,156.99,151.70,143.65,141.98,14 1.56,125.25,125.13,118.95,110.82,109.21,108.46,64.48,46.15,19.14.

[0148] The obtained compound was determined to be compound I-6 based on the spectral results.

[0149] Example 8, N-(5,6-dimethoxybenzo[d]thiazol-2-yl)-2-((5-methylbenzo[d]oxazol-2-yl)thio)acetamide (Compound I-7)

[0150] Prepared by referring to the method of Example 2, except replacing 2-((5-fluorobenzo[d]oxazol-2-yl)thio)acetic acid with (5-methyl-[1,3]-benzoxazol-2-thio)acetic acid, other conditions were the same, to obtain 80 mg, with a yield of 6-60%.

[0151] 1 H NMR(400MHz,DMSO-d6)δ12.63(s,1H),7.57-7.49(m,2H),7.42(s,1H),7.34( s,1H),7.16-7.10(m,1H),4.50(s,2H),3.82(d,J=14.4Hz,6H),2.38(s,3H). 13 C NMR(100MHz,DMSO-d6)δ166.36,163.87,150.13,149.44,147.54,142.98,141.80 ,134.62,125.70,123.35,118.71,110.13,104.16,104.08,56.40,56.20,21.37.

[0152] The obtained compound was determined to be compound I-7 based on the spectral results.

[0153] Example 9, N-(6,7-dihydro-[1,4]dioxane[2',3':4,5]benzo[1,2-d]thiazol-2-yl)-2-((5-methylbenzo[d]oxazol-2-yl)thio)acetamide (Compound I-8)

[0154] Prepared by referring to the method of Example 2, except that 2-((5-fluorobenzo[d]oxazol-2-yl)thio)acetic acid was replaced by (5-methyl-[1,3]-benzoxazol-2-thio)acetic acid, and 5,6-dimethoxy-1,3-benzothiazol-2-amine was replaced by 6,7-dihydro-[1,4]dioxane[2',3':4,5]benzo[1,2-d]thiazol-2-amine (seller: Bid Pharmaceuticals), other conditions were the same, and 50 mg was obtained with a yield of 6 to 60%.

[0155] 1 H NMR(400MHz,DMSO-d6)δ12.63(s,1H),7.52(d,J=8.3Hz,1H),7.45(s,1H),7.41(s,1 H),7.24(s,1H),7.13(dd,J=8.3,1.1Hz,1H),4.49(s,2H),4.27(s,4H),2.38(s,3H). 13 C NMR(100MHz,DMSO-d6)δ166.58,163.83,157.11,150.13,143.62,141.90,141.79 ,134.61,125.70,124.66,118.71,110.13,109.19,108.45,64.48,36.00,21.36.

[0156] The obtained compound was determined to be compound I-8 based on the spectral results.

[0157] Example 10, 2-((5-fluorobenzo[d]oxazol-2-yl)thio)-N-(5-methoxybenzo[d]thiazol-2-yl)acetamide (Compound I-9)

[0158] Prepared by referring to the method of Example 2, except that 5,6-dimethoxy-1,3-benzothiazol-2-amine was replaced by 5-methoxy-1,3-benzothiazol-2-amine (sold by Bid Pharmaceuticals), other conditions were the same, and 67 mg was obtained with a yield of 6-60%.

[0159] 1H NMR (400MHz, DMSO-d6) δ12.77(s,1H),7.84(d,J=8.7Hz,1H),7.70(dd,J=8.9,4.3Hz,1H),7.53(dd,J=8.8,2.6Hz ,1H),7.32(d,J=2.3Hz,1H),7.19(td,J=9.4,2.6Hz,1H),6.95(dd,J=8.7,2.5Hz,1H),4.53(s,2H),3.83(s,3H). 13 C NMR (100MHz, DMSO-d6) δ166.63,166.21,159.17,148.41,122.63,113.48,112.22,111.96,111.55,111.44,105.70,105.44,104.64,55.91,36.14.

[0160] The obtained compound was determined to be compound I-9 based on the spectral results.

[0161] Example 11, 2-(Benzo[d]oxazole-2-thio)-N-(5-methoxybenzo[d]thiazol-2-yl)propionamide (Compound I-10)

[0162] Prepared by referring to the method of Example 2, except that 5,6-dimethoxy-1,3-benzothiazol-2-amine was replaced by 5-methoxy-1,3-benzothiazol-2-amine (seller: Bid Pharmaceuticals), and (5-fluoro-[1,3]-benzoxazole-2-thio)acetic acid was replaced by 2-([1,3]-benzoxazole-2-thio)propionic acid, other conditions were the same, and 58 mg was obtained with a yield of 6-60%.

[0163] 1 H NMR (400MHz, DMSO-d6) δ12.84 (s, 1H), 7.85 (d, J = 8.7Hz, 1H), 7.71-7.61 (m, 2H), 7.40-7.29 (m ,3H),6.97(dd,J=8.7,2.5Hz,1H),4.95(q,J=7.0Hz,1H),3.83(s,3H),1.76(d,J=7.1Hz,3H). 13 C NMR (100MHz, DMSO-d6) δ170.23,162.88,159.17,151.71,141.55,125.27,125.16,122.66,118.96,113.55,110.83,104.63,55.91,46.14,19.09.

[0164] The obtained compound was determined to be compound I-10 based on the spectral results.

[0165] Example 12, 2-(Benzo[d]oxazole-2-oxo)-N-(5-methoxybenzo[d]thiazol-2-yl)acetamide (Compound I-11)

[0166] Prepared by referring to the method of Example 2, except that 2-((5-fluorobenzo[d]oxazol-2-yl)thio)acetic acid was replaced by 2-(benzo[d]oxazol-2-oxo)acetic acid, and 5,6-dimethoxy-1,3-benzothiazol-2-amine was replaced by 5-methoxy-2-benzothiazolamine (seller: Bid Pharmaceuticals), other conditions were the same, and 46 mg was obtained with a yield of 6-60%.

[0167] 1 H NMR (400MHz, DMSO-d6) δ12.83(s,1H),7.84(d,J=8.7Hz,1H),7.40(d,J=7.8Hz,1H),7.34(d,J=7 .9Hz,2H),7.20(dt,J=24.1,7.7Hz,2H),6.96(dd,J=8.7,2.4Hz,1H),4.93(s,2H),3.83(s,3H). 13 C NMR(100MHz,DMSO-d6)δ166.49,159.17,158.93,154.45,150.35,142.48,131.79 ,124.50,123.63,123.01,122.66,113.54,110.25,110.08,104.67,55.91,44.70.

[0168] The obtained compound was determined to be compound I-11 based on the spectral results.

[0169] Example 13, N-(5-methoxybenzo[d]oxazol-2-yl)-2-((5-methylbenzo[d]thiazol-2-yl)thio)acetamide (Compound I-12)

[0170] Prepared by referring to the method of Example 2, except that 2-((5-fluorobenzo[d]oxazol-2-yl)thio)acetic acid was replaced by 2-((5-methylbenzo[d]oxazol-2-yl)thio)acetic acid, and 5,6-dimethoxy-1,3-benzothiazol-2-amine was replaced by 5-methoxy-2-benzothiazolamine (seller: Bid Pharmaceuticals), other conditions were the same, and 43 mg was obtained with a yield of 6-60%.

[0171] 1H NMR (400MHz, DMSO-d6) δ12.75(s,1H),7.83(d,J=8.4Hz,1H),7.52(d,J=7.8Hz,1H),7.41(s,1H), 7.33(s,1H),7.13(d,J=7.3Hz,1H),6.95(d,J=7.2Hz,1H),4.51(s,2H),3.83(s,3H),2.38(s,3H). 13 C NMR(100MHz,DMSO-d6)δ166.89,163.80,159.15,150.11,141.74,134.67,12 5.74,123.59,122.62,118.68,113.45,110.12,104.58,55.90,35.98,21.34.

[0172] The obtained compound was determined to be compound I-12 based on the spectral results.

[0173] Example 14, 2-((5-methoxybenzo[d]oxazol-2-yl)thio)-N-(5-methoxybenzo[d]thiazol-2-yl)acetamide (Compound I-13)

[0174] Prepared by referring to the method of Example 2, except that 2-((5-fluorobenzo[d]oxazol-2-yl)thio)acetic acid was replaced by 2-((5-methoxybenzo[d]oxazol-2-yl)thio)acetic acid, and 5,6-dimethoxy-1,3-benzothiazol-2-amine was replaced by 5-methoxy-2-benzothiazolamine (seller: Bid Pharmaceuticals), other conditions were the same, and 33 mg was obtained with a yield of 6-60%.

[0175] 1 H NMR (400MHz, DMSO-d6) δ12.75(s,1H),7.84(d,J=8.7Hz,1H),7.55(d,J=8.9Hz,1H),7.32(d,J=2.4Hz,1H),7.18( d,J=2.5Hz,1H),6.95(dd,J=8.7,2.5Hz,1H),6.89(dd,J=8.9,2.6Hz,1H),4.51(s,2H),3.83(s,3H),3.77(s,3H). 13 C NMR(100MHz,DMSO-d6)δ166.78,164.44,159.16,157.47,150.38,146.44,142.55 ,123.63,122.62,113.45,112.48,110.87,104.62,102.61,56.25,55.91,36.07.

[0176] The obtained compound was determined to be compound I-13 based on the spectral results.

[0177] Example 15, N-(6,7-dihydro-[1,4]dioxane[2',3':4,5]benzo[1,2-d]thiazol-2-yl)-2-((5-methoxybenzo[d]oxazol-2-yl)thio)acetamide (Compound I-14)

[0178] Prepared by referring to the method of Example 2, except that 2-((5-fluorobenzo[d]oxazol-2-yl)thio)acetic acid was replaced by 2-((5-methoxybenzo[d]oxazol-2-yl)thio)acetic acid, and 5,6-dimethoxy-1,3-benzothiazol-2-amine was replaced by 6,7-dihydro-[1,4]dioxane[2',3':4,5]benzo[1,2-d]thiazol-2-amine (seller: Bid Pharmaceuticals), other conditions were the same, and 53 mg was obtained with a yield of 6-60%.

[0179] 1 H NMR(400MHz,DMSO-d6)δ12.63(s,1H),7.54(d,J=8.9Hz,1H),7.46(s,1H),7.24(s,1H),7 .18(d,J=2.5Hz,1H),6.89(dd,J=8.9,2.6Hz,1H),4.48(s,2H),4.27(s,4H),3.77(s,3H). 13 C NMR(100MHz,DMSO-d6)δ166.52,164.44,157.46,146.43,143.62,142.55,141.9 1,124.65,112.48,110.87,109.20,108.44,102.61,64.48,56.26,36.01,14.55.

[0180] The obtained compound was determined to be compound I-14 based on the spectral results.

[0181] Example 16, N-(5,6-dimethoxybenzo[d]thiazol-2-yl)-2-((5-methoxybenzo[d]oxazol-2-yl)thio)acetamide (Compound I-15)

[0182] Prepared by referring to the method of Example 2, except replacing 2-((5-fluorobenzo[d]oxazol-2-yl)thio)acetic acid with 2-((5-methoxybenzo[d]oxazol-2-yl)thio)acetic acid, other conditions were the same, to obtain 34 mg, with a yield of 6-60%.

[0183] 1H NMR (400MHz, DMSO-d6) δ12.64(s,1H),7.55(t,J=4.3Hz,2H),7.34(s,1H),7.18(d,J=2.5 Hz,1H),6.89(dd,J=8.9,2.5Hz,1H),4.49(s,2H),3.84(s,3H),3.80(s,3H),3.77(s,3H). 13 C NMR(100MHz,DMSO-d6)δ166.32,164.47,157.47,156.54,149.44,147.54,146.43,142.9 7,142.56,123.35,112.47,110.86,104.16,104.08,102.61,56.40,56.25,56.20,35.98.

[0184] The obtained compound was determined to be compound I-15 based on the spectral results.

[0185] Example 17, 2-(Benzo[d]oxazole-2-thio)-N-(6,7-dihydro-[1,4]dioxane[2',3':4,5]benzo[1,2-d]thiazol-2-yl)acetamide (Compound I-16)

[0186] Prepared by referring to the method of Example 2, except that 2-((5-fluorobenzo[d]oxazol-2-yl)thio)acetic acid was replaced by 2-(benzo[d]oxazole-2-thio)acetic acid, and 5,6-dimethoxy-1,3-benzothiazol-2-amine was replaced by 6,7-dihydro-[1,4]dioxane[2',3':4,5]benzo[1,2-d]thiazol-2-amine (seller: Bid Pharmaceuticals), other conditions were the same, and 90 mg was obtained with a yield of 6-60%.

[0187] 1 H NMR (400MHz, DMSO-d6) δ = 12.65 (s, 1H), 7.68-7.60 (m, 2H), 7.45 (s, 1H), 7.35-7.31 (m, 2H), 7.24 (s, 1H), 4.50 (s, 2H), 4.27 (s, 4H). 13 C NMR(100MHz,DMSO-d6)δ166.54,163.98,157.12,151.87,143.62,143.57,141.91 ,141.60,125.20,124.91,124.66,118.79,110.76,109.19,108.46,64.48,36.03.

[0188] The obtained compound was determined to be compound I-16 based on the spectral results.

[0189] Example 18, 2-(Benzo[d]oxazole-2-thio)-N-(5-methoxybenzo[d]thiazol-2-yl)acetamide (Compound I-17)

[0190] Prepared by referring to the method of Example 2, except that 2-((5-fluorobenzo[d]oxazol-2-yl)thio)acetic acid was replaced by 2-(benzo[d]oxazole-2-thio)acetic acid, and 5,6-dimethoxy-1,3-benzothiazol-2-amine was replaced by 5-methoxy-2-benzothiazolamine (seller: Bid Pharmaceuticals), other conditions were the same, and 51 mg was obtained with a yield of 6-60%.

[0191] 1 H NMR (400MHz, DMSO-d6) δ12.77(s,1H),7.85-7.82,7.69(d,1H),7.68-7.60(m,2H),7.35-7.31(m,3H),6.97-6.94(m,1H),4.53(s,2H),3.83(s,3H). 13 C NMR (100MHz, DMSO-d6) δ166.84,163.95,159.15,151.85,141.54,125.24,124.96,123.60,122.63,118.77,113.47,110.76,104.59,55.90,36.01.

[0192] According to the spectral results, the obtained compound was determined to be compound I-17.

[0193] Example 19, 2-(Benzo[d]oxazole-2-thio)-N-(5,6-dimethoxybenzo[d]thiazol-2-yl)acetamide (Compound I-18)

[0194] Prepared by referring to the method of Example 2, except replacing 2-((5-fluorobenzo[d]oxazol-2-yl)thio)acetic acid with 2-(benzo[d]oxazole-2-thio)acetic acid, other conditions were the same, to obtain 44 mg, with a yield of 6-60%.

[0195] 1 H NMR (400MHz, DMSO-d6) δ12.65(s,1H),7.68-7.61(m,2H),7.55(s,1H),7.36-7.31(m,3H),4.52(s,2H),3.84(s,3H),3.80(s,3H). 13C NMR(100MHz,DMSO-d6)δ166.41,163.98,156.57,151.84,149.42,147.53,142.87,14 1.55,125.24,124.96,123.33,118.76,110.75,104.09,104.00,56.38,56.19,35.93.

[0196] The obtained compound was determined to be compound I-18 based on the spectral results.

[0197] Example 20, N-(5,6-dimethoxybenzo[d]thiazol-2-yl)-2-((5-chlorobenzo[d]oxazol-2-yl)thio)acetamide (Compound I-19)

[0198] Prepared by referring to the method of Example 2, except replacing 2-((5-fluorobenzo[d]oxazol-2-yl)thio)acetic acid with 2-((5-chlorobenzo[d]oxazol-2-yl)thio)acetic acid, other conditions were the same, to obtain 17 mg, with a yield of 6-60%.

[0199] 1 H NMR(400MHz,DMSO-d6)δ12.65(s,1H),7.75(m,1H),7.72-7.69(m,1H),7.5 5(s,1H),7.39-7.36(m,1H),7.34(s,1H),4.53(s,2H),3.84-3.80(m,6H). 13 C NMR (100MHz, DMSO-d6) δ166.10,150.67,149.45,147.55,142.90,129.50,124.86,123.34,118.59,112.06,104.17,56.40,56.21,36.13.

[0200] The obtained compound was determined to be compound I-19 based on the spectral results.

[0201] Example 21, 2-((5-chlorobenzo[d]oxazol-2-yl)thio)-N-(5-methoxybenzo[d]thiazol-2-yl)acetamide (Compound I-20)

[0202] Prepared by referring to the method of Example 2, except that 2-((5-fluorobenzo[d]oxazol-2-yl)thio)acetic acid was replaced by 2-((5-chlorobenzo[d]oxazol-2-yl)thio)acetic acid, and 5,6-dimethoxy-1,3-benzothiazol-2-amine was replaced by 5-methoxy-2-benzothiazolamine (seller: Bid Pharmaceuticals), other conditions were the same, and 22 mg was obtained with a yield of 6-60%.

[0203] 1 H NMR(400MHz,DMSO-d6)δ12.77(s,1H),7.85-7.83(m,1H),7.75-7.74(m,1H),7.71-7.69(m ,1H),7.39-7.36(m,1H),7.33-7.32(m,1H),6.97-6.94(m,1H),4.54(s,2H),3.83(s,3H). 13 C NMR (100MHz, DMSO-d6) δ166.06,159.17,150.68,142.88,129.50,124.86,122.63,118.59,113.47,112.06,104.62,55.91,36.22.

[0204] The obtained compound was determined to be compound I-20 based on the spectral results.

[0205] Example 22, 2-((5-chlorobenzo[d]oxazol-2-yl)thio)-N-(6,7-dihydro-[1,4]dioxane[2',3':4,5]benzo[1,2-d]thiazol-2-yl)acetamide (Compound I-21)

[0206] Prepared by referring to the method of Example 2, except that 2-((5-fluorobenzo[d]oxazol-2-yl)thio)acetic acid was replaced by 2-((5-chlorobenzo[d]oxazol-2-yl)thio)acetic acid, and 5,6-dimethoxy-1,3-benzothiazol-2-amine was replaced by 6,7-dihydro-[1,4]dioxane[2',3':4,5]benzo[1,2-d]thiazol-2-amine (seller: Bid Pharmaceuticals), other conditions were the same, and 53 mg was obtained with a yield of 6-60%.

[0207] 1 H NMR(400MHz,DMSO-d6)δ12.65(s,1H),7.75-7.74(m,1H),7.71-7.69(m,1H) ),7.46(s,1H),7.39-7.36(m,1H),7.24(s,1H),4.51(s,2H),4.27(s,4H). 13 C NMR(100MHz,DMSO-d6)δ166.06,150.67,143.63,142.88,141.92,129.49,124.85 ,124.64,121.99,118.59,112.05,111.29,110.30,109.21,108.43,64.48,36.16.

[0208] The obtained compound was determined to be compound I-21 based on the spectral results.

[0209] Example 23, 2-((5-bromobenzo[d]oxazol-2-yl)thio)-N-(6,7-dihydro-[1,4]dioxane[2',3':4,5]benzo[1,2-d]thiazol-2-yl)acetamide (Compound I-22)

[0210] Prepared by referring to the method of Example 2, except that 2-((5-fluorobenzo[d]oxazol-2-yl)thio)acetic acid was replaced by 2-((5-bromobenzo[d]oxazol-2-yl)thio)acetic acid, and 5,6-dimethoxy-1,3-benzothiazol-2-amine was replaced by 6,7-dihydro-[1,4]dioxane[2',3':4,5]benzo[1,2-d]thiazol-2-amine (seller: Bid Pharmaceuticals), other conditions were the same, and 27 mg was obtained with a yield of 6-60%.

[0211] 1 H NMR (400MHz, DMSO-d6) δ12.65(s,1H),7.87-7.86(m,1H),7.66-7.64(m,1H),7.51-7.46(m,2H),7.24(s,1H),4.51(s,2H),4.27(s,4H). 13 C NMR (100MHz, DMSO-d6) δ165.87,157.08,151.05,143.63,143.33,141.92,127.59,124.66,121.45,117.23,112.54,109.20,108.46,64.48,36.16.

[0212] The obtained compound was determined to be compound I-22 based on the spectral results.

[0213] Example 24, 2-((5-bromobenzo[d]oxazol-2-yl)thio)-N-(5,6-dimethoxybenzo[d]thiazol-2-yl)acetamide (Compound I-23)

[0214] Prepared by referring to the method of Example 2, except replacing 2-((5-fluorobenzo[d]oxazol-2-yl)thio)acetic acid with 2-((5-bromobenzo[d]oxazol-2-yl)thio)acetic acid, other conditions were the same, to obtain 29 mg, with a yield of 6-60%.

[0215] 1H NMR(400MHz,DMSO-d6)δ12.65(s,1H),7.88-7.87(m,1H),7.67-7.65(m,1H), 7.55(s,1H),7.51-7.48(m,1H),7.34(s,1H),4.53(s,2H),3.84-3.80(m,6H). 13 C NMR(100MHz,DMSO-d6)δ166.20,165.90,156.54,151.03,149.43,147.54,143.30,142.86,1 27.61,123.33,121.40,117.23,112.54,104.10,104.00,56.39,56.19,36.05,31.59,30.27

[0216] The obtained compound was determined to be compound I-23 based on the spectral results.

[0217] Example 25, 2-((5-bromobenzo[d]oxazol-2-yl)thio)-N-(5-methoxybenzo[d]thiazol-2-yl)acetamide (Compound I-24)

[0218] Prepared by referring to the method of Example 2, except that 2-((5-fluorobenzo[d]oxazol-2-yl)thio)acetic acid was replaced by 2-((5-bromobenzo[d]oxazol-2-yl)thio)acetic acid, and 5,6-dimethoxy-1,3-benzothiazol-2-amine was replaced by 5-methoxy-2-benzothiazolamine (seller: Bid Pharmaceuticals), other conditions were the same, and 31 mg was obtained with a yield of 6-60%.

[0219] 1 H NMR(400MHz,DMSO-d6)δ12.77(s,1H),7.87-7.83(m,2H),7.67-7.64(m,1H),7.5 1-7.48(m,1H),7.33-7.32(m,1H),6.97-6.94(m,1H),4.54(s,2H),3.83(s,3H). 13 C NMR(100MHz,DMSO-d6)δ166.66,165.86,159.15,151.03,150.28,143.29,127.61 ,123.59,122.63,121.41,117.23,113.48,112.54,104.58,55.90,36.14,31.59.

[0220] The obtained compound was determined to be compound I-24 based on the spectral results.

[0221] Example 26, 3-(Benzo[d]oxazole-2-thio)-N-(5-methoxybenzo[d]thiazol-2-yl)-2,2-dimethylpropionamide (Compound I-25)

[0222] Prepared by referring to the method of Example 2, except that 2-((5-fluorobenzo[d]oxazol-2-yl)thio)acetic acid was replaced by 3-(benzo[d]oxazol-2-thio)-2,2-dimethylpropionic acid, and 5,6-dimethoxy-1,3-benzothiazol-2-amine was replaced by 5-methoxy-2-benzothiazolamine (sold by Bid Pharmaceuticals), other conditions were the same, and 56 mg was obtained with a yield of 6-60%.

[0223] 1 H NMR(400MHz,DMSO-d6)δ12.34(s,1H),7.84-7.82(m,1H),7.63-7.55(m,2H), 7.32-7.25(m,3H),6.97-6.94(m,1H),3.83(s,3H),3.79(s,2H),1.46(s,6H). 13 C NMR(100MHz,DMSO-d6)δ175.52,165.17,159.97,159.07,151.74,150.25,141.54,125.0 4,124.70,123.74,122.52,118.58,113.11,110.62,104.43,55.89,44.34,41.15,24.08.

[0224] The obtained compound was determined to be compound I-25 based on the spectral results.

[0225] Example 27, 2-((5-(tert-butyl)benzo[d]oxazol-2-yl)thio)-N-(5-methoxybenzo[d]thiazol-2-yl)acetamide (Compound I-26)

[0226] Prepared by referring to the method of Example 2, except that 2-((5-fluorobenzo[d]oxazol-2-yl)thio)acetic acid was replaced by 2-((5-tert-butylbenzo[d]oxazol-2-yl)thio)acetic acid, and 5,6-dimethoxy-1,3-benzothiazol-2-amine was replaced by 5-methoxy-2-benzothiazolamine (seller: Bid Pharmaceuticals), other conditions were the same, and 49 mg was obtained with a yield of 6-60%.

[0227] 1H NMR(400MHz,DMSO-d6)δ12.76(s,1H),7.85-7.83(m,1H),7.59-7.54(m,2H),7.3 8-7.32(m,2H),6.97-6.94(m,1H),4.51(s,2H),3.84(s,3H),1.31-1.30(m,9H). 13 C NMR(100MHz,DMSO-d6)δ166.81,163.82,159.15,149.90,148.25,141.54,123.5 9,122.63,122.30,115.26,113.45,109.88,104.59,55.90,36.02,35.09,31.92.

[0228] According to the spectral results, the obtained compound was determined to be compound I-26.

[0229] Example 28, N-(5-methoxybenzo[d]thiazol-2-yl)-2-((5-methylbenzo[d]oxazol-2-yl)thio)propionamide (Compound I-27)

[0230] Prepared by referring to the method of Example 2, except that 2-((5-fluorobenzo[d]oxazol-2-yl)thio)acetic acid was replaced by 2-((5-methylbenzo[d]oxazol-2-yl)thio)propionic acid, and 5,6-dimethoxy-1,3-benzothiazol-2-amine was replaced by 5-methoxy-2-benzothiazolamine (seller: Bid Pharmaceuticals), other conditions were the same, and 29 mg was obtained with a yield of 6-60%.

[0231] 1 H NMR(400MHz,DMSO-d6)δ12.81(s,1H),7.86-7.84(m,1H),7.54-7.52(m,1H),7.45(s,1H),7.32-7.31(m,1 H),7.16-7.14(m,1H),6.98-6.95(m,1H),4.95-4.90(m,1H),3.83(s,3H),2.39(s,3H),1.75-1.73(m,3H). 13 C NMR(100MHz,DMSO-d6)δ170.39,162.69,159.16,149.95,141.70,134.76,126.01,123.67,122.67,118.8 3,113.55,110.20,104.57,55.91,46.00,40.45,40.24,40.03,39.82,39.61,39.41,39.20,21.34,18.94.

[0232] The obtained compound was determined to be compound I-27 based on the spectral results.

[0233] Example 29, N-(6,7-dihydro-[1,4]dioxane[2',3':4,5]benzo[1,2-d]thiazol-2-yl)-2-((5-methylbenzo[d]thiazol-2-yl)thio)propionamide (Compound I-28)

[0234] Prepared by referring to the method of Example 2, except that 2-((5-fluorobenzo[d]oxazol-2-yl)thio)acetic acid was replaced by 2-((5-methylbenzo[d]oxazol-2-yl)thio)propionic acid, and 5,6-dimethoxy-1,3-benzothiazol-2-amine was replaced by 6,7-dihydro-[1,4]dioxane[2',3':4,5]benzo[1,2-d]thiazol-2-amine (seller: Bid Pharmaceuticals), other conditions were the same, and 32 mg was obtained with a yield of 6-60%.

[0235] 1 H NMR(400MHz,DMSO-d6)δ12.70(s,1H),7.53-7.51(m,1H),7.47(s,1H),7.45(s,1H),7.24( s,1H),7.16-7.14(m,1H),4.93-4.87(m,1H),4.28(s,4H),2.39(s,3H),1.74-1.72(m,3H). 13 C NMR(100MHz,DMSO-d6)δ170.11,162.71,157.07,149.94,143.64,141.97,141.70,13 4.74,125.99,124.71,118.83,110.19,109.21,108.41,64.48,46.01,21.34,19.00.

[0236] The obtained compound was determined to be compound I-28 based on the spectral results.

[0237] Example 30, 3-(Benzo[d]oxazole-2-thio)-N-(6,7-dihydro-[1,4]dioxane[2',3':4,5]benzo[1,2-d]thiazol-2-yl)-2,2-dimethylpropionamide (Compound I-29)

[0238] Prepared by referring to the method of Example 2, except that 2-((5-fluorobenzo[d]oxazol-2-yl)thio)acetic acid was replaced by 3-(benzo[d]oxazol-2-thio)-2,2-dimethylpropionic acid, and 5,6-dimethoxy-1,3-benzothiazol-2-amine was replaced by 6,7-dihydro-[1,4]dioxane[2',3':4,5]benzo[1,2-d]thiazol-2-amine (seller: Bid Pharmaceuticals), other conditions were the same, and 30 mg was obtained with a yield of 6 to 60%.

[0239] 1 H NMR (400MHz, DMSO-d6) δ12.22(s,1H),7.61-7.57(m,2H),7.44(s,1H),7.33-7.28(m,2H),7.20(s,1H),4.28(s,4H),3.77(s,2H),1.45(s,6H). 13 C NMR(100MHz,DMSO-d6)δ175.30,165.18,157.85,151.72,143.52,141.78,141.5 1,125.05,124.72,118.57,110.61,109.06,108.12,64.48,44.24,41.15,24.09.

[0240] The obtained compound was determined to be compound I-29 based on the spectral results.

[0241] Example 31, 2-((5-(tert-butyl)benzo[d]thiazol-2-yl)thio)-N-(6,7-dihydro-[1,4]dioxane[2',3':4,5]benzo[1,2-d]thiazol-2-yl)acetamide (Compound I-30)

[0242] Prepared by referring to the method of Example 2, except that 2-((5-fluorobenzo[d]oxazol-2-yl)thio)acetic acid was replaced by 2-((5-tert-butylbenzo[d]oxazol-2-yl)thio)acetic acid, and 5,6-dimethoxy-1,3-benzothiazol-2-amine was replaced by 6,7-dihydro-[1,4]dioxane[2',3':4,5]benzo[1,2-d]thiazol-2-amine (seller: Bid Pharmaceuticals), other conditions were the same, and 22 mg was obtained with a yield of 6 to 60%.

[0243] 1H NMR(400MHz,DMSO-d6)δ12.64(s,1H),7.59-7.58(m,1H),7.56-7.53(m,1H),7.4 5(s,1H),7.38-7.36(m,1H),7.24(s,1H),4.48(s,2H),4.27(s,4H),1.31(s,9H). 13 C NMR(100MHz,DMSO-d6)δ166.59,163.82,157.21,149.90,148.26,147.64,143.61,141.90,141.53,1 30.12,124.61,122.30,115.25,109.88,109.19,108.40,64.48,35.09,31.92,31.59,30.27,14.40.

[0244] The obtained compound was determined to be compound I-30 based on the spectral results.

[0245] Example 32. Determination of the dose-effect relationship of compounds in the OPG expression regulation screening model

[0246] A high-throughput screening model for OPG expression upregulators constructed in the laboratory was used for high-throughput screening. This model was constructed by transfecting U-2OS cells with the OPG promoter-regulated firefly luciferase expression reporter gene plasmid pGL4.17-OPGp to construct a stably transfected cell line [Development of a High-Throughput Screening Strategy for Upregulators of the OPG / RANKL Ratio with the Potential for Antiosteoporosis Effects. J Biomol Screen. 2016 Aug; 21(7): 738-48. doi: 10.1177 / 1087057116654657] named UOP. This stably transfected cell line can use the expression of luciferase to reflect the degree of promoter activation or inhibition in the cell, thereby being used to screen compounds with the potential to upregulate OPG. The model cells UOP were inoculated into 96-well transparent bottom white plates. After cells adhere, discard the culture medium and add 198 μL of serum-free Mccoy's 5A medium and 2 μL of the test sample (compound concentration ranges from 0 to 100 μM) to each well. Use 1% DMSO as a blank control. After 18-24 hours, aspirate the culture medium and measure luciferase activity using the Luciferase Assay System.

[0247] Osteoprotegerin (OPG) plays an important role in regulating bone remodeling. OPG is produced by bone marrow mesenchymal stem cells and osteoblasts, etc. It can act as a soluble decoy receptor for RANKL, competitively binding to RANKL, preventing RANKL from binding to RANK, and then blocking osteoblast-induced osteoclast precursor differentiation and fusion, thereby inhibiting osteoclast differentiation and further inhibiting bone resorption. As can be seen from Table 2, compounds I-2, I-4, I-8, I-10, I-11, I-14, I-16, and I-17 have the activity of upregulating OPG expression, indicating that these compounds have the ability to promote osteogenesis and inhibit osteoclastogenesis and regulate bone metabolism.

[0248] Table 2 Some compounds upregulate the activity of OPG

[0249] Cpds. Compound number Maximum increase rate (Max%) I-2 170 I-4 146 I-8 206 I-10 315 I-11 160 I-14 170 I-16 196 I-17 143

[0250] Example 33. Determination of the dose-effect relationship of compounds in the BMP2 expression regulation screening model

[0251] A stable transfected cell model expressing the mouse BMP-2 upstream regulatory sequence-luciferase reporter gene was constructed and named PMB [Identification of upregulators of BMP2 expression via high-throughput screening of a synthetic and natural compound library, J Biomol Screen. 2009 Dec; 14(10): 1251-6. doi: 10.1177 / 1087057109346446]. This model was used to screen compounds with the potential to upregulate BMP2. The PMB model cells were seeded into 96-well clear-bottom white plates. After the cells adhered, the original culture medium was discarded, and 198 μL of serum-free DMEM culture medium and 2 μL of the test sample (compound concentration 0-100 μM) were added to each well. 1% DMSO was used as a blank control. After 18-24 hours, the culture medium was aspirated and the luciferase activity was detected using the Luciferase Assay System.

[0252] As can be seen from Table 3, compounds I-2, I-4, I-9, I-12, I-13, I-15, I-16, I-26, I-27, and I-29 have the activity of upregulating BMP2 expression.

[0253] Table 3 Some compounds upregulate BMP2 activity

[0254] Cpds. Compound number Maximum increase rate (Max%) I-2 139 I-4 145 I-9 190 I-12 236 I-13 143 I-15 171 I-16 180 I-26 330 I-27 205 I-29 135

[0255] Example 34. Testing of Compounds for PPARδ Agonist Activity

[0256] One day before transfection, 5 × 10 4 COS-7 cells were seeded in 96-well plates at a density of 100 μL / well. A monolayer of cells approximately 90% confluent was achieved by transfection. A total of 200 ng of plasmid was diluted with 25 μL of culture medium per well. 18 ng of pBIND-hPPARδ-LBD plasmid and 180 ng of GAL4UAS-Luc luciferase reporter gene plasmid were diluted at a 1:10 ratio in serum-free α-MEM medium. [A pan-PPAR agonist E17241 ameliorates hyperglycaemia and diabetic dyslipidemia in KKAy mice via up-regulating ABCA1 in islet, liver, and white adipose tissue, Biomed Pharmacother. 2024 Feb 2:172:116220. doi:10.1016 / j.biopha.2024.116220] Dilute 0.5 μL of Lipofectamine into each well with 25 μL of culture medium. TM Dilute liposome Lipofectamine with serum-free Opti-MEM medium to a volume of 2000 TM 2000, incubate at room temperature for 5 minutes. Gently mix the above-mentioned plasmid and liposome dilutions together and incubate at room temperature for 20-30 minutes. The complex can be stable at room temperature for 6 hours. Add complete culture medium to the mixture of plasmid and liposome at a volume of 100 μL per well and mix gently. Aspirate the original culture medium in the 96-well plate and add the transfection mixture obtained in the previous step at a volume of 150 μL per well. After 4 to 6 hours, aspirate the transfection mixture and add the compound diluted to 10 μM with serum-free culture medium. Incubate at 37°C for 18 to 24 hours, and then measure the expression of the luciferase reporter gene.

[0257] Activation of PPARδ can regulate bone metabolism. As shown in Table 4, compounds I-2, I-9, I-10, I-16, I-24, and I-26 have the activity of stimulating PPARδ, indicating that these compounds may have the effect of regulating bone metabolism. The agonist activity of compound I-16 on PPARδ in MC3T3-E1 cells is shown in Table 4. Figure 1 shown.

[0258] Table 4. PPARδ agonist activity of some compounds

[0259] Cpds. Compound number PPARδ agonist activity (relative to control) I-2 1.94 I-9 1.91 I-10 2.14 I-16 1.58 I-24 1.42 I-26 1.55

[0260] Example 35. Detection of cell alkaline phosphatase (ALP) activity after compound action

[0261] MC3T3-E1 cells were seeded in a 6-well plate and cultured at 37°C, 5% CO₂ for 24 hours. Once confluent, the supernatant was aspirated and replaced with osteogenic differentiation induction medium containing various concentrations of compound or a blank control. Culture was continued for 7 or 12 days, with the induction medium changed every 3 days. The supernatant was aspirated and the cells were washed twice with ice-cold PBS, followed by a rinse with 1 mL of ultrapure water. The cells were scraped off in 1 mL of ice-cold ultrapure water and transferred to a new Eppendorf tube. On ice, sonicate at 10 W for 30 seconds each, for a total of two sonication cycles. Centrifuge at 12,000 rpm for 15 minutes at 4°C, and transfer the supernatant to a new pre-chilled Eppendorf tube. To prepare a 1 mg / mL PNPP buffer, add 5 mg of PNPP to 5 mL of a solution containing 1 mol / L diethanolamine and 0.5 mmol / L MgCl₂. Incubate 100 μL of protein supernatant with 100 μL of PNPP solution and 100 μL of BCA reaction solution at 37°C for 30 min. Terminate the reaction by adding 3 mol / L NaOH. Measure ALP and protein absorbance using a microplate reader at 405 nm and 570 nm, respectively. Calculate the ALP value and protein concentration for each well using the standard curve. The ratio of the ALP value to the total protein concentration in each well is the standardized ALP.

[0262] Alkaline phosphatase (ALP) is an exoenzyme of osteoblasts, and its expression activity is a significant characteristic of osteoblast differentiation. Compound I-16 can dose-dependently upregulate ALP activity in MC3T3-E1 cells ( Figure 2 ), indicating that compound I-16 can promote osteoblast differentiation in vitro.

[0263] Example 36. Alizarin red (ARS) staining and quantification of cells after compound action

[0264] MC3T3-E1 cells were seeded in 6-well or 12-well plates. When cells reached over 80% growth, the medium was replaced with osteogenic differentiation induction medium containing 10 mM β-glycerophosphate and 50 μg / mL L-ascorbic acid supplemented with the compound. Incubate at 37°C, 5% CO₂ for 21 days, with fresh medium replaced every 3 days. After incubation, the culture medium was discarded and the cells were rinsed twice with PBS. Fix the cells with 95% ethanol for 10 minutes and rinse three times with double-distilled water. Stain the cells with 40 mM Alizarin Red S solution (pH 4.2) for 10 minutes at room temperature. Rinse three times with distilled water and photograph the cells. Prepare a 100 mM cetylpyridinium chloride solution in double-distilled water and add an equal amount of cetylpyridinium chloride to each well. Incubate at room temperature for 1 hour. Aspirate the supernatant and measure absorbance at 562 nm. Zero the absorbance using the supernatant without Alizarin Red. Repeat three times for each sample, and calculate the average value.

[0265] Alizarin Red S staining (ARS) is a commonly used staining method, which is widely used in the study of osteoblast differentiation, bone cells or tissue pathophysiology. Figure 3 As shown, after 21 days of treatment with compound I-16 on MC3T3-E1 cells, the number of calcified nodules formed by differentiated MC3T3-E1 cells increased significantly in a dose-dependent manner. After quantification, the effect of compound I-16 at 100 nM on the formation of calcified nodules was 3.5 times greater, further indicating that compound I-16 can significantly promote osteoblast differentiation in vitro.

[0266] Example 37. Tartrate-resistant acid phosphatase (TRAP) staining of cells after compound action

[0267] Prepare the fixative in advance: 25 mL of citrate diluent + 65 mL of acetone + 8 mL of 37% formaldehyde. Prepare ample 37°C deionized water and check the temperature before use. Allow the fixative to cool to room temperature and fix for 30 seconds. Rinse with deionized water, making sure the wells do not dry out, and aspirate thoroughly. Add 0.5 mL of Rapid Garnet GBC Base Solution and 0.5 mL of Sodium Nitrite Solution to two test tubes. Gently invert and mix for 30 seconds, then let stand for 2 minutes. Label two 100 mL beakers A and B. In Beaker A, add 45 mL of 37°C deionized water + 1.0 mL of the Diazo Rapid Garnet GBC Solution prepared in Step 4 + 0.5 mL of Naphthol AS-BI Phosphate Solution + 2.0 mL of Acetic Acid Solution. Prepare the solution in Beaker B similar to that in Beaker A and add 1.0 mL of Tartaric Acid Solution. Combine the solutions in Slide Staining Jars A and B into one jar and place it in a 37°C water bath. Ensure the solution is at 37°C before staining. Place the slide in a staining jar, protect from light, and stain at 37°C for 1 hour. Wash the slide with deionized water and counterstain with hematoxylin for 2 minutes. Rinse with tap water for a few minutes. Allow to dry naturally and observe under a microscope to investigate the direct effects of compounds I-2, I-8, I-10, I-11, I-14, and I-16 on osteoclast differentiation, as well as the indirect effects of the compounds on osteoclast differentiation after acting on MC3T3-E1 cells. First, MC3T3-E1 cells were induced to differentiate into osteoblasts using osteoblast differentiation medium. After 3 days, the medium was changed to osteoblast differentiation medium containing a certain concentration of compounds. After 6 days of action, the cell culture supernatant (Culture Supernatant, CS) was collected for subsequent experiments. RAW264.7 cells were induced to differentiate into osteoclasts using 50 ng / mL RANKL. After 3 days, the above culture supernatant was mixed with osteoclast induction medium at a ratio of 1:1 and added to RAW264.7 cells. At the same time, a group of osteogenic differentiation medium with only compound added and a mixture of osteoclast induction medium were set up and added to RAW264.7. After 3 days of treatment, Trap staining was used to detect osteoclast formation.

[0268] Tartrate-resistant acid phosphatase (Trap) is a marker enzyme for osteoclasts. It is specifically distributed in osteoclasts and is unique to osteoclasts. It is usually used as an important marker for identifying osteoclasts. Trap staining is a staining method for detecting characteristic substances in bone tissue and osteocytes, making osteoclasts red and the background green or blue. As can be seen from Table 5, compounds I-2, I-8, I-10, I-11, and I-14 have the activity of inhibiting RANKL-induced osteoclasts at concentrations of 0.1 μM, 1 μM, and 10 μM.

[0269] Given that compound I-16 at 100 nM significantly increased OPG levels and inhibited RANKL levels, the direct effect of 100 nM compound I-16 on osteoclast differentiation and the indirect effect of 100 nM compound I-16 on osteoclast differentiation in MC3T3-E1 cells were investigated. Figure 4 As shown in the results, after induction with 50 ng / mL RANKL, RAW264.7 cells were able to differentiate into mature osteoclasts. Under the direct action of 100 nM compound I-16, the number of osteoclasts did not decrease significantly. However, after adding compound I-16 to the culture supernatant of MC3T3-E1 cells under 50 ng / mL RANKL induction (100 nM compound I-16CS), the number of osteoclasts was significantly reduced. The above results indicate that compound I-16 can inhibit the differentiation of osteoclasts through osteoblasts.

[0270] Table 5 TRAP staining assay for the inhibitory activity of some compounds on osteoclast differentiation

[0271] Cpds. Compound number Inhibition rate % (0.1 μM) Inhibition rate % (1 μM) Inhibition rate % (10 μM) I-2 65.7 65.3 62.1 I-8 57.0 53.0 49.7 I-10 61.6 62.9 63.2 I-11 61.6 62.9 63.2 I-14 75.4 70.9 53.5

[0272] Example 38. Effects of Compounds on Bone Resorption Lacunae in Grinded Bone Slices

[0273] Take a sterile 12-well plate, place one bone slice in each well, and then inoculate 10 4 0.8 mL of RAW264.7 cells were added to the culture medium. After 24 hours, the culture medium was discarded and replaced with culture medium containing 50 ng / mL RANKL. The medium was changed every three days for 6 consecutive days. On the 7th day, culture medium containing various concentrations of the compound or other conditioned medium was added. After incubation for 13 days, bone slices were collected and subjected to the following procedures: fixation with 2.5% glutaraldehyde for 7 minutes, ultrasonic cleaning with 0.25 mol / L ammonium hydroxide for 1 minute three times, dehydration with a series of ethanol solutions, and air-drying. Staining with 1% toluidine blue for 3-4 minutes at room temperature was performed, followed by rinsing with distilled water. Bone lacunae were counted under a light microscope and expressed as the number of lacunae per number of bone slices.

[0274] like Figure 5 It can be seen that in the absence of RANKL-induced RAW264.7 cells, almost no bone lacunae were formed on the bone grinding slices, and the osteocytes were arranged neatly. After the action of 50ng / mL RANKL-induced RAW264.7 cells, a large number of bone lacunae were formed, and the osteocytes were arranged in a disorderly manner. After the direct action of 100nM compound I-16, the number of bone lacunae decreased to a certain extent, but the arrangement of osteocytes was not restored. After the intervention of 100nM compound I-16 in the culture supernatant of MC3T3-E1 cells (100nM compound I-16CS), the number of bone lacunae was significantly reduced, with statistical significance, and the osteocytes were arranged neatly.

[0275] Example 39. Detection of the Effect of Compounds on Target Gene Protein Levels

[0276] MC3T3-E1 cells were cultured at 1×10 5 U-2OS cells were counted as 10 / well or 4× as 10 5 Cells were seeded into 6-well plates at 37°C, 5% CO2 for 24 hours. Compounds were diluted in serum-free medium. In addition, serum-free medium containing the same final concentration of DMSO as the compound was added to the blank control wells and incubated at 37°C, 5% CO2 for 24 hours. Cells were collected, samples were prepared, and SDS-PAGE gel electrophoresis was performed. The membranes were transferred using a semi-dry transfer apparatus. Blocked with 5% (w / v) skim milk powder blocking solution at room temperature for 1-2 hours. The corresponding primary antibody was added and incubated at room temperature for 2 hours. Rinse three times in 1×TBST. Incubate with the secondary antibody at room temperature for 1 hour. Rinse the membrane three times in 1×TBST. Substrate luminescent solution was added and the membrane was exposed to light for color development. Image J software was used to perform grayscale scanning of the colored bands and quantify the protein levels.

[0277] like Figure 6 As shown, compound I-16 can increase the level of OPG protein in MC3T3-E1 cells in a dose-dependent manner at 1-100 nM, has no significant effect on RANKL protein, and increases the OPG / RANKL ratio. In addition, compound I-16 can increase the level of OPG protein in U-2OS cells at 1-100 nM, but has no significant effect on RANKL protein level. Ratio calculation shows that compound I-16 can increase the OPG / RANKL ratio in U-2OS cells and MC3T3-E1 cells in a dose-dependent manner.

[0278] Example 40. Determination of OPG secretion level by enzyme-linked immunosorbent assay (ELISA)

[0279] The R&D Mouse OPG ELISA Kit (#MOP00) was used to measure changes in OPG protein expression in the supernatant of MC3T3-E1 cell culture. The specific steps are as follows: Before use, equilibrate all reagents and samples at room temperature (18-25°C). Prepare the sample diluent and plate wash buffer according to the manufacturer's instructions. Redissolve the mouse OPG standard with Calibrator Diluent RD5-3 to obtain a 2000 pg / mL stock solution. Add 50 μL of Assay Diluent RD1-21 to each well. Add 50 μL of standard or sample to the test wells and incubate at room temperature for 2 hours. Aspirate the wells and wash the plate five times with Wash Buffer. Add 100 μL of OPG conjugate to each well and incubate at room temperature for 2 hours. Aspirate the wells and wash the plate again five times. Add 100 μL of substrate solution to each well. Incubate at room temperature for 30 minutes in the dark. Add 100 μL of Stop Solution to each well. The absorbance at 450 nm was read using a microplate reader. The concentration of OPG in the sample was calculated based on the standard curve of the standard.

[0280] The results are as follows Figure 7 As shown in the results, after treatment with 1-100 nM compound I-16, the concentration of OPG in the culture supernatant of MC3T3-E1 cells increased in a dose-dependent manner, with the highest increase being 3-fold. This result indicates that compound I-16 can promote the secretion of OPG protein in MC3T3-E1 cells. Based on this, we also speculate that compound I-16 may have the effect of inhibiting osteoclast differentiation, and can inhibit osteoclast differentiation by binding to RANKL on the surface of osteoclasts through the secreted OPG.

[0281] Example 41. Detection of the Effect of Compounds on Target Gene mRNA Levels

[0282] MC3T3-E1 cells were cultured at 5×10 5 Cells were seeded at a density of 100 cells / well in 6-well plates and cultured at 37°C, 5% CO₂ for 18-24 hours. The culture medium was then replaced with serum-free medium containing various concentrations of compound and a blank control and cultured at 37°C, 5% CO₂ for 18-24 hours. Total RNA was extracted and reverse transcribed into cDNA. The reverse-transcribed cDNA was used as a template to amplify the Runx2, Osx, Alp, and Bglap genes from untreated and I-16-treated mouse MC3T3-E1 cells. The Gapdh gene was used as an internal control.

[0283] like Figure 8As shown, compound I-16 significantly increased the levels of osteogenesis-promoting genes Runx2, Alp, Bglap, etc. in MC3T3-E1 cells, further indicating that compound I-16 can promote osteoblast differentiation in vitro.

[0284] Example 42. Pharmacodynamic evaluation of ovariectomized rats induced by combined dexamethasone (OVX-D)

[0285] (1) Establishment of an osteoporosis model in rats induced by ovariectomy combined with dexamethasone

[0286] Rats were anesthetized with 40 mg / kg body weight of 2% sodium pentobarbital via intraperitoneal injection. After general anesthesia, the rats were placed in the supine position, immobilized, and disinfected with iodine. An abdominal incision was made, and the abdominal muscles separated. The pale pink ovaries and surrounding fat were visible within the abdominal cavity. The fat mass was gently lifted with forceps to expose the ovaries. The fallopian tubes at the lower end of the ovaries were ligated with silk thread, and both ovaries were removed after ligation. The incisions were sutured and disinfected. A sham-operated control experiment was conducted in the same ovariectomy procedure as the ovariectomy procedure. After anesthesia, a sham ovariectomy was performed. The ovaries were located but not removed, and the incisions were sutured. Dexamethasone was injected one week after surgery. This was administered twice weekly for four weeks.

[0287] (2) Experimental design, grouping, and dosing regimen

[0288] Rats were randomly divided into 5 groups according to their body weight, namely, sham operation group (Sham), ovariectomy (OVX) group, positive drug (alendronate) group, low-dose group of compound I-16 (I-16-L group), and high-dose group of compound I-16 (I-16-H group). Rats in the sham operation group (Sham group) underwent sham ovariectomy without ovarian removal and were given 3% sodium carboxymethylcellulose (CMC-Na) solution by gavage after surgery. Rats in the ovariectomy group (OVX group) underwent bilateral ovariectomy and were given 3% CMC-Na solution by gavage after surgery. Rats in the positive drug group (Aln group) underwent bilateral ovariectomy and were given 3 mg / kg / d of alendronate by gavage after surgery. Rats in the low-dose group (I-16-L group) underwent bilateral ovariectomy and were given 5 mg / kg / d of compound I-16 by gavage after surgery. Rats in the high-dose group (I-16-H) underwent bilateral ovariectomy and were gavaged with 20 mg / kg / d of compound I-16. All rats underwent the first bone density test 40 days after administration, and continued to receive the drug for 90 days.

[0289] (3) Sampling and bone specimen preparation

[0290] Rats received a subcutaneous injection of 10 mg / kg body weight of calcein in the neck 14 and 13 days before the end of the experiment, and 4 and 3 days before the end of the experiment, respectively. The two fluorescent labeling injections were separated by 10 days. After administration, the rats were anesthetized and blood was collected from the femoral artery. After standing, the whole blood was centrifuged at 2000 rpm for 10 minutes, and serum was obtained for subsequent serum marker analysis. Afterwards, the rats in each group were sacrificed, and the right femur was removed, the surrounding tissue was removed, and the surrounding tissue was wrapped with gauze soaked in saline and stored at -80°C for subsequent bone density analysis and micro-CT analysis. Bone tissue sections were then prepared. The proximal third of the left femur was removed and the surrounding tissue removed. After fixation with 4% paraformaldehyde (0.1 M PBS) for 24 hours, the sections were thoroughly rinsed with 0.1 M PBS, and then decalcified with 10% EDTA·Na2 (0.1 M PBS) at 4°C. The medium was changed every 1 day, and decalcification was completed after 5 weeks for the preparation of decalcified bone paraffin sections. The heart, liver, spleen, lung and kidney of rats were fixed with 10% formaldehyde and paraffin sections were prepared.

[0291] (4) Preparation of bone and hard tissue sections

[0292] The right femur of rats was fixed with 10% formaldehyde and dehydrated in a gradient ethanol solution: 80% ethanol overnight, 95% ethanol for 2 hours, anhydrous ethanol I for 1 hour, anhydrous ethanol II for 30 minutes, xylene I for 40 minutes, and xylene II for 60 minutes. The embedding solution was then infiltrated with a gradient of embedding solutions and embedded in resin blocks. Slices were cut into 200 μm thick sections using a slow saw, glued together, ground to approximately 50 μm using a microtome, polished, stained with iron hematoxylin for 5 minutes, rinsed with running water to reverse the blue, stained with iris red for 5 minutes, rinsed with running water, stained with phosphotungstic acid solution, immersed in aniline blue for 5 minutes, eluted with acetic acid solution, dehydrated with a gradient of ethanol, cleared with xylene, and mounted.

[0293] (5) Preparation of paraffin sections

[0294] The decalcified femurs were thoroughly rinsed three times with PBS and dehydrated with graded ethanol: 80% ethanol overnight, 95% ethanol for 2 h, anhydrous ethanol I for 1 h, anhydrous ethanol II for 30 min, xylene I for 40 min, xylene II for 60 min, immersed in wax solutions I, II, and III for 1 h each, embedded in paraffin with a melting point of 62°C, cut into 5 μm thick longitudinal bone sections, placed on poly-lysine-coated slides, and baked at 42°C for 2 days.

[0295] (6) Bone density test

[0296] The rats were anesthetized by intraperitoneal injection of 2% sodium pentobarbital solution, and the femur of each rat was scanned by dual-energy X-ray absorptiometry to calculate the femoral density.

[0297] According to the results obtained from Micro-CT analysis, Figure 9As shown, compared with the sham group, the BMD of the OVX group was significantly reduced. After treatment with low-dose (5 mg / kg / d) and high-dose (20 mg / kg / d) Compound I-16, BMD was significantly improved in a dose-dependent manner. In addition, BMD was significantly improved after treatment with alendronate sodium (3 mg / kg / d).

[0298] (7) Micro-CT

[0299] The distal femur of rats was scanned using an Inveon Micro PET / CT scanner. The scanner was set at an operating voltage of 50 kV, an operating current of 400 μA, and a resolution of 10 μm / pixel. Three-dimensional reconstruction was performed after the scan, and the data was analyzed using the Inveon analysis workstation to analyze bone morphological indicators. Bone morphological measurements include bone volume fraction (BV / TV), trabecular thickness (Tb.Th), bone surface to bone volume ratio (BS / BV), trabecular separation (Tb.Sp), and trabecular number (Tb.N).

[0300] After 12 weeks of drug administration, the distal femur of the right side of each group of rats was scanned by Micro-CT and three-dimensional image reconstruction was performed. The results showed that ( Figure 10 Compared with the sham group, the femoral trabeculae in the OVX group were reduced in number, structurally broken, and disorganized. Furthermore, the trabeculae degenerated and thinned, and the spaces between them significantly widened and enlarged, demonstrating clear osteoporosis symptoms. After 12 weeks of treatment with compound I-16 (5 mg / kg / d I-16-L, 20 mg / kg / d I-16-H), trabecular thickness and trabecular structure were significantly restored. Treatment with the active drug alendronate (3 mg / kg / d) significantly increased the number of trabeculae and reduced the degree of trabecular loss.

[0301] like Figure 11 As shown in the results, compared with the Sham group, the trabecular bone volume fraction (BV / TV), trabecular thickness (Tb.Th), and trabecular number (Tb.N) of the OVX group rats were significantly reduced, and the trabecular spacing (Tb.Sp) and bone surface area to bone volume ratio (BS / BV) increased. However, we found that intervention with compound I-16 and alendronate sodium can effectively inhibit bone loss in OVX rats. Figure 11As shown, the BV / TV and Tb.N of the compound I-16 group and the alendronate sodium (Aln) group were significantly increased compared with the OVX group, and the high-dose I-16 had the most obvious effect. In addition, both high-dose and low-dose compound I-16 could significantly increase Tb.Th in OVX rats, while no significant effect was observed in the Aln group. At the same time, we also found that Tb.Sp and BV / TV decreased in the I-16-H group and the Aln group. In summary, both compound I-16 and alendronate sodium treatment can improve the bone mass of OVX rats, and the high-dose compound I-16 is more effective than alendronate sodium. In addition, as Figure 11 As shown in the results, compound I-16 is more effective than alendronate in improving trabecular thickness.

[0302] (8) HE staining

[0303] Dewax the paraffin sections, stain with hematoxylin for 10 minutes, rinse with tap water to remove excess color, and then differentiate in differentiation solution for 2 seconds. Soak in tap water for 5 minutes to turn blue. Stain in eosin solution for 30 seconds. Dehydrate the sections and mount them with neutral gum.

[0304] After HE staining, compared with the Sham group, the trabeculae in the OVX group were significantly sparse and thinner, with a large number of trabecular fractures and structural disorder. After 12 weeks of treatment with compound I-16 and positive drug sodium alendronate, the trabecular structure was significantly restored ( Figure 12 ).

[0305] (9) Toluidine blue staining and osteoblast counting

[0306] Dewax the sections and stain with toluidine blue for 10 minutes. Differentiate with 95% alcohol. Dry with a hair dryer or in an oven. Clear with xylene for several minutes. Mount the sections with neutral gum. Count the osteoblasts in five randomly selected fields based on microscopic observation, and calculate the average value.

[0307] Depend on Figure 13 As can be seen, the number of osteoblasts in the OVX group increased compared to the Sham group, indicating that osteoblast activity in ovariectomized rats also increased. We found that high doses of compound I-16 significantly increased the number of osteoblasts, with statistical significance, indicating that compound I-16 can both inhibit bone resorption and promote bone formation in ovariectomized rats in vivo, consistent with its in vitro activity.

[0308] (10) TRAP staining of paraffin sections

[0309] Dewaxed sections. TRAP staining ( Figure 14TRAP staining of femoral sections showed a significant increase in osteoclast numbers in the OVX group compared with the Sham group, indicating increased osteoclast activity, active bone resorption, and accelerated osteolysis. However, treatment with compound I-16 and alendronate significantly reduced osteoclast numbers. This result is consistent with the in vitro activity results.

[0310] (11) Calcein double labeling experiment

[0311] Bone hard tissue sections were observed under a fluorescence microscope. Since rats were injected with calcein at two time points, two calcein deposition lines were produced at these two time points. The mineral apposition rate (MAR) was obtained by dividing the width of the two calcein deposition lines by the number of days between the two injections.

[0312] like Figure 15 As shown, the mineralization width of ovariectomized rats was significantly reduced, while the mineralization width was significantly increased after treatment with compound I-16, and the mineralization deposition rate increased in a dose-dependent manner, indicating that compound I-16 can promote bone formation.

[0313] (12) Determination of serum OPG, RANKL, and osteocalcin levels

[0314] The ELISA kits produced by Senbeijia Biotechnology Co., Ltd. were used to determine the levels of OPG, RANKL and osteocalcin in rat serum.

[0315] The results showed that ( Figure 16 ), OPG levels in the serum of OVX rats decreased and RANKL levels increased significantly. After 12 weeks of compound I-16 intervention, OPG levels in the serum of OVX rats increased significantly, while RANKL levels decreased significantly, thereby increasing their OPG / RANKL ratio. After the treatment with sodium alendronate, OPG levels increased to a certain extent, while RANKL levels decreased to a certain extent, but there was no statistical significance. However, OPG / RANKL levels were significantly increased. In addition, high-dose compound I-16 was able to increase the level of osteocalcin in the serum of OVX rats.

[0316] Industrial applicability

[0317] The heterocyclic compounds of the present invention can specifically excite PPARδ, upregulate the activity of BMP2 expression, upregulate the activity of OPG expression, promote osteoblast differentiation and bone formation, inhibit osteoclast differentiation and bone resorption, thereby achieving excellent effects in treating osteoporosis and bone metabolism-related diseases, and can be used to prepare therapeutic agents for osteoporosis and bone metabolism-related diseases.

Claims

1. A heterocyclic compound represented by general formula I or a pharmaceutically acceptable salt or solvate thereof: in, A is an aryl group which may have a substituent or a heteroaryl group which may have a substituent; B is absent or selected from a cycloalkyl group optionally having a substituent, a heterocycloalkyl group optionally having a substituent, an aryl group optionally having a substituent, or a heteroaryl group optionally having a substituent; X is -(CR 4 R 5 ) p -, -(CR 4 R 5 ) p -O-, -(CR 4 R 5 ) p -S-, -(CR 4 R 5 ) p -NR 6 -, -O-(CR 4 R 5 ) p -, -S-(CR 4 R 5 ) p - or -N(R 6 )-(CR 4 R 5 ) p -; Y is -(CR 4 R 5 ) g -, -CONR 6 -, -(CR 4 R 5 ) g -CONR 6 -, -CONR 6 -(CR 4 R 5 ) g -, -CONR 6 SO2-, -CONR 6 SO2-(CR 4 R 5 ) g - or -(CR 4 R 5 ) g -CONR 6 SO2-; J and Q are each independently selected from carbon and nitrogen; W is selected from carbon and nitrogen; Z is selected from carbon, oxygen, sulfur, and nitrogen; R 1 、R 2 、R 3 each independently selected from hydrogen, fluorine, chlorine, bromine, iodine, nitro, cyano, aldehyde, ester, an alkylcarbonyl group optionally having a substituent, an amino group, an aryl group optionally having a substituent, an aryloxy group optionally having a substituent, a linear or branched alkyl group having 1 to 6 carbon atoms optionally having a substituent, a linear or branched alkoxy group having 1 to 6 carbon atoms optionally having a substituent, a linear or branched oxaalkyl group having 1 to 6 carbon atoms optionally having a substituent, a linear or branched azaalkyl group having 1 to 6 carbon atoms optionally having a substituent, a linear or branched aliphatic carboxyl group having 1 to 6 carbon atoms optionally having a substituent, a cycloalkyl group having 3 to 6 carbon atoms optionally having a substituent, and an alicyclic carboxyl group having 4 to 6 carbon atoms optionally having a substituent; R 4 、R 5 、R 6 Each is independently selected from hydrogen, fluorine, chlorine, bromine, iodine, and a linear or branched alkyl group having 1 to 6 carbon atoms which may have a substituent; m is an integer selected from 0, 1, 2, 3, 4, 5; n is an integer selected from 0, 1, 2, 3, 4; k is an integer selected from 0, 1, 2, and 3; p is an integer selected from 0, 1, 2, 3, 4, 5, 6; g is an integer selected from 0, 1, 2, 3, 4, 5, and 6.

2. The heterocyclic compound or pharmaceutically acceptable salt or solvate thereof according to claim 1, characterized in that: In the compound of formula I: A is an aryl group optionally having a substituent, or a heteroaryl group optionally having a substituent selected from benzothiazolyl, benzoxazolyl, thiazolyl, oxazolyl, phenyl or pyridyl; B is absent or selected from a cycloalkyl group optionally having a substituent, an oxacycloalkyl group optionally having a substituent, an azacycloalkyl group optionally having a substituent, a thiacycloalkyl group optionally having a substituent, an aryl group optionally having a substituent, or a heteroaryl group optionally having a substituent; X is -(CR 4 R 5 ) p -, -(CR 4 R 5 ) p -O-, -(CR 4 R 5 ) p -S-, -(CR 4 R 5 ) p -NR 6 -, -O-(CR 4 R 5 ) p -, -S-(CR 4 R 5 ) p -, -N(R 6 )-(CR 4 R 5 ) p -; Y is -(CR 4 R 5 ) g -, -CONR 6 -, -(CR 4 R 5 ) g -CONR 6 -, -CONR 6 -(CR 4 R 5 ) g -, -CONR 6 SO2-, -CONR 6 SO2-(CR 4 R 5 ) g - or -(CR 4 R 5 ) g -CONR 6 SO2-; J and Q are each independently selected from carbon and nitrogen; W is selected from carbon and nitrogen; Z is selected from carbon, oxygen, sulfur, and nitrogen; R 1 、R 2 、R 3 each independently selected from hydrogen, fluorine, chlorine, bromine, iodine, nitro, cyano, aldehyde, ester, an alkylcarbonyl group optionally having a substituent, an amino group, an aryl group optionally having a substituent, an aryloxy group optionally having a substituent, a linear or branched alkyl group having 1 to 6 carbon atoms optionally having a substituent, a linear or branched alkoxy group having 1 to 6 carbon atoms optionally having a substituent, a linear or branched oxaalkyl group having 1 to 6 carbon atoms optionally having a substituent, a linear or branched azaalkyl group having 1 to 6 carbon atoms optionally having a substituent, a linear or branched aliphatic carboxyl group having 1 to 6 carbon atoms optionally having a substituent, a cycloalkyl group having 3 to 6 carbon atoms optionally having a substituent, and an alicyclic carboxyl group having 4 to 6 carbon atoms optionally having a substituent; R 4 、R 5 、R 6 Each is independently selected from hydrogen, fluorine, chlorine, bromine, iodine, and a linear or branched alkyl group having 1 to 6 carbon atoms which may have a substituent; m is an integer selected from 0, 1, 2, 3, 4, 5; n is an integer selected from 0, 1, 2, 3, 4; k is an integer selected from 0, 1, 2, and 3; p is an integer selected from 0, 1, 2, 3, 4, 5, 6; g is an integer selected from 0, 1, 2, 3, 4, 5, and 6.

3. The heterocyclic compound or pharmaceutically acceptable salt or solvate thereof according to claim 2, characterized in that: In the compound of formula I: When B is absent, n is an integer selected from 0, 1, 2, 3, 4; R 2 selected from hydrogen, fluorine, chlorine, bromine, nitro, aldehyde, methyl formate, methyl acetate, ethyl formate, ethyl acetate, an alkylcarbonyl group which may have a substituent, an amino group, a linear or branched alkyl group having 1 to 6 carbon atoms which may have a substituent, a linear or branched alkoxy group having 1 to 6 carbon atoms which may have a substituent, a linear or branched oxaalkyl group having 1 to 6 carbon atoms which may have a substituent, a linear or branched aliphatic carboxyl group having 1 to 6 carbon atoms which may have a substituent, and a cycloalkyl group having 3 to 6 carbon atoms which may have a substituent.

4. The heterocyclic compound according to claim 1 or a pharmaceutically acceptable salt or solvate thereof, which is the following compound: N-(5,6-dimethoxybenzo[d]thiazol-2-yl)-2-((5-fluorobenzo[d]oxazol-2-yl)thio)acetamide; 2-(Benzo[d]oxazole-2-thio)-N-(5,6-dimethoxybenzo[d]thiazol-2-yl)propionamide; 2-(Benzo[d]oxazole-2-oxo)-N-(5,6-dimethoxybenzo[d]thiazol-2-yl)acetamide; 2-(Benzo[d]oxazole-2-oxo)-N-(6,7-dihydro-[1,4]dioxanyl[2',3':4,5]benzo[1,2-d]thiazol-2-yl)acetamide; N-(6,7-Dihydro-[1,4]dioxano[2',3':4,5]benzo[1,2-d]thiazol-2-yl)-2-((5-fluorobenzo[d]oxazol-2-yl)thio)acetamide; 2-(Benzo[d]oxazole-2-thio)-N-(6,7-dihydro-[1,4]dioxane[2',3':4,5]benzo[1,2-d]thiazol-2-yl)propionamide; N-(5,6-dimethoxybenzo[d]thiazol-2-yl)-2-((5-methylbenzo[d]oxazol-2-yl)thio)acetamide; N-(6,7-Dihydro-[1,4]dioxanyl[2',3':4,5]benzo[1,2-d]thiazol-2-yl)-2-((5-methylbenzo[d]oxazol-2-yl)thio)acetamide; 2-((5-fluorobenzo[d]oxazol-2-yl)thio)-N-(5-methoxybenzo[d]thiazol-2-yl)acetamide; 2-(Benzo[d]oxazole-2-thio)-N-(5-methoxybenzo[d]thiazol-2-yl)propionamide; 2-(Benzo[d]oxazole-2-oxo)-N-(5-methoxybenzo[d]thiazol-2-yl)acetamide; N-(5-methoxybenzo[d]thiazol-2-yl)-2-((5-methylbenzo[d]oxazol-2-yl)thio)acetamide; 2-((5-methoxybenzo[d]oxazol-2-yl)thio)-N-(5-methoxybenzo[d]thiazol-2-yl)acetamide; N-(6,7-dihydro-[1,4]dioxano[2',3':4,5]benzo[1,2-d]thiazol-2-yl)-2-((5-methoxybenzo[d]oxazol-2-yl)thio)acetamide; N-(5,6-dimethoxybenzo[d]thiazol-2-yl)-2-((5-methoxybenzo[d]oxazol-2-yl)thio)acetamide; 2-(Benzo[d]oxazole-2-thio)-N-(6,7-dihydro-[1,4]dioxanyl[2',3':4,5]benzo[1,2-d]thiazol-2-yl)acetamide; 2-(Benzo[d]oxazole-2-thio)-N-(5-methoxyoxybenzo[d]thiazol-2-yl)acetamide; 2-(Benzo[d]oxazole-2-thio)-N-(5,6-dimethoxybenzo[d]thiazol-2-yl)acetamide; N-(5,6-dimethoxybenzo[d]thiazol-2-yl)-2-((5-chlorobenzo[d]oxazol-2-yl)thio)acetamide; 2-((5-chlorobenzo[d]oxazol-2-yl)thio)-N-(5-methoxybenzo[d]thiazol-2-yl)acetamide; 2-((5-chlorobenzo[d]oxazol-2-yl)thio)-N-(6,7-dihydro-[1,4]dioxanyl[2',3':4,5]benzo[1,2-d]thiazol-2-yl)acetamide; 2-((5-bromobenzo[d]oxazol-2-yl)thio)-N-(6,7-dihydro-[1,4]dioxanyl[2',3':4,5]benzo[1,2-d]thiazol-2-yl)acetamide; 2-((5-bromobenzo[d]oxazol-2-yl)thio)-N-(5,6-dimethoxybenzo[d]thiazol-2-yl)acetamide; 2-((5-bromobenzo[d]oxazol-2-yl)thio)-N-(5-methoxybenzo[d]thiazol-2-yl)acetamide; 3-(Benzo[d]oxazole-2-thio)-N-(5-methoxybenzo[d]thiazol-2-yl)-2,2-dimethylpropionamide; 2-((5-(tert-Butyl)benzo[d]oxazol-2-yl)thio)-N-(5-methoxybenzo[d]thiazol-2-yl)acetamide; N-(5-methoxybenzo[d]thiazol-2-yl)-2-((5-methylbenzo[d]oxazol-2-yl)thio)propanamide; N-(6,7-dihydro-[1,4]dioxane-[2',3':4,5]benzo[1,2-d]thiazol-2-yl)-2-((5-methylbenzo[d]oxazol-2-yl)thio)propionamide; 3-(Benzo[d]oxazole-2-thio)-N-(6,7-dihydro-[1,4]dioxane[2',3':4,5]benzo[1,2-d]thiazol-2-yl)-2,2-dimethylpropionamide; 2-((5-(tert-Butyl)benzo[d]oxazol-2-yl)thio)-N-(6,7-dihydro-[1,4]dioxanyl[2',3':4,5]benzo[1,2-d]thiazol-2-yl)acetamide.

5. The method for preparing the heterocyclic compound represented by general formula I according to claim 1, comprising the reaction represented by the following formula II: The specific steps include: (1) In formula II, compound 1 undergoes a nucleophilic substitution reaction with a halogen-substituted carboxylic acid compound to obtain intermediate 2; (2) In formula II, intermediate 2 is condensed with compound 3 to form the compound shown in formula I; In the above reaction formula, A, B, W, Z, J, Q, X, Y, R 1 、R 2 、R 3 The definitions of m, n, and k are the same as those of the heterocyclic compound represented by Formula I.

6. A pharmaceutical composition comprising a therapeutically effective amount of a heterocyclic compound represented by the following general formula I and one or more pharmaceutically acceptable carriers, in, A is an aryl group which may have a substituent or a heteroaryl group which may have a substituent; B is absent or selected from a cycloalkyl group optionally having a substituent, a heterocycloalkyl group optionally having a substituent, an aryl group optionally having a substituent, or a heteroaryl group optionally having a substituent; X is -(CR 4 R 5 ) p -, -(CR 4 R 5 ) p -O-, -(CR 4 R 5 ) p -S-, -(CR 4 R 5 ) p -NR 6 -, -O-(CR 4 R 5 ) p -, -S-(CR 4 R 5 ) p - or -N(R 6 )-(CR 4 R 5 ) p -; Y is -(CR 4 R 5 ) g -, -CONR 6 -, -(CR 4 R 5 ) g -CONR 6 -, -CONR 6 -(CR 4 R 5 ) g -, -CONR 6 SO2-, -CONR 6 SO2-(CR 4 R 5 ) g - or -(CR 4 R 5 ) g -CONR 6 SO2-; J and Q are each independently selected from carbon and nitrogen; W is selected from carbon and nitrogen; Z is selected from carbon, oxygen, sulfur, and nitrogen; R 1 、R 2 、R 3 each independently selected from hydrogen, fluorine, chlorine, bromine, iodine, nitro, cyano, aldehyde, ester, an alkylcarbonyl group optionally having a substituent, an amino group, an aryl group optionally having a substituent, an aryloxy group optionally having a substituent, a linear or branched alkyl group having 1 to 6 carbon atoms optionally having a substituent, a linear or branched alkoxy group having 1 to 6 carbon atoms optionally having a substituent, a linear or branched oxaalkyl group having 1 to 6 carbon atoms optionally having a substituent, a linear or branched azaalkyl group having 1 to 6 carbon atoms optionally having a substituent, a linear or branched aliphatic carboxyl group having 1 to 6 carbon atoms optionally having a substituent, a cycloalkyl group having 3 to 6 carbon atoms optionally having a substituent, and an alicyclic carboxyl group having 4 to 6 carbon atoms optionally having a substituent; R 4 、R 5 、R 6 Each is independently selected from hydrogen, fluorine, chlorine, bromine, iodine, and a linear or branched alkyl group having 1 to 6 carbon atoms; m is an integer selected from 0, 1, 2, 3, 4, 5; n is an integer selected from 0, 1, 2, 3, 4; k is an integer selected from 0, 1, 2, and 3; p is an integer selected from 0, 1, 2, 3, 4, 5, 6; g is an integer selected from 0, 1, 2, 3, 4, 5, and 6.

7. A pharmaceutical preparation comprising a therapeutically effective amount of a heterocyclic compound represented by the following general formula I and one or more pharmaceutically acceptable carriers. wherein A is an aryl group optionally having a substituent, or a heteroaryl group optionally having a substituent; B is absent or selected from a cycloalkyl group optionally having a substituent, a heterocycloalkyl group optionally having a substituent, an aryl group optionally having a substituent, or a heteroaryl group optionally having a substituent; X is -(CR 4 R 5 ) p -, -(CR 4 R 5 ) p -O-, -(CR 4 R 5 ) p -S-, -(CR 4 R 5 ) p -NR 6 -, -O-(CR 4 R 5 ) p -, -S-(CR 4 R 5 ) p - or -N(R 6 )-(CR 4 R 5 ) p -; Y is -(CR 4 R 5 ) g -, -CONR 6 -, -(CR 4 R 5 ) g -CONR 6 -, -CONR 6 -(CR 4 R 5 ) g -, -CONR 6 SO2-, -CONR 6 SO2-(CR 4 R 5 ) g - or -(CR 4 R 5 ) g -CONR 6 SO2-; J and Q are each independently selected from carbon and nitrogen; W is selected from carbon and nitrogen; Z is selected from carbon, oxygen, sulfur, and nitrogen; R 1 、R 2 、R 3 each independently selected from hydrogen, fluorine, chlorine, bromine, iodine, an optionally substituted linear or branched alkyl group having 1 to 6 carbon atoms, an optionally substituted linear or branched alkoxy group having 1 to 6 carbon atoms, an optionally substituted linear or branched oxaalkyl group having 1 to 6 carbon atoms, an optionally substituted linear or branched azaalkyl group having 1 to 6 carbon atoms, an optionally substituted linear or branched aliphatic carboxyl group having 1 to 6 carbon atoms, an optionally substituted cycloalkyl group having 3 to 6 carbon atoms, an optionally substituted alicyclic carboxyl group having 4 to 6 carbon atoms, a nitro group, a cyano group, an ester group, an aldehyde group, an optionally substituted alkylcarbonyl group, an amino group, an optionally substituted aryl group, and an optionally substituted aryloxy group; R 4 、R 5 、R 6 Each is independently selected from hydrogen, fluorine, chlorine, bromine, iodine, and a linear or branched alkyl group having 1 to 6 carbon atoms; m is an integer selected from 0, 1, 2, 3, 4, 5; n is an integer selected from 0, 1, 2, 3, 4; k is an integer selected from 0, 1, 2, and 3; p is an integer selected from 0, 1, 2, 3, 4, 5, 6; g is an integer selected from 0, 1, 2, 3, 4, 5, and 6. The pharmaceutical preparation according to claim 7 , which is an oral preparation.

9. Use of the heterocyclic compound represented by general formula I or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 4 in the preparation of a therapeutic agent for osteoporosis and bone metabolism-related diseases.

10. The use according to claim 9, characterized in that The osteoporosis is selected from postmenopausal osteoporosis, senile osteoporosis, idiopathic osteoporosis and bone loss caused by other reasons.

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