A group of anti-osteoporosis compounds and their applications

Abstract

The present invention relates to a group of anti-osteoporosis compounds and their applications. These compounds can upregulate OPG expression, inhibit inflammatory pathway activation, inhibit osteoclast differentiation or bone resorption, and promote osteoblast differentiation or bone formation; and have high oral bioavailability and anti-osteoporosis activity.

Description

Technical Field

[0001] The present invention belongs to the field of medical biotechnology, and in particular relates to a group of anti-osteoporosis compounds and applications thereof. Background Art

[0002] Currently, anti-osteoporosis drugs are mainly divided into drugs that inhibit bone resorption, drugs that promote bone formation, and drugs with multiple mechanisms of action [doi:10.1038 / nrd3299; doi:10.1016 / j.bone.2011.02.011]. Drugs that inhibit bone resorption include bisphosphonates [doi:10.1016 / s0889-857x(05)70194-0.], estrogen, selective estrogen receptor modulators, calcitonin, monoclonal antibodies against RANKL (such as denosumab), and sclerostin monoclonal antibodies such as romosozumab [doi:10.1126 / science.289.5484.1508; doi:10.1016 / j.bone.2011.02.011; doi:10.4158 / GL-2020-0524SUPPL; doi:10.1056 / NEJMoa1607948; doi:10.1056 / NEJMoa1708322; doi:10.1080 / 14712598.2017.1280455]. Bone-forming drugs, such as teriparatide (parathyroid hormone), are generally used in patients at high risk of fracture, such as those with glucocorticoid-induced osteoporosis [doi:10.1126 / science.289.5484.1508]. Strontium ranelate is believed to have properties that inhibit bone resorption and promote bone formation, potentially rebalancing bone turnover in favor of bone formation [doi:10.2147 / CIA.S141753].

[0003] Although currently available osteoporosis medications are effective, most have limitations and side effects. The toxic side effects of existing osteoporosis drugs restrict their clinical application [doi:10.7326 / M15-1361]. For example, long-term use of bisphosphonates can excessively inhibit bone turnover, impair bone strength, cause atypical fractures, and increase the risk of acute myocardial infarction. They also have gastrointestinal and other side effects [doi:https: / / doi.org / 10.1136 / bmj.c4444]. Estrogen replacement therapy for osteoporosis can cause breast cancer, uterine bleeding, and cardiovascular disease, and is no longer routinely used. Parathyroid hormone can cause hypercalcemia and renal side effects and is generally reserved for patients with severe osteoporosis or intolerance to other osteoporosis medications. Discontinuation of the monoclonal antibody drug denosumab can lead to a further decrease in bone density, significantly increasing the risk of fractures. Therefore, the development of new, safe and effective osteoporosis medications is urgently needed.

[0004] The OPG-RANKL-RANK pathway is an important component of the bone metabolism signal regulation network and plays an important role in bone remodeling and osteoporosis [doi:10.1038 / nrdp.2016.69, doi:10.1056 / NEJMcp1513724]. Receptor Activator for Nuclear Factor-κB Ligand (RANKL) and osteoprotegerin (OPG) are cytokines secreted by osteoblasts that mediate the growth, differentiation, and function of osteoclasts, playing an important role in bone metabolism [doi:10.1056 / NEJMcp1513724; doi:10.1517 / 14728222.2010.511179; doi:10.1007 / s11154-014-9308-6; doi:10.1517 / 14728222.2010.511179]. RANK is the receptor for RANKL and is present on the cell membrane of osteoclasts and other cells. When RANKL binds to RANK, osteoclast differentiation is activated, inducing osteoclast precursor cells to form mature osteoclasts, leading to enhanced osteoclast differentiation [doi:10.1073 / pnas.95.7.3597]. OPG is a soluble decoy receptor for RANKL, which can prevent RANKL from binding to RANK, thereby inhibiting osteoclast differentiation [doi:10.1016 / s0092-8674(00)80209-3]. In addition, RANKL binding to RANK can activate inflammatory pathways (such as the nuclear receptor NF-κB (nuclear factor-κB), p38 mitogen-activated protein kinase (p38 mitogen-activated protein kinase), and PI3K / AKT pathway), promoting osteoclast differentiation. [DOI:10.1016 / j.phrs.2015.05.006; DOI:10.1002 / jbmr.4215; doi:10.1074 / jbc.M410480200]. Therefore, using small molecules to upregulate OPG expression in bone tissue to regulate the balance between OPG and RANKL and inhibit inflammatory pathways is an important strategy for developing drugs to treat osteoporosis. The applicant's prior patent, ZL201910090949.8, has already discussed the therapeutic effects of small molecule compounds that modulate this pathway on osteoporosis. However, identifying drug / prodrug molecules with improved drugability and more convenient administration methods to further develop drugs more suitable for the treatment and prevention of osteoporosis and chronic inflammatory diseases remains a key challenge. Summary of the Invention

[0005] The present invention relates to a group of compounds having the following functions:

[0006] (1) Upregulate the expression of OPG;

[0007] (2) inhibiting the activation of inflammatory pathways;

[0008] (3) Inhibit osteoclast differentiation and promote osteoblast differentiation;

[0009] (4) Treatment of osteoporosis;

[0010] The compound has the structure shown in the following general formula (1):

[0011]

[0012] in,

[0013] R1 is a hydrogen atom, a methyl group, a nitro group or a halogen;

[0014] A1 is O, S, NH, NMe;

[0015] A2 is CH or N;

[0016] X is NH, NMe or O;

[0017] M is a straight-chain alkane of 0-4 carbon atoms, or a straight-chain alkane in which one carbon atom is replaced by O;

[0018] R2 is alkyl-substituted pyridine or phenyl, and alkyl refers to C1-C6 alkyl;

[0019] and,

[0020] When R1 is a chlorine atom, A1 is an oxygen atom, A2 is CH, X is NH, and M is 0, then R2 cannot be a substituted pyridine.

[0021] in,

[0022] Halogen refers to: fluorine, chlorine, bromine, iodine;

[0023] C1-C6 alkyl refers to: straight-chain and branched saturated hydrocarbon groups containing 1-6 carbon atoms, such as methyl, ethyl, isopropyl, n-butyl, pentyl or hexyl; it also includes cycloalkyl, that is, cyclic C3-C6 hydrocarbon groups, such as cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl.

[0024] Preferably, the compound is a compound as shown in the following table

[0025]

[0026]

[0027]

[0028] The present invention also relates to a pharmaceutical composition comprising a compound of formula (1), wherein the pharmaceutical composition comprises a therapeutically effective amount of the compound of formula (1) and one or more pharmaceutically acceptable carriers. Preferably, the pharmaceutical composition is an oral preparation, and more preferably, the oral preparation is an oral tablet.

[0029] The present invention also relates to the use of the compound represented by general formula (1) in the preparation of medicines, wherein the medicines are used to treat the following diseases:

[0030] (1) Diseases caused by abnormal osteoclasts or bone resorption;

[0031] (2) Diseases caused by insufficient osteoblast differentiation;

[0032] (3) Diseases caused by inflammation;

[0033] (4) Diseases caused by abnormalities in the OPG-RANKL-RANK pathway;

[0034] (5) Osteoporosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 , compounds promote osteoblast formation (1A: compound 3c; 1B: compound 3v).

[0036] Figure 2 , the effects of compounds on the expression of osteoclast formation-related marker genes (2A: compound 3c; 2B: compound 3i1).

[0037] Figure 3 , Effects of compounds on OPG expression (3A: compound 3c; 3B: compound 3v; 3C: compound 3i1).

[0038] Figure 4 Effects of compounds on OPG secretion (4A: compound 3c; 4B: compound 3v; 4C: compound 3i1) DETAILED DESCRIPTION

[0039] Example 1. Synthesis of 5-methyl-N-(4-methylpyridin-2-yl)furan-2-carboxamide (3c)

[0040]

[0041] 5-Methyl-2-carboxyfuran (1 mmol) was dissolved in 10 ml of chloroform, followed by the addition of 5 ml of thionyl chloride and a catalytic amount of DMF (0.01 mmol). The mixture was refluxed for 3 hours. After completion of the reaction, the reaction solution was concentrated under reduced pressure and directly proceeded to the next step without further purification. The reduced pressure residue was dissolved in 10 ml of dichloromethane, followed by the addition of triethylamine (1.5 mmol) and stirring at room temperature for 0.5 h. 2-Amino-4-methylpyridine (1.1 mmol) was then added and stirring continued at room temperature for 4 hours. The reaction solution was washed sequentially with saturated sodium chloride and distilled water. The dichloromethane organic layer was separated and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (CH2Cl2:MeOH=100:1) to obtain compound 3c as a white solid in a yield of 44.0%.

[0042] 1 H NMR (600Mz, CDCl3): δ8.73 (s, 1H), 8.22-8.02 (m, 2H), 7.13 (d, J = 3.3Hz, 1H), 6.8 5(dd,J=4.9,1.4Hz,1H),6.13(dd,J=3.4,1.1Hz,1H),2.35(s,3H),2.34(s,3H). 13 CNMR(150MHz, CDCl3): δ156.31,151.32,149.92,147.63,145.89,121.02,117.14,114.63,109.18,21.47,13.90.MS-ESI(m / z):217.2(M+H) + ..

[0043] Example 2. Synthesis of N-(4-methylpyridin-2-yl)-5-nitrofuran-2-carboxamide (3d)

[0044]

[0045] 5-Nitro-2-carboxyfuran (1 mmol) was dissolved in 10 ml of chloroform, followed by the addition of 5 ml of thionyl chloride and a catalytic amount of DMF (0.01 mmol). The mixture was refluxed for 3 hours. After completion of the reaction, the reaction solution was concentrated under reduced pressure and directly proceeded to the next step without further purification. The reduced pressure residue was dissolved in 10 ml of dichloromethane, followed by the addition of triethylamine (1.5 mmol) and stirring at room temperature for 0.5 h. 2-Amino-4-methylpyridine (1.1 mmol) was then added and stirring continued at room temperature for 4 hours. The reaction solution was washed sequentially with saturated sodium chloride and distilled water. The dichloromethane organic layer was separated and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (CH2Cl2:MeOH=100:1) to obtain Compound 3d as a white solid in a yield of 73.0%.

[0046] 1 H NMR (600Mz, CDCl3): δ8.91(s,1H),8.21(dd,J=5.1,0.8Hz,1H),8.11(dd,J=1.5,0.8Hz,1H),7.43-7.35(m,2H),7.01-6.92(m,1H),2.41(s,3H). 13 C NMR (150MHz, CDCl3): δ154.31,150.36,150.30,147.90,147.52,122.11,117.20,115.25,112.49,21.52.MS-ESI(m / z):248.2(M+H) + ..

[0047] Example 3. Synthesis of 5-bromo-N-(4-methylpyridin-2-yl)furan-2-carboxamide (3e)

[0048]

[0049] 5-Bromo-2-carboxyfuran (1 mmol) was dissolved in 10 ml of chloroform, followed by the addition of 5 ml of thionyl chloride and a catalytic amount of DMF (0.01 mmol). The mixture was refluxed for 3 hours. After completion of the reaction, the reaction solution was concentrated under reduced pressure and directly proceeded to the next step without further purification. The reduced pressure residue was dissolved in 10 ml of dichloromethane, followed by the addition of triethylamine (1.5 mmol) and stirring at room temperature for 0.5 h. 2-Amino-4-methylpyridine (1.1 mmol) was then added and stirring continued at room temperature for 4 hours. The reaction solution was washed sequentially with saturated sodium chloride and distilled water. The dichloromethane organic layer was separated and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (CH2Cl2:MeOH=100:1) to obtain compound 3e as a white solid in a yield of 73.0%.

[0050] 1 H NMR (500Mz, CDCl3): δ8.73(s,1H),8.16(d,J=4.8Hz,1H),8.12(s,1H),7.19(d,J=2.6Hz,1H),6.91-6.83(m,1H),6.54-6.45(m,1H),2.38(s,3H). 13 C NMR (125MHz, CDCl3): δ155.05,150.92,150.11,149.02,147.77,125.61,121.46,118.10,114.82,114.76,21.53.MS-ESI(m / z):281.2(M+H) + ..

[0051] Example 4. Synthesis of 4-methylpyridin-2-yl-5-chlorofuran-2-methyl ester (3m)

[0052]

[0053] 5-Chloro-2-carboxyfuran (1 mmol) was dissolved in 10 ml of chloroform, followed by the addition of 5 ml of thionyl chloride and a catalytic amount of DMF (0.01 mmol). The mixture was refluxed for 3 hours. After completion of the reaction, the reaction solution was concentrated under reduced pressure and directly proceeded to the next step without further purification. The reduced pressure residue was dissolved in 10 ml of dichloromethane, followed by the addition of triethylamine (1.5 mmol) and stirring at room temperature for 0.5 h. 2-hydroxy-4-methylpyridine (1.1 mmol) was then added and stirring continued at room temperature for 4 hours. The reaction solution was washed sequentially with saturated sodium chloride and distilled water, and the dichloromethane organic layer was separated and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (CH2Cl2:MeOH=100:1) to obtain compound 3m as a white solid in a yield of 51.0%.

[0054] 1 H NMR (500Mz, CDCl3): δ8.27(d,J=5.1Hz,1H),7.39-7.38(m,1H),7.09-7.06(m,1H),7.01(s,1H),6.41-6.37(m,1H),2.41(s,3H). 13 C NMR(125MHz, CDCl3): δ148.30,137.99,123.71,122.14,118.24,116.94,109.51,108.30,21.19.MS-ESI(m / z):238.2(M+H) + ..

[0055] Example 5. Synthesis of pyridin-2-yl-5-chlorofuran-2-methyl ester (3n)

[0056]

[0057] 5-Chloro-2-carboxyfuran (1 mmol) was dissolved in 10 ml of chloroform, followed by the addition of 5 ml of thionyl chloride and a catalytic amount of DMF (0.01 mmol). The mixture was refluxed for 3 hours. After completion of the reaction, the reaction solution was concentrated under reduced pressure and directly proceeded to the next step without further purification. The reduced pressure residue was dissolved in 10 ml of dichloromethane, followed by the addition of triethylamine (1.5 mmol) and stirring at room temperature for 0.5 h. 2-Hydroxypyridine (1.1 mmol) was then added and stirring continued at room temperature for 4 hours. The reaction solution was washed sequentially with saturated sodium chloride and distilled water. The dichloromethane organic layer was separated and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (CH2Cl2:MeOH=100:1) to obtain compound 3n as a white solid in a yield of 46.0%.

[0058] 1 H NMR (600Mz, CDCl3): δ8.42 (ddd, J=4.9, 2.0, 0.7Hz, 1H), 7.82 (ddd, J=8.1, 7.4, 2.0Hz, 1H), 7 .39(d,J=3.6Hz,1H),7.29-7.23(m,1H),7.20(dt,J=8.1,0.9Hz,1H),6.39(d,J=3.6Hz,1H). 13 C NMR (150MHz, CDCl3): δ157.45,155.28,148.81,142.95,142.74,139.73,122.47,122.20,116.48,109.52.MS-ESI(m / z):224.2(M+H) + ..

[0059] Example 6. Synthesis of pyridin-3-yl-5-chlorofuran-2-methyl ester (3o)

[0060]

[0061] 5-Chloro-2-carboxyfuran (1 mmol) was dissolved in 10 ml of chloroform, followed by the addition of 5 ml of thionyl chloride and a catalytic amount of DMF (0.01 mmol). The mixture was refluxed for 3 hours. After completion of the reaction, the reaction solution was concentrated under reduced pressure and directly proceeded to the next step without further purification. The reduced pressure residue was dissolved in 10 ml of dichloromethane, followed by the addition of triethylamine (1.5 mmol) and stirring at room temperature for 0.5 h. 3-Hydroxypyridine (1.1 mmol) was then added and stirring continued at room temperature for 4 hours. The reaction solution was washed sequentially with saturated sodium chloride and distilled water. The dichloromethane organic layer was separated and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (CH2Cl2:MeOH=100:1) to obtain compound 3o as a white solid in a yield of 49.0%.

[0062] 1H NMR (600Mz, CDCl3): δ8.54 (d, J=2.7Hz, 1H), 8.52 (dd, J=4.7, 1.4Hz, 1H), 7.60 (ddd, J=8.3, 2.7,1.4Hz,1H),7.39(d,J=3.6Hz,1H),7.37(dd,J=8.3,4.7Hz,1H),6.42(d,J=3.5Hz,1H). 13 C NMR (150MHz, CDCl3): δ155.35,147.45,147.00,143.49,142.90,142.60,129.27,124.09,122.34,109.63.MS-ESI(m / z):224.2(M+H) + ..

[0063] Example 7. Synthesis of pyridin-4-yl-5-chlorofuran-2-methyl ester (3p)

[0064]

[0065] 5-Chloro-2-carboxyfuran (1 mmol) was dissolved in 10 ml of chloroform, followed by the addition of 5 ml of thionyl chloride and a catalytic amount of DMF (0.01 mmol). The mixture was refluxed for 3 hours. After completion of the reaction, the reaction solution was concentrated under reduced pressure and directly proceeded to the next step without further purification. The reduced pressure residue was dissolved in 10 ml of dichloromethane, followed by the addition of triethylamine (1.5 mmol) and stirring at room temperature for 0.5 h. 4-Hydroxypyridine (1.1 mmol) was then added and stirring continued at room temperature for 4 hours. The reaction solution was washed sequentially with saturated sodium chloride and distilled water. The dichloromethane organic layer was separated and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (CH2Cl2:MeOH=100:1) to obtain compound 3p as a white solid in a yield of 47.0%.

[0066] 1 H NMR (600Mz, CDCl3): δ8.69-8.63(m,2H),7.39(d,J=3.6Hz,1H),7.25-7.20(m,2H),6.43(d,J=3.6Hz,1H). 13 C NMR(150MHz, CDCl3): δ157.13,154.51,151.77,143.14,142.56,122.54,116.85,109.70.MS-ESI(m / z):224.2(M+H) + .

[0067] Example 8. Synthesis of 5-chloro-N-phenylfuran-2-carboxamide (3t)

[0068]

[0069] 5-Chloro-2-carboxyfuran (1 mmol) was dissolved in 10 ml of chloroform, followed by the addition of 5 ml of thionyl chloride and a catalytic amount of DMF (0.01 mmol). The mixture was refluxed for 3 hours. After completion of the reaction, the reaction solution was concentrated under reduced pressure and directly proceeded to the next step without further purification. The reduced pressure residue was dissolved in 10 ml of dichloromethane, followed by the addition of triethylamine (1.5 mmol) and stirring at room temperature for 0.5 h. Aniline (1.1 mmol) was then added and stirring continued at room temperature for 4 hours. The reaction solution was washed sequentially with saturated sodium chloride and distilled water. The dichloromethane organic layer was separated and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (CH2Cl2:MeOH=100:1) to obtain compound 3t as a white solid in a yield of 51.0%.

[0070] 1 H NMR (500Mz, CDCl3): δ7.97 (s, 1H), 7.64 (d, J = 8.0Hz, 2H), 7.36 (t, J = 7.6Hz, 2H), 7.21 (d, J = 3.0Hz, 1H), 7.15 (t, J = 7.2Hz, 1H), 6.38-6.34 (m, 1H). 13 C NMR (125MHz, CDCl3): δ155.06,147.05,138.85,

[0071] 137.23,129.26,124.88,120.19,117.51,109.74.MS-ESI(m / z):222.2(M+H) + ..

[0072] Example 9. Synthesis of N-benzyl-5-chlorofuran-2-carboxamide (3u)

[0073]

[0074] 5-Chloro-2-carboxyfuran (1 mmol) was dissolved in 10 ml of chloroform, followed by the addition of 5 ml of thionyl chloride and a catalytic amount of DMF (0.01 mmol). The mixture was refluxed for 3 hours. After completion of the reaction, the reaction solution was concentrated under reduced pressure and directly proceeded to the next step without further purification. The reduced pressure residue was dissolved in 10 ml of dichloromethane, followed by the addition of triethylamine (1.5 mmol) and stirring at room temperature for 0.5 h. Benzylamine (1.1 mmol) was then added and stirring continued at room temperature for 4 hours. The reaction solution was washed sequentially with saturated sodium chloride and distilled water. The dichloromethane organic layer was separated and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (CH2Cl2:MeOH=100:1) to obtain compound 3u as a white solid in a yield of 72.0%.

[0075] 1 H NMR (600Mz, CDCl3): δ7.33-7.27(m,4H),7.26-7.22(m,1H),7.05(d,J=3.6Hz,1H),6.90(s,1H),6.23(d,J=3.6Hz,1H),4.54(d,J=6.1Hz,2H). 13 C NMR (150MHz, CDCl3): δ157.25,147.08,138.33,137.90,128.66,127.83,127.54,116.41,109.03,43.14.MS-ESI(m / z):236.2(M+H) + ..

[0076] Example 10: Synthesis of 5-chloro-N-phenylethylfuran-2-carboxamide (3v)

[0077]

[0078] 5-Chloro-2-carboxyfuran (1 mmol) was dissolved in 10 ml of chloroform, followed by the addition of 5 ml of thionyl chloride and a catalytic amount of DMF (0.01 mmol). The mixture was refluxed for 3 hours. After completion of the reaction, the reaction solution was concentrated under reduced pressure and directly proceeded to the next step without further purification. The reduced pressure residue was dissolved in 10 ml of dichloromethane, followed by the addition of triethylamine (1.5 mmol) and stirring at room temperature for 0.5 h. Phenethylamine (1.1 mmol) was then added and stirring continued at room temperature for 4 hours. The reaction solution was washed sequentially with saturated sodium chloride and distilled water. The dichloromethane organic layer was separated and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (CH2Cl2:MeOH=100:1) to obtain Compound 3v as a white solid in a yield of 60.2%.

[0079] 1 H NMR (600Mz, CDCl3): δ7.28-7.25(m,2H),7.19-7.16(m,3H),7.04(d,J=3.5Hz,1H),6.43(t,J=5.9 Hz,1H),6.25(d,J=3.5Hz,1H),3.43(td,J=7.2,6.0Hz,2H),2.71-2.61(m,2H),1.94-1.90(m,2H). 13 C NMR (150MHz, CDCl3): δ157.34,147.33,141.31,138.11,128.50,128.35,126.07,116.04,109.02,38.95,33.32,31.11.MS-ESI(m / z):264.2(M+H) + .

[0080] Example 11. Synthesis of 5-chloro-N-(3-phenylpropyl)furan-2-carboxamide (3w)

[0081]

[0082] 5-Chloro-2-carboxyfuran (1 mmol) was dissolved in 10 ml of chloroform, followed by the addition of 5 ml of thionyl chloride and a catalytic amount of DMF (0.01 mmol). The mixture was refluxed for 3 hours. After completion of the reaction, the reaction solution was concentrated under reduced pressure and directly proceeded to the next step without further purification. The reduced pressure residue was dissolved in 10 ml of dichloromethane, followed by the addition of triethylamine (1.5 mmol) and stirring at room temperature for 0.5 h. Amphetamine (1.1 mmol) was then added and stirring continued at room temperature for 4 hours. The reaction solution was washed sequentially with saturated sodium chloride and distilled water, and the dichloromethane organic layer was separated and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (CH2Cl2:MeOH=100:1) to obtain Compound 3w as a white solid in a yield of 60.2%.

[0083] 1H NMR (600Mz, CDCl3): δ7.28-7.25(m,2H),7.19-7.16(m,3H),7.04(d,J=3.5Hz,1H),6.4 3(t,J=5.9Hz,1H),6.25(d,J=3.5Hz,1H),3.43(td,J=7.2,6.0Hz,2H),2.71-2.61(m,2H ),1.94-1.90(m,2H).13CNMR(150MHz,CDCl3):δ157.34,147.33,141.31,138.11,128.5 0,128.35,126.07,116.04,109.02,38.95,33.32,31.11.MS-ESI(m / z):264.2(M+H)+.

[0084] Example 12: Synthesis of 5-chloro-N-(4-phenylbutyl)furan-2-carboxamide (3x)

[0085]

[0086] 5-Chloro-2-carboxyfuran (1 mmol) was dissolved in 10 ml of chloroform, followed by the addition of 5 ml of thionyl chloride and a catalytic amount of DMF (0.01 mmol). The mixture was refluxed for 3 hours. After completion of the reaction, the reaction solution was concentrated under reduced pressure and directly proceeded to the next step without further purification. The reduced pressure residue was dissolved in 10 ml of dichloromethane, followed by the addition of triethylamine (1.5 mmol) and stirring at room temperature for 0.5 h. Phenbutylamine (1.1 mmol) was then added and stirring continued at room temperature for 4 hours. The reaction solution was washed sequentially with saturated sodium chloride and distilled water. The dichloromethane organic layer was separated and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (CH2Cl2:MeOH=100:1) to obtain Compound 3x as a white solid in a yield of 63.5%.

[0087] 1 H NMR (600Mz, CDCl3): δ7.26-7.23(m,2H),7.17-7.13(m,3H),7.03(d,J=3.5Hz,1H),6.56(t,J=5.9Hz ,1H),6.22(d,J=3.5Hz,1H),3.39(q,J=6.9Hz,2H),2.61(t,J=7.5Hz,2H),1.70-1.63(m,2H),1.63-

[0088] 1.53 (m, 2H). 13 C NMR (150MHz, CDCl3): δ157.28,147.32,141.93,137.99,128.31,128.26,125 .75,115.92,108.91,39.02,35.39,29.13,28.53.MS-ESI(m / z):278.2(M+H) + ..

[0089] Example 13. Synthesis of 5-chloro-N-(4-phenoxyethyl)furan-2-carboxamide (3y)

[0090]

[0091] 5-Chloro-2-carboxyfuran (1 mmol) was dissolved in 10 ml of chloroform, followed by the addition of 5 ml of thionyl chloride and a catalytic amount of DMF (0.01 mmol). The mixture was refluxed for 3 hours. After completion of the reaction, the reaction solution was concentrated under reduced pressure and directly proceeded to the next step without further purification. The reduced pressure residue was dissolved in 10 ml of dichloromethane, followed by the addition of triethylamine (1.5 mmol) and stirring at room temperature for 0.5 h. Phenoxyethylamine (1.1 mmol) was then added and stirring continued at room temperature for 4 hours. The reaction solution was washed sequentially with saturated sodium chloride and distilled water. The dichloromethane organic layer was separated and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (CH2Cl2:MeOH=100:1) to obtain compound 3y as a white solid in a yield of 61.5%.

[0092] 1 H NMR (500Mz, CDCl3): δ7.44 (t, J = 7.7Hz, 2H), 7.26 (d, J = 3.6Hz, 1H), 7.09 (dd, J = 22. 0,7.7Hz,4H),6.42(d,J=3.5Hz,1H),4.26(t,J=5.2Hz,2H),3.98(t,J=5.5Hz,2H). 13 C NMR (125MHz, CDCl3): δ158.37,157.44,146.96,138.37,129.51,121.15,116.43,114.45,109.05,66.40,38.74.MS-ESI(m / z):266.2(M+H) + ..

[0093] Example 14. Synthesis of 5-chloro-N-(4-fluorobenzyl)furan-2-carboxamide (3z)

[0094]

[0095] 5-Chloro-2-carboxyfuran (1 mmol) was dissolved in 10 ml of chloroform, followed by the addition of 5 ml of thionyl chloride and a catalytic amount of DMF (0.01 mmol). The mixture was refluxed for 3 hours. After completion of the reaction, the reaction solution was concentrated under reduced pressure and directly proceeded to the next step without further purification. The reduced pressure residue was dissolved in 10 ml of dichloromethane, followed by the addition of triethylamine (1.5 mmol) and stirring at room temperature for 0.5 h. 4-Fluorobenzylamine (1.1 mmol) was then added and stirring continued at room temperature for 4 hours. The reaction solution was washed sequentially with saturated sodium chloride and distilled water. The dichloromethane organic layer was separated and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (CH2Cl2:MeOH=100:1) to obtain compound 3z as a white solid in a yield of 51.7%.

[0096] 1H NMR (500Mz, CDCl3): δ7.44(t,J=6.5Hz,2H),7.24(d,J=3.5Hz,1H),7.14(t,J=8 .5Hz,2H),7.02(t,J=6.5Hz,1H),6.43(d,J=6.5Hz,1H), 4.68(d,J=6.1Hz,2H). 13 CNMR (125MHz, CDCl3): δ162.27 (d, J = 245.8Hz) 157.30, 146.99, 138.49, 133.80, 129.63 (d,J=8.1Hz),116.64,115.56(d,J=21.6Hz),109.17,42.47.MS-ESI(m / z):254.2(M+H) + ..

[0097] Example 15. Synthesis of 5-chloro-N-(4-fluorobenzyl)-N-methylfuran-2-carboxamide (3a1)

[0098]

[0099] 5-Chloro-2-carboxyfuran (1 mmol) was dissolved in 10 ml of chloroform, followed by the addition of 5 ml of thionyl chloride and a catalytic amount of DMF (0.01 mmol). The mixture was refluxed for 3 hours. After completion of the reaction, the reaction solution was concentrated under reduced pressure and directly proceeded to the next step without further purification. The reduced pressure residue was dissolved in 10 ml of dichloromethane, followed by the addition of triethylamine (1.5 mmol) and stirring at room temperature for 0.5 h. N-methyl-4-fluorobenzylamine (1.1 mmol) was then added and stirring continued at room temperature for 4 hours. The reaction solution was washed sequentially with saturated sodium chloride and distilled water. The dichloromethane organic layer was separated and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (CH2Cl2:MeOH=100:1) to obtain Compound 3a1 as a white solid in a yield of 51.5%.

[0100] 1 H NMR (500Mz, CDCl3): δ7.31 (brs, 2H), 7.06 (t, J = 8.8Hz, 3H), 6.31 (brs, 1H), 4.79 (s, 0.72H), 4.70 (s, 1.27H), 3.21 (s, 2.03H), 3.04 (s, 1.02H). 13C NMR (125MHz, CDCl3): δ162.16 (d, J = 245.8Hz), 158.99, 147.01, 138.52, 132.43, 129.89, 128.66,118.96,115.51(d,J=21.4Hz),108.28,51.20,35.61.MS-ESI(m / z):267.2(M+H) + ..

[0101] Example 16. Synthesis of 5-chloro-N-(3-fluorobenzyl)-N-methylfuran-2-carboxamide (3b1)

[0102]

[0103] 5-Chloro-2-carboxyfuran (1 mmol) was dissolved in 10 ml of chloroform, followed by the addition of 5 ml of thionyl chloride and a catalytic amount of DMF (0.01 mmol). The mixture was refluxed for 3 hours. After completion of the reaction, the reaction solution was concentrated under reduced pressure and directly proceeded to the next step without further purification. The reduced pressure residue was dissolved in 10 ml of dichloromethane, followed by the addition of triethylamine (1.5 mmol) and stirring at room temperature for 0.5 h. N-methyl-3-fluorobenzylamine (1.1 mmol) was then added and stirring continued at room temperature for 4 hours. The reaction solution was washed sequentially with saturated sodium chloride and distilled water. The dichloromethane organic layer was separated and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (CH2Cl2:MeOH=100:1) to obtain compound 3b1 as a white solid in a yield of 52.3%.

[0104] 1 H NMR (500Mz, CDCl3): δ7.34-7.30(m,1H),7.09-6.99(m,4H),6.31(brs,1H),4.75(s,2H),3.22(s,1.92H),3.10(s,1.25H). 13 C NMR (125MHz, CDCl3): δ163.25 (d, J = 247.1Hz), 159.36, 147.21, 139.53 (d, J = 6.7Hz), 13 8.81,130.43,123.75,119.19,114.78,108.50,51.82,36.08.MS-ESI(m / z):268.2(M+H) + ..

[0105] Example 17. Synthesis of 5-chloro-N-methyl-N-(3-methylbenzyl)furan-2-carboxamide (3c1)

[0106]

[0107] 5-Chloro-2-carboxyfuran (1 mmol) was dissolved in 10 ml of chloroform, followed by the addition of 5 ml of thionyl chloride and a catalytic amount of DMF (0.01 mmol). The mixture was refluxed for 3 hours. After completion of the reaction, the reaction solution was concentrated under reduced pressure and directly proceeded to the next step without further purification. The reduced pressure residue was dissolved in 10 ml of dichloromethane, followed by the addition of triethylamine (1.5 mmol) and stirring at room temperature for 0.5 h. N-methyl-3-methylbenzylamine (1.1 mmol) was then added and stirring continued at room temperature for 4 hours. The reaction solution was washed sequentially with saturated sodium chloride and distilled water. The dichloromethane organic layer was separated and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (CH2Cl2:MeOH=100:1) to obtain compound 3c1 as a white solid in a yield of 51.3%.

[0108] 1 H NMR (500Mz, CDCl3): δ7.43-7.24(m,4H),7.24-7.12(m,1H),6.42-6.41(m,1H),4.85(s,2H),3.31(s,1.62H),3.19(s,1.43H),2.50(s,3H). 13 C NMR (125MHz, CDCl3): δ159.13,147.36,138.48,137.32,133.61,129.44,128.26, 126.99,118.89,118.08,108.25,51.93,35.72,21.11.MS-ESI(m / z):264.2(M+H) + ..

[0109] Example 18. Synthesis of 5-methyl-N-(pyridin-2-yl)furan-2-carboxamide (3d1)

[0110]

[0111] 5-Methyl-2-carboxyfuran (1 mmol) was dissolved in 10 ml of chloroform, followed by the addition of 5 ml of thionyl chloride and a catalytic amount of DMF (0.01 mmol). The mixture was refluxed for 3 hours. After completion of the reaction, the reaction solution was concentrated under reduced pressure and directly proceeded to the next step without further purification. The reduced pressure residue was dissolved in 10 ml of dichloromethane, followed by the addition of triethylamine (1.5 mmol) and stirring at room temperature for 0.5 h. 2-Aminopyridine (1.1 mmol) was then added and stirring continued at room temperature for 4 hours. The reaction solution was washed sequentially with saturated sodium chloride and distilled water, and the dichloromethane organic layer was separated and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (CH2Cl2:MeOH=100:1) to obtain Compound 3d1 as a white solid in a yield of 45.5%.

[0112] 1 H NMR (500Mz, CDCl3): δ8.68(brs,1H),8.32(d,J=8.0Hz,2H),7.73(t,J=8.0Hz,1H),7.16(s,1H),7.09-7.01(m,1H),6.17(s,1H),2.39(s,3H). 13 C NMR(125MHz, CDCl3): δ148.12,138.56,119.92,117.36,114.18,109.33,14.03.MS-ESI(m / z):203.2(M+H) + ..

[0113] Example 19. Synthesis of 5-methyl-N-(5-methylpyridin-2-yl)furan-2-carboxamide (3e1)

[0114]

[0115] 5-Methyl-2-carboxyfuran (1 mmol) was dissolved in 10 ml of chloroform, followed by the addition of 5 ml of thionyl chloride and a catalytic amount of DMF (0.01 mmol). The mixture was refluxed for 3 hours. After completion of the reaction, the reaction solution was concentrated under reduced pressure and directly proceeded to the next step without further purification. The reduced pressure residue was dissolved in 10 ml of dichloromethane, followed by the addition of triethylamine (1.5 mmol) and stirring at room temperature for 0.5 h. 5-methyl-2-aminopyridine (1.1 mmol) was then added and stirring continued at room temperature for 4 hours. The reaction solution was washed sequentially with saturated sodium chloride and distilled water. The dichloromethane organic layer was separated and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (CH2Cl2:MeOH=100:1) to obtain compound 3e1 as a white solid in a yield of 45.5%.

[0116] 1 H NMR (500Mz, CDCl3): δ8.69(s,1H),8.20(d,J=8.4Hz,1H),8.11(s,1H),7.52(d,J=8.1Hz,1H),7.12(s,1H),6.14(s,1H),2.35(s,3H),2.28(s,3H). 13 C NMR (125MHz, CDCl3): δ156.25,155.47,149.12,147.95,145.92,139.04,129.20,117.04,113.64,109.18,17.92,13.93.MS-ESI(m / z):217.2(M+H) + ..

[0117] Example 20. Synthesis of 5-methyl-N-(6-methylpyridin-2-yl)furan-2-carboxamide (3f1)

[0118]

[0119] 5-Methyl-2-carboxyfuran (1 mmol) was dissolved in 10 ml of chloroform, followed by the addition of 5 ml of thionyl chloride and a catalytic amount of DMF (0.01 mmol), and the mixture was refluxed for 3 hours. After completion of the reaction, the reaction solution was concentrated under reduced pressure and directly proceeded to the next step without further purification. The reduced pressure residue was dissolved in 10 ml of dichloromethane, followed by the addition of triethylamine (1.5 mmol) and stirring at room temperature for 0.5 h. 6-methyl-2-aminopyridine (1.1 mmol) was then added and stirring continued at room temperature for 4 hours. The reaction solution was washed sequentially with saturated sodium chloride and distilled water, the dichloromethane organic layer was separated, and the mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (CH2Cl2:MeOH=100:1) to obtain compound 3f1 as a white solid in a yield of 43.5%.

[0120] 1 H NMR (500Mz, CDCl3): δ8.60 (brs, 1H), 8.12 (d, J = 8.2Hz, 1H), 7.67-7.56 (m, 1H ),7.15(s,1H),6.91(d,J=7.4Hz,1H),6.15(s,1H),2.48(s,3H),2.38(s,3H). 13 C NMR (125MHz, CDCl3): δ157.06,156.36,155.57,150.62,145.96,138.81,119.40,117.20,111.03,109.26,24.21,14.01.MS-ESI(m / z):217.2(M+H) + ..

[0121] Example 21. Synthesis of N-(4-methoxypyridin-2-yl)-5-methylfuran-2-carboxamide (3g1)

[0122]

[0123] 5-Methyl-2-carboxyfuran (1 mmol) was dissolved in 10 ml of chloroform, followed by the addition of 5 ml of thionyl chloride and a catalytic amount of DMF (0.01 mmol). The mixture was refluxed for 3 hours. After completion of the reaction, the reaction solution was concentrated under reduced pressure and directly proceeded to the next step without further purification. The reduced pressure residue was dissolved in 10 ml of dichloromethane, followed by the addition of triethylamine (1.5 mmol) and stirring at room temperature for 0.5 h. 4-methoxy-2-aminopyridine (1.1 mmol) was then added and stirring continued at room temperature for 4 hours. The reaction solution was washed sequentially with saturated sodium chloride and distilled water, the dichloromethane organic layer was separated, and the mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (CH2Cl2:MeOH=100:1) to obtain compound 3g1 as a white solid in a yield of 38.5%.

[0124] 1 H NMR (500Mz, CDCl3): δ8.85 (s, 1H), 8.19 (d, J = 9.0Hz, 1H), 7.92-7.89 (m, 1H), 7.20 (d d,J=8.9,2.5Hz,1H),7.05(d,J=2.9Hz,1H),6.06(s,1H),3.74(s,3H),2.25(s,3H). 13 C NMR (125MHz,CDCl3):δ

[0125] 155.89,155.21,152.68,145.80,144.84,134.49,123.17,116.67,114.51,108.92,55.78,13.70.MS-ESI(m / z):233.2(M+H) + ..

[0126] Example 22. Synthesis of N-(4-ethylpyridin-2-yl)-5-methylfuran-2-carboxamide (3h1)

[0127]

[0128] 5-Methyl-2-carboxyfuran (1 mmol) was dissolved in 10 ml of chloroform, followed by the addition of 5 ml of thionyl chloride and a catalytic amount of DMF (0.01 mmol). The mixture was refluxed for 3 hours. After completion of the reaction, the reaction solution was concentrated under reduced pressure and directly proceeded to the next step without further purification. The reduced pressure residue was dissolved in 10 ml of dichloromethane, followed by the addition of triethylamine (1.5 mmol) and stirring at room temperature for 0.5 h. 4-ethyl-2-aminopyridine (1.1 mmol) was then added and stirring continued at room temperature for 4 hours. The reaction solution was washed sequentially with saturated sodium chloride and distilled water, and the dichloromethane organic layer was separated and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (CH2Cl2:MeOH=100:1) to obtain Compound 3h1 as a white solid in a yield of 37.6%.

[0129] 1 H NMR (500Mz, CDCl3): δ8.71(s,1H),8.19(s,1H),8.17(d,J=4.9Hz,1H),7.14(s,1H),6.89 (d,J=4.5Hz,1H),6.15(s,1H),2.67(q,J=7.5Hz,2H),2.36(s,3H),1.26(t,J=7.5Hz,3H). 13 C NMR (125MHz, CDCl3): δ156.37,156.00,155.58,151.43,147.78,145.92,119 .84,117.18,113.51,109.24,28.70,14.47,13.96.MS-ESI(m / z):231.2(M+H) + ..

[0130] Example 23. Synthesis of N-(4-cyclopropylpyridin-2-yl)-5-methylfuran-2-carboxamide (3i1)

[0131]

[0132] 5-Methyl-2-carboxyfuran (1 mmol) was dissolved in 10 ml of chloroform, followed by the addition of 5 ml of thionyl chloride and a catalytic amount of DMF (0.01 mmol). The mixture was refluxed for 3 hours. After completion of the reaction, the reaction solution was concentrated under reduced pressure and directly proceeded to the next step without further purification. The reduced pressure residue was dissolved in 10 ml of dichloromethane, followed by the addition of triethylamine (1.5 mmol) and stirring at room temperature for 0.5 h. 4-cyclopropyl-2-aminopyridine (1.1 mmol) was then added and stirring continued at room temperature for 4 hours. The reaction solution was washed sequentially with saturated sodium chloride and distilled water, and the dichloromethane organic layer was separated and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (CH2Cl2:MeOH=100:1) to obtain compound 3i1 as a white solid in a yield of 39.5%.

[0133] 1 H NMR (500Mz, CDCl3): δ8.68(s,1H),8.10(d,J=5.3Hz,1H),8.04(s,1H),7.13(s,1H),6.71(d,J=5.0Hz,1 H), 6.15 (s, 1H), 2.36 (s, 3H), 1.90 (tt, J = 9.0, 5.0Hz, 1H), 1.07 (d, J = 7.8Hz, 2H), 0.84 (d, J = 5.3Hz, 2H). 13 C NMR (125MHz, CDCl3): δ156.81,156.38,155.59,151.33,147.58,145.89,117 .22,117.16,110.77,109.23,15.55,13.95,10.78.MS-ESI(m / z):243.2(M+H) + .HRMS-ESI(m / z):Calcd.for C 14 H 15 N2O2(M+H) + :243.1128; Found:243.1126.

[0134] Example 24. Synthesis of N-(4-fluoropyridin-2-yl)-5-methylfuran-2-carboxamide (3j1)

[0135]

[0136] 5-Methyl-2-carboxyfuran (1 mmol) was dissolved in 10 ml of chloroform, followed by the addition of 5 ml of thionyl chloride and a catalytic amount of DMF (0.01 mmol). The mixture was refluxed for 3 hours. After completion of the reaction, the reaction solution was concentrated under reduced pressure and directly proceeded to the next step without further purification. The reduced pressure residue was dissolved in 10 ml of dichloromethane, followed by the addition of triethylamine (1.5 mmol) and stirring at room temperature for 0.5 h. 4-Fluoro-2-aminopyridine (1.1 mmol) was then added and stirring continued at room temperature for 4 hours. The reaction solution was washed sequentially with saturated sodium chloride and distilled water, and the dichloromethane organic layer was separated and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (CH2Cl2:MeOH=100:1) to obtain Compound 3j1 as a white solid in a yield of 47.5%.

[0137] 1H NMR (500Mz, CDCl3): δ8.81(s,1H),8.32(dd,J=9.2,4.2Hz,1H),8.12(s,1H),7.43(t,J=9.5Hz,1H),7.14(d,J=2.9Hz,1H),6.13(s,1H),2.33(s,3H). 13 C NMR (125MHz, CDCl3): δ157.44, 156.08, 155.69, 155.45, 146.57 (d, J = 248.1Hz), 135.49 (d, J =25.6Hz),125.31(d,J=19.3Hz),117.41,114.91,109.27,13.90.MS-ESI(m / z):221.2(M+H) + ..

[0138] Example 25. Synthesis of 2-methyl-4-methylpyridin-2-yl-5-methylfuran-2-yl ester (3k1)

[0139]

[0140] 5-Methyl-2-carboxyfuran (1 mmol) was dissolved in 10 ml of chloroform, followed by the addition of 5 ml of thionyl chloride and a catalytic amount of DMF (0.01 mmol). The mixture was refluxed for 3 hours. After completion of the reaction, the reaction solution was concentrated under reduced pressure and directly proceeded to the next step without further purification. The reduced pressure residue was dissolved in 10 ml of dichloromethane, followed by the addition of triethylamine (1.5 mmol) and stirring at room temperature for 0.5 h. 4-methyl-2-hydroxypyridine (1.1 mmol) was then added and stirring continued at room temperature for 4 hours. The reaction solution was washed sequentially with saturated sodium chloride and distilled water. The dichloromethane organic layer was separated and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (CH2Cl2:MeOH=100:1) to obtain Compound 3k1 as a white solid in a yield of 39.6%.

[0141] 1 H NMR (500Mz, CDCl3): δ8.27(d,J=4.9Hz,1H),7.38-7.28(m,1H),7.06(d,J=4.5Hz,1H),7.02(s,1H),6.00(s,1H),2.42(s,3H),2.40(s,3H). 13 C NMR (125MHz, CDCl3): δ158.82,157.94,156.58,151.41,148.26,142.17,123.44,121.82,117.10,109.10,21.19,14.26.MS-ESI(m / z):218.2(M+H)+ ..

[0142] Example 26: Cell culture

[0143] Both mouse osteoblast precursor cells MC3T3-E1 and human osteosarcoma cells U-2OS are adherent cells. Once cells have grown confluently, discard the old culture medium, rinse with 1 ml of PBS, and discard. Add 1 ml of trypsin and digest. Discard the digestion solution and immediately add α-MEM or Mcoy's 5A complete medium supplemented with 10% FBS. Remove the cells by pipetting to completely detach them from the bottom of the dish and disperse them into a single cell suspension. The cell suspension is then seeded into a new dish at a 1:3 ratio, supplemented with the appropriate amount of complete medium, and placed in an incubator for continued culture. Culture conditions were 37°C, 5% CO2. Mouse monocyte-macrophage RAW264.7 cells were passaged at a ratio of 1:4-1:6 using DMEM complete medium. Other culture conditions were the same as for MC3T3-E1.

[0144] Example 27: Determination of the Dose-Effect Relationship of N-Methylpyridine Furanamide Derivatives in an OPG Expression Regulation Screening Model

[0145] A high-throughput screening model for OPG expression upregulators, previously constructed in the laboratory, was used for high-throughput screening. This model involves transfecting U-2OS cells with the pGL4.17-OPGp plasmid, which contains the firefly luciferase reporter gene and regulates the expression of the human OPG promoter (-5917 to +19). The resulting stably transfected cell line is called OPG-Luc U-2OS (abbreviated as UOP) (see CN109651345B and CN104845936B).

[0146] The specific screening method is as follows: the UOP of the model cells in the logarithmic growth phase is adjusted to 5×10 4 The cells were seeded into a 96-well transparent bottom white plate at a density of 100 cells / well. After the cells were fully attached to the wall, the original culture medium was discarded and 198 μL of Mcoy's 5A culture medium with 5% FBS and 2 μL of the sample to be tested were added to each well. DMSO with the same concentration as the sample to be tested was used as a blank control. After 18 to 24 hours, the culture medium was aspirated and the cells were lysed by adding lysis buffer. Cell luciferase activity was measured using the Reporter Assay System. The upregulation rate of the compound on luciferase activity was calculated according to the following formula: Upregulation rate = Firefly luciferase expression activity of cells after compound treatment / Firefly luciferase expression activity of cells after DMSO treatment × 100%. The regulation rate of each compound was calculated, and compounds with a regulation rate ≥ 150% were defined as compounds with the potential to upregulate OPG. Graphpad Prism software was used to generate the graphs, and the EC values were obtained. 50and the maximum rate of increase (Max).

[0147] The screening results of each compound are shown in Table 1.

[0148] Table 1. Upregulation activity of some compounds in the OPG expression upregulator screening model

[0149]

[0150]

[0151]

[0152] Example 28: Effects of Compounds on Osteoclast Differentiation

[0153] Tartrate-resistant acid phosphatase (TRAP) is a specific marker enzyme for osteoclasts, and its expression and secretion are closely related to osteoclast function. Azo-coupled histochemical analysis uses naphthol diphosphate as a substrate and azo-parafuchsin as a chromogen. In the presence of potassium sodium tartrate, TRAP hydrolyzes naphthol AS-BI phosphate to form naphthol AS-BI, which then binds to the chromogen at the enzyme's active site to form an insoluble red precipitate that turns purple upon counterstaining with hematoxylin.

[0154] Mouse macrophage RAW264.7 cells were cultured at 2 × 10 cells per well. 2 Cells were seeded in 12-well plates. After cells adhered, the medium was changed to osteoclast differentiation induction medium. Induction was carried out at 37°C under 5% CO2 for 3 days, followed by addition of the compound for 3 days. Prepare the fixative in advance: 25 mL of citrate diluent + 65 mL of acetone + 8 mL of 37% formaldehyde. Prepare sufficient deionized water in a 37°C water bath and check the temperature before use. Bring the fixative to room temperature, add 200 μL per well, fix for 30 seconds, rinse with 37°C deionized water, and aspirate thoroughly, avoiding any drying of the wells. Add 0.5 mL of Rapid Garnet GBC Base Solution + 0.5 mL of sodium nitrite solution to two test tubes, gently invert to mix for 30 seconds, and let stand for 2 minutes. Label two 50mL BD tubes A and B. To tube A, add 45mL of 37°C deionized water, 1.0mL of the diazo rapid garnet GBC solution prepared in step 4, 0.5mL of naphthol AS-BI phosphoric acid solution, and 2.0mL of acetic acid solution. To tube B, prepare the solution according to the solution in beaker A and add 1.0mL of tartaric acid solution. Combine the solutions in tubes A and B into one tube and place it in a 37°C water bath. Before staining, ensure the solution temperature is at 37°C. Add the staining solution to the fixed cell plate, protect from light, and stain at 37°C for 1 hour. After rinsing with distilled water, counterstain with hematoxylin for 2 minutes, and rinse with tap water for several minutes. Allow to air dry and observe under a microscope. Select TRAP-positive cells with ≥3 nuclei as osteoclasts and count them.

[0155] Compounds with significant OPG upregulation activity (3c, 3e, 3g, 3q, 3r, 3t, 3v, 3y, 3d1, 3e1, 3j1, 3g1, 3h1, and 3i1) were evaluated for their inhibitory activity against RANKL-induced osteoclast differentiation in mouse macrophage RAW264.7 cells using TRAP staining. The inhibitory activity of each compound at 1 μM and 10 μM was determined. As shown in Table 2, the inhibitory effects on TRAP activity for these compounds ranged from 37% to 72%, indicating that these compounds exhibited significant inhibitory activity against osteoclast differentiation.

[0156] Table 2 TRAP staining assay for the inhibitory activity of compounds on osteoclast differentiation

[0157]

[0158] Example 29: Effects of Compounds 3c and 3v on the Formation of Calcified Nodules, an Indicator of Late Mineralization in MC3T3-E1 Cells

[0159] Alizarin Red S staining is a classic technique that uses chelation to form a complex between calcium ions and Alizarin Red S to analyze the orange-red calcium deposition phenomenon in fixed cell samples.

[0160] The specific steps are as follows: MC3T3-E1 cells were seeded in 6-well or 12-well plates. When the cells grew to more than 80%, the culture medium was replaced with osteogenic differentiation induction medium containing 10 mM β-glycerophosphate and 50 μg / mL L-ascorbic acid supplemented with the compound. Co-culture was carried out at 37°C, 5% CO2 for 21 days, and fresh culture medium was replaced every 3 days. After the incubation period, the culture medium was discarded and the cells were rinsed twice with PBS. The cells were fixed with 95% ethanol for 10 minutes and rinsed three times with double-distilled water. The cells were stained with 0.2% Alizarin Red S solution (pH 4.2) at room temperature for 10 minutes. The cells were rinsed three times with distilled water. The cells were counterstained with hematoxylin for 1 minute, rinsed three times with distilled water, and photographed. The cells were photographed under a microscope, and the calcification nodules were quantified using Image J software.

[0161] The formation of calcified nodules is an indicator of late osteoblast differentiation. MC3T3-E1 cells were induced to differentiate using osteogenic differentiation medium and treated with different concentrations of 3c and 3v for 21 days. Alizarin red staining was then used to detect the formation of calcified nodules. Figure 1As shown, after 21 days of treatment with 3c and 3v, the number of calcified nodules formed by MC3T3-E1 cell differentiation increased significantly, and was dose-dependent. After quantification, compound 3c had a significant increasing effect on the formation of calcified nodules at 0.1, 1, and 10 μM, and compound 3v had a significant increasing effect on the formation of calcified nodules at 1 and 10 μM. This further demonstrates that compounds 3c and 3v can significantly promote osteoblast differentiation in vitro.

[0162] Example 30: Effects of Compounds 3c and 3il on Osteoclast Differentiation-Related Proteins

[0163] During the formation of osteoclasts, RANKL can induce the expression of osteoclast-related marker genes NFATc1 and matrix metalloproteinase-9 (MMP-9). Both are osteoclast-specific transcription factors and markers, so we detected the protein levels of NFATc1 and MMP9 in RAW264.7 cells after RANKL induction. Mouse macrophages RAW264.7 were seeded in 6-well plates, and after the cells adhered, they were replaced with osteoclast differentiation induction medium. The cells were induced at 37°C and 5% CO2 for 3 days, and then the compound was added for 1 day. The protein expression levels of NFATc1 and MMP-9 were detected by Western blot, as shown in Figure 2. Figure 2 As shown in Figure 3, compounds 3c and 3i1 significantly reduced the expression levels of NFATc1 and MMP-9 proteins in RANKL-induced RAW264.7 cells in a dose-dependent manner. These results confirm that compounds 3c and 3i1 have an inhibitory effect on osteoclast differentiation.

[0164] Example 31: Effects of Compounds 3c, 3v, and 3i1 on OPG Protein Expression in Mouse Osteoblasts MC3T3-E1 Cells

[0165] MC3T3-E1 cells were cultured at 1×10 5Cells were seeded into 6-well plates at 100 μg / well and incubated at 37°C, 5% CO₂ for 24 hours. Compounds were diluted to a specific concentration. DMSO was added to blank control wells at the same final concentration as in the compound-containing wells and incubated at 37°C, 5% CO₂ for 24 hours. Cells were harvested by centrifugation at 900 rpm for 4 minutes. RIPA lysis buffer containing protease and phosphatase inhibitors was added and cells were lysed on ice for 30 minutes. The supernatant was collected and transferred to a fresh EP tube for protein quantification using the BCA protein assay. All samples were adjusted to the same concentration using RIPA buffer and the appropriate amount of 5× protein loading buffer was added. Protein samples were boiled in a boiling water bath for 10 minutes and stored at -80°C before Western blot analysis. Primary antibodies and corresponding HRP-conjugated secondary antibodies were added for incubation. Enhanced HRP substrate chemiluminescence (ECL) was used for color development.

[0166] Western blot was used to detect the expression of OPG protein in MC3T3-E1 cells after treatment with different concentrations of 3c, 3v, and 3i1 for 24 hours. The imaging results were grayscale scanned and quantified. Figure 3 As shown, 3c, 3v, and 3i1 could dose-dependently upregulate OPG protein levels.

[0167] Example 32: Effects of Compounds 3c, 3v, and 3i1 on OPG Protein Secretion Levels in MC3T3-E1 Cells

[0168] MC3T3-E1 cells were treated with different concentrations of compounds for 48 h, and the cell supernatants were collected. 4°C, 2000-3000 r·min -1 After centrifugation for 20 minutes, the supernatant was collected. Detection was performed using an ELISA kit (Nanjing Sen Bei Jia Biotechnology Co., Ltd., Nanjing, China). A blank well, a test sample well, and a standard well were set up. 40 μL of sample diluent was first added to each well, followed by 10 μL of the test sample. After sealing the plate, the plate was incubated at 37°C for 30 minutes. The sealing film was removed, the liquid was discarded, and 300 μL of washing solution was added to each well. After standing for 30 seconds, the solution was discarded. Repeat this process five times. 50 μL of enzyme-labeled reagent was added to each well and the plate was incubated at 37°C for 30 minutes. The plate was washed five times. 50 μL of color developer A was added, followed by 50 μL of color developer B. The plate was developed at 37°C in the dark for 15 minutes. 50 μL of stop solution was added to each well. The absorbance of each well was measured at a wavelength of 450 nm. The assay should be performed within 15 minutes after the addition of the stop solution. The concentration of OPG in the test sample was calculated based on the standard curve of the standard.

[0169] The results are as follows Figure 4As shown, 3c, 3v, and 3i1 can significantly promote the secretion level of OPG in MC3T3-E1 cells. The above results indicate that 3c, 3v, and 3i1 can increase the expression and secretion of OPG, a key protein for osteogenic differentiation, in MC3T3-E1 cells in vitro.

[0170] Example 33: In vivo pharmacokinetic (PK) study of the compound administered intravenously or orally to SD rats

[0171] Male SD rats (300-400 g) were selected. They were randomly divided into groups, housed in a standard environment, and had free access to water and food. The drug was administered by intravenous injection (IV) (1 mg / kg) and oral gavage (PO) (20 mg / kg). The rats were weighed before administration, and the dosage was calculated based on body weight. The animals were fasted for 10-16 hours before administration and resumed eating 4 hours after the end of administration. The animals were free to eat during the rest of the experiment. Blood was collected from the submandibular vein 0.083 h, 0.25 h, 0.5 h, 1 h, 2 h, 4 h, 8 h, and 24 h after intravenous administration, and 0.25 h, 0.5 h, 1 h, 2 h, 4 h, 6 h, 8 h, and 24 h after oral gavage administration. Approximately 200 μL of each sample was collected, anticoagulated with sodium heparin, placed on ice after collection, and centrifuged within 2 hours to separate the plasma (centrifugation conditions: centrifugal force 6800 g, 6 min, 2-8 ° C). The collected plasma samples were stored in a -70°C refrigerator before analysis. After analysis, the remaining plasma samples were kept in a -70°C refrigerator for one month. The concentration of the test substance in the plasma samples was detected. The accuracy of the quality control samples was evaluated while the samples were analyzed. It was required that the accuracy of more than 66% of the quality control samples was between 80-120%. The blood drug concentration at each time point was collected, the data was processed, and the pharmacokinetic parameters were calculated (calculation method: WinNonlin), such as the area under the drug concentration-time curve (AUC (0-t) ), distribution half-life (T 1 / 2 ), maximum blood concentration (C max ), peak time (T max ) and oral bioavailability (F), etc.

[0172] As can be seen from Table 3, at the same dose, the area under the drug-time curve (AUC) of compound 3c is 0-∞ (h·ng·mL -1 ) and bioavailability F% were 89.21 and 2.46, respectively. The area under the drug-time curve (AUC) of compound 3i1 was 0-∞ (h·ng·mL -1 ) and bioavailability F% were 120.78 and 7.33, respectively. The area under the drug-time curve (AUC) of compound 3g1 was 0-∞ (h·ng·mL -1) and bioavailability F% were 460.26 and 12.73, respectively, while the area under the concentration-time curve (AUC) of 3a was 0-∞ (h·ng·mL -1 ) and bioavailability F% were 25.06 and 0.89. Compared with 3a, compounds 3c, 3i1, and 3g1 could significantly improve the bioavailability.

[0173] Table 3. In vivo pharmacokinetic data of compounds

[0174]

[0175] Finally, it should be noted that the above embodiments are only used to help those skilled in the art understand the essence of the present invention and are not used to limit the scope of protection of the present invention.

Claims

1. A compound having a structure represented by the following formula (1), 2. A pharmaceutical composition comprising the compound according to claim 1, characterized in that The pharmaceutical composition comprises: (1) a therapeutically effective amount of the compound of claim 1; and (2) one or more pharmaceutically acceptable carriers.

3. The pharmaceutical composition according to claim 2, characterized in that The pharmaceutical composition is an oral preparation.

4. The pharmaceutical composition according to claim 3, characterized in that The pharmaceutical composition is a tablet for oral administration.

5. Use of the compound according to claim 1 in the preparation of a medicament for treating osteoporosis.

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