Alfalfa furan analogs, methods of making and use in the treatment of osteoporosis

Abstract

This invention belongs to the field of chemical pharmaceutical raw material technology, specifically relating to an alfalfa furan analogue, its preparation method, and its use in the treatment of osteoporosis. The structure of the alfalfa furan analogue is shown in Formula I. Experimental verification has shown that the alfalfa furan analogue provided by this invention has a good therapeutic effect on osteoporosis, laying the foundation for the development of drugs for treating osteoporosis. Formula I

Description

Technical Field

[0001] This invention belongs to the field of chemical pharmaceutical raw material technology, specifically relating to an alfalfa furan analogue, its preparation method, and its use in the treatment of osteoporosis. Background Technology

[0002] Osteoporosis (OP) is a systemic skeletal metabolic disorder characterized by low bone mass, deterioration of bone microstructure, increased bone fragility, and elevated fracture risk. With the increasing aging of the global population, the incidence of osteoporosis in the elderly is exploding, becoming a major public health challenge posing a serious threat to the health and quality of life of middle-aged and elderly people. From the perspective of bone biology mechanisms, bone remodeling depends on a precise dynamic balance between osteoclast-mediated bone resorption and osteoblast-mediated bone formation. However, in the development of osteoporosis in the elderly, the decline in the differentiation potential of bone marrow mesenchymal stem cells (BMSCs) into osteoblasts and the increase in osteoblast apoptosis lead to severe bone formation insufficiency and a metabolic imbalance of high / low turnover rate.

[0003] Currently, first-line anti-osteoporosis drugs in clinical practice are mainly divided into two categories: 1. Bone resorption inhibitors: such as bisphosphonates, calcitonin, and denosumab. Although these drugs can slow down bone loss, they cannot rebuild the lost bone microstructure, and long-term inhibition of bone turnover can easily lead to serious complications such as atypical femoral fractures (AFF) and osteonecrosis of the jaw (ONJ); 2. Bone formation promoters: such as parathyroid hormone receptor agonists: teriparatide, abalotide, and sclerostin monoclonal antibody romozolumab. Although these drugs have the ability to reverse bone loss, they are limited by drug-like defects—including the need for long-term subcutaneous injection of peptide / monoclonus antibody drugs, low bioavailability, high production costs, and some drugs have the risk of increased cardiovascular events or black box warnings, such as the potential risk of osteosarcoma in rats. Therefore, based on key osteogenic signaling pathways, the development of novel small-molecule bone formation promoters that are highly efficient, low in toxicity, and have good oral bioavailability is a highly challenging and clinically valuable frontier direction in the field of medicinal chemistry and new drug discovery.

[0004] Bone morphogenetic protein-2 (BMP-2), a core member of the TGF-β superfamily, is currently the most potent known signaling factor for inducing bone formation in vivo. BMP-2 activates the downstream Smad1 / 5 / 8 phosphorylation cascade by binding to membrane receptors, or cross-activates non-Smad-dependent pathways such as p38 MAPK, thereby precisely upregulating the expression of core osteogenic transcription factors such as Runx2 (Runt-related transcription factor 2) and Osterix (OSX), ultimately driving the directed differentiation of bone mesenchymal stem cells (BMSCs) into osteoblasts and promoting bone matrix mineralization. However, recombinant human BMP-2 protein, when applied topically in clinical settings, is highly susceptible to nonspecific ectopic ossification, tissue edema, and immunogenicity, and systemic administration is not feasible. Therefore, the search for small molecule compounds that can endogenously upregulate or allosterically activate the BMP-2 pathway represents a new breakthrough for small-molecule bone-promoting therapies.

[0005] Natural products and their advantageous skeletons are a natural treasure trove for lead compound discovery due to their unique stereochemistry and multi-target pharmacological characteristics. The unique ecological environment of the Ili Valley in Xinjiang, characterized by strong sunlight, extreme temperature differences, and high altitude, has nurtured alfalfa (Medicago sativa L.) resources with unique metabolic phenotypes. Studies have shown that alfalfafuran, an active isoflavone natural product with a 2-phenylbenzofuran-like skeleton extracted and isolated from alfalfafuran, exhibits certain anti-osteoporosis bioactivity. However, there is still room for improvement in the therapeutic effect of natural alfalfafuran molecules on osteoporosis. Based on this, the inventors' team has filed this application.

[0006]

[0007] Alfalfa furan Summary of the Invention

[0008] The technical problem to be solved by the present invention is to provide an alfalfa furan analogue with good therapeutic effect on osteoporosis, its preparation method and application.

[0009] To solve the above problems, the technical solution adopted by the present invention is as follows:

[0010] An alfalfa furan analog, its pharmaceutically acceptable salt, or its stereoisomer, the key feature of which is that the structure of the compound is shown in Formula I:

[0011]

[0012] Formula I

[0013] R1 and R2 are independently selected from hydrogen, C1-C8 alkyl, C1-C8 alkoxy, halogen, nitro, cyano, 1-3 halogen-substituted C1-C8 alkyl, 1-3 halogen-substituted C1-C8 alkoxy, phenyl, benzyl, and benzyloxy.

[0014] As a further improvement of the present invention, R1 and R2 are independently selected from hydrogen, C1-C5 alkyl, C1-C5 alkoxy, halogen, nitro, cyano, C1-C5 alkyl with 1-3 halogen substituted, C1-C5 alkoxy with 1-3 halogen substituted, phenyl, benzyl, and benzyloxy.

[0015] As a further improvement of the present invention, the C1-C5 alkyl group is selected from one of the following structures: —CH3, —CH2CH3, —CH(CH3)2, —(CH2)2CH3, —(CH2)3CH3, —CH2CH(CH3)2, —C(CH3)3, —CH(CH3)CH2CH3, —(CH2)4CH3, —CH(CH3)(CH2)2CH3, —CH(CH2CH3)2, —CH2C(CH3)3, —CH2CH(CH3)CH2CH3, —C(CH3)2CH2CH3, —CH(CH3)CH(CH3)2, —(CH2)2CH(CH3)2;

[0016] The C1-C5 alkoxy group is selected from one of the following structures: —OCH3, —OCH2CH3, —OCH(CH3)2, —O(CH2)2CH3, —O(CH2)3CH3, —OCH2CH(CH3)2, —OC(CH3)3, —OCH(CH3)CH2CH3, —O(CH2)4CH3, —OCH(CH3)(CH2)2CH3, —OCH(CH2CH3)2, —OCH2C(CH3)3, —OCH2CH(CH3)CH2CH3, —OC(CH3)2CH2CH3, —OCH(CH3)CH(CH3)2, —O(CH2)2CH(CH3)2;

[0017] The halogen is selected from F, Cl, Br or I;

[0018] The 1-3 halogen-substituted C1-C5 alkyl groups are selected from -CH2F, -CHF2, -CF3, -CH2CF3, -CH2CH2CF3, -CH2CH2CH2CF3, and -CH2CH2CH2CH2CF3;

[0019] The 1-3 halogen-substituted C1-C5 alkoxy groups are selected from -OCF3, -OCH2CF3, -OCH2CH2CF3, -OCH2CH2CH2CF3, and -OCH2CH2CH2CH2CF3.

[0020] As a further improvement of the present invention, R1 and R2 are selected from one of the following structures: H, nitro, cyano, phenyl, benzyl, benzyloxy, -F, -Cl, -Br, -CH3, -CH2CH3, -CH(CH3)2, -OCH2CH3, -C(CH3)3, -CH2CH2CH3, -OCH3, -CHF2, -CF3, -OCF3, -CH2CH(CH3)2.

[0021] As a further improvement of the present invention, the compound is selected from one of the following structures:

[0022] , , ,

[0023] Compound 1 Compound 2 Compound 3

[0024] , , ,

[0025] Compound 4, Compound 5, Compound 6

[0026] , , ,

[0027] Compound 7, Compound 8, Compound 9

[0028] , , ,

[0029] Compound 10, Compound 11, Compound 12

[0030] , , ,

[0031] Compound 13, Compound 14, Compound 15

[0032] , , ,

[0033] Compound 16, Compound 17, Compound 18

[0034] , , ,

[0035] Compound 19, Compound 20, Compound 21

[0036] , , ,

[0037] Compound 22, Compound 23, Compound 24

[0038] , , ,

[0039] Compound 25, Compound 26, Compound 27

[0040] , , ,

[0041] Compound 28, Compound 29, Compound 30

[0042] , , ,

[0043] Compound 31, Compound 32, Compound 33

[0044] , , ,

[0045] Compound 34, Compound 35, Compound 36

[0046] , , ,

[0047] Compound 37, Compound 38, Compound 39

[0048] , , ,

[0049] Compound 40, Compound 41, Compound 42

[0050] , , ,

[0051] Compound 43, Compound 44, Compound 45

[0052] , , ,

[0053] Compound 46, Compound 47, Compound 48

[0054] ,

[0055] Compound 49 Compound 50.

[0056] Technical Theme Two

[0057] A method for preparing the compound as described in Technical Subject 1, which is carried out via the following route:

[0058] S1: ;

[0059] S2: ;

[0060] S3: .

[0061] Technical Theme 3

[0062] A pharmaceutical composition comprising the alfalfa furan analogue described in Technical Subject 1, a pharmaceutically acceptable salt thereof, or a stereoisomer thereof.

[0063] Furthermore, the "pharmaceutical composition" may also contain one or more pharmaceutically acceptable carriers or excipients, and be prepared in the form of tablets, capsules, granules, powders, suspensions, emulsions, powders, solutions, gels, syrups, pills, tinctures, tinctures, decoctions, lozenges, mixtures, suppositories, injections, inhalants, or sprays.

[0064] As used herein, “pharmaceutically acceptable carriers or excipients” include: diluents, fillers, binders, disintegrants, lubricants, flow aids, granulators, coating agents, wetting agents, solvents, co-solvents, suspending agents, emulsifiers, sweeteners, flavoring agents, taste masking agents, colorants, anti-caking agents, humectants, chelating agents, plasticizers, thickeners, antioxidants, preservatives, stabilizers, surfactants, and buffers. Those skilled in the art will understand that some pharmaceutically acceptable excipients may be used for more than one function and for alternative functions, depending on the amount of said excipient present in the formulation and what other ingredients are present in the formulation.

[0065] For example, when intended for oral administration, it can be formulated into oral preparations such as tablets (including regular tablets, enteric-coated tablets, lozenges, dispersible tablets, chewable tablets, effervescent tablets, and orally disintegrating tablets), capsules (including hard capsules, soft capsules, and enteric-coated capsules), granules, and pills, containing fillers (e.g., sugar derivatives such as lactose, sucrose, glucose, mannitol, and sorbitol; starch derivatives such as corn starch, potato starch, dextrin, and carboxymethyl starch; cellulose derivatives such as crystalline cellulose, hydroxypropyl cellulose, carboxymethyl cellulose, calcium carboxymethyl cellulose, and sodium carboxymethyl cellulose); Arabica... Gum; dextran; silicate derivatives such as magnesium aluminum metasilicate; phosphate derivatives such as calcium phosphate; carbonate derivatives such as calcium carbonate; sulfate derivatives such as calcium sulfate, etc.), binders (e.g., gelatin, polyvinylpyrrolidone, and polyethylene glycol), disintegrants (e.g., cellulose derivatives such as sodium carboxymethyl cellulose and polyvinylpyrrolidone), lubricants (e.g., talc, calcium stearate, magnesium stearate, cetyl, boric acid, sodium benzoate, leucine), stabilizers (methylparaben, propylparaben, etc.), flavoring agents (e.g., commonly used sweeteners, acidulants, and flavorings, etc.).

[0066] When used parenterally, the drug can be formulated as an injectable preparation, including sterile powder for injection and solvent for injection. The carrier or excipients used may include sterile water, Ringer's solution, and isotonic sodium chloride solution. Appropriate excipients such as antioxidants, buffers, antibacterial agents, solubilizers, co-solvents, pH adjusters, and osmotic pressure regulators may be added depending on the properties of the drug. Solubilizers or co-solvents may include poloxamer, lecithin, hydroxypropyl-β-cyclodextrin, etc.; pH adjusters may include phosphates, acetates, hydrochloric acid, sodium hydroxide, etc.; and osmotic pressure regulators may include sodium chloride, mannitol, glucose, phosphates, acetates, etc. If preparing a lyophilized powder for injection, mannitol, glucose, etc., may also be added as a support agent. When used rectally, the drug can be formulated as suppositories, etc.

[0067] When intended for pulmonary administration, the drug may be formulated as an inhaler or spray, etc. Numerous resources available to those skilled in the art describe pharmaceutically acceptable excipients and can be used to select appropriate pharmaceutically acceptable excipients, such as books like *Remington's Complete Pharmacy*, *Chinese Pharmaceutical Yearbook*, and *Pharmaceutics*.

[0068] The compounds of this invention and their pharmaceutically acceptable salts can be formulated into conventional formulations, as well as sustained-release formulations, controlled-release formulations, targeted formulations, and various microparticle delivery systems.

[0069] Technical Theme 4

[0070] The use of an alfalfa furan analogue as described in Technical Subject 1, a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, in the preparation of a medicament for treating osteoporosis.

[0071] Technology Theme 5

[0072] The use of an alfalfa furan analogue as described in Technical Subject 1, a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, in the preparation of a drug that upregulates bone morphogenetic protein-2 expression levels.

[0073] The beneficial effects of adopting the above technical solution are as follows:

[0074] This invention provides an alfalfa furan analogue, its preparation method, and its use in the treatment of osteoporosis. Experimental verification has shown that the alfalfa furan analogue provided by this invention has good therapeutic effects on osteoporosis, laying the foundation for the development of drugs for treating osteoporosis. Attached Figure Description

[0075] Figure 1 This is a structural diagram of the pGL4.17(Luc2 / Neo) Vector plasmid;

[0076] Figure 2 This is a schematic diagram of partial vertebral fluorescence in wild-type AB strain zebrafish given different drugs in Example 2;

[0077] Figure 3 These are representative mouse femoral CT images from Example 3, which, from left to right, are the overall three-dimensional reconstruction of the femur and three cross-sectional reconstruction images of the femur taken from the proximal to the distal end along the long axis of the femur.

[0078] Figure 4 This is a quantitative result image of bone microstructure detected by CT in Example 3, including bone volume (BV), bone volume fraction (BV / TV), trabecular thickness (Tb.Th), number of trabecular bones (Tb.N), structural model index (SMI), and bone mineral density (BMD). Detailed Implementation

[0079] To make the objectives, technical solutions, and advantages of the present invention clearer, the invention will be described clearly and completely below in conjunction with specific embodiments.

[0080] The compounds provided by this invention are prepared by the following general synthetic method:

[0081]

[0082] A benzyl alcohol compound with different substituents (1.00 mmol) was dissolved in acetone (3.0 mL) and added to a round-bottom flask. CuCl (0.10 mmol), TEMPO (0.10 mmol), and 2,2'-bipyridine (0.10 mmol) were added sequentially, and the mixture was stirred openly at room temperature for approximately 3 hours. After the reaction was complete, the mixture was quenched with saturated NH4Cl aqueous solution, extracted three times with ethyl acetate, and the organic phases were combined. The mixture was washed three times with saturated NaCl aqueous solution and dried over anhydrous Na2SO4. The mixture was filtered, and the filtrate was concentrated under reduced pressure. The crude product was purified by silica gel column chromatography, and the product fraction was collected and concentrated to obtain the final product.

[0083]

[0084] Under an argon atmosphere, zinc powder (12.0 mmol) was added to a three-necked flask containing tetrahydrofuran (40 mL). After the internal temperature dropped to −5 °C, titanium tetrachloride (6.0 mmol) was added, and the mixture was stirred at room temperature for 0.5 h, then refluxed and stirred for 2.5 h. The internal temperature was then lowered to −5 °C, and a tetrahydrofuran solution containing 2.0 mmol of 6-hydroxybenzo[d][1,3]dioxane-5-carboxaldehyde and 2.4 mmol of benzaldehyde compounds with different substituents was added. The mixture was refluxed and stirred for approximately 9 h. After the reaction was complete, the mixture was quenched with 10% NaHCO3 aqueous solution, extracted three times with dichloromethane, and the organic phases were combined. The mixture was washed three times with saturated NaCl aqueous solution and dried over anhydrous Na2SO4. The mixture was filtered, the filtrate was concentrated under reduced pressure, and the crude product was purified by silica gel column chromatography. The product fraction was collected and concentrated to obtain the final product.

[0085]

[0086] 2.0 mmol of 6-(2-styryl)-1,3-phenyldioxy-5-ol compounds with different substituents were dissolved in tetrahydrofuran (20 mL) and added to a round-bottom flask. K₂CO₃ (10.0 mmol) was added, and the mixture was stirred at room temperature for 10 minutes. Iodine (10.0 mmol) was then added, and stirring continued at room temperature for approximately 5 hours. After the reaction was complete, the reaction mixture was poured into a saturated NaHCO₃ aqueous solution, followed by the addition of a saturated NaHSO₃ aqueous solution. The mixture was extracted three times with ethyl acetate, and the combined organic phases were washed three times with a saturated NaCl aqueous solution and dried over anhydrous Na₂SO₄. The mixture was filtered, and the filtrate was concentrated under reduced pressure. The crude product was purified by silica gel column chromatography, and the product fraction was collected and concentrated to obtain the final product.

[0087] Example 1: Preparation of Compound 1

[0088]

[0089] Its synthesis method is the same as the general synthesis method, and the synthetic route includes the following:

[0090] The structural characterization results of the obtained product are as follows: 1 H NMR (500 MHz, DMSO-d6) δ 7.67–7.61 (m, 1H), 7.46–7.38 (m, 1H), 7.23 (d, J = 2.1 Hz, 0H), 7.20–7.11 (m, 1H), 5.97 (s, 1H).

[0091] HRMS: [M + H] + 257.0608

[0092] Example 2 Preparation of Compound 2

[0093]

[0094] Its synthesis method is the same as the general synthesis method, and the synthetic route includes the following:

[0095]

[0096] The structural characterization results of the obtained product are as follows: 1 H NMR (500 MHz, DMSO-d6) δ 7.76–7.72 (m,0H), 7.71–7.61 (m, 1H), 7.42–7.34 (m, 1H), 7.24–7.17 (m, 1H), 5.97 (s, 1H).

[0097] HRMS: [M + H] + 273.0313.

[0098] Example 3 Preparation of Compound 3

[0099]

[0100] Its synthesis method is the same as the general synthesis method, and the synthetic route includes the following:

[0101]

[0102] The structural characterization results of the obtained product are as follows: 1H NMR (500 MHz, DMSO-d6) δ 7.86 (t, J =2.1 Hz, 2H), 7.72 (ddd, J = 9.1, 2.2, 1.2 Hz, 2H), 7.63 (d, J = 2.1 Hz, 2H), 7.52 (ddd, J = 8.1, 2.2, 1.2 Hz, 2H), 7.33 (dd, J = 9.1, 7.9 Hz, 2H), 7.24(d, J = 2.1 Hz, 2H), 7.18 (s, 2H), 5.97 (s, 3H).HRMS: [M + H] + 316.9808.

[0103] Example 4: Preparation of Compound 4

[0104]

[0105] Its synthesis method is the same as the general synthesis method, and the synthetic route includes the following:

[0106]

[0107] The structural characterization results of the obtained product are as follows: 1 H NMR (500 MHz, DMSO-d6) δ 7.68–7.59 (m,2H), 7.27 (dd, J = 8.8, 7.7 Hz, 1H), 7.23–7.15 (m, 2H), 5.97 (s, 1H), 2.38(d, J = 0.6 Hz, 1H).HRMS: [M + H] + 253.0859.

[0108] Example 5: Preparation of Compound 5

[0109]

[0110] Its synthesis method is the same as the general synthesis method, and the synthetic route includes the following:

[0111]

[0112] The structural characterization results of the obtained product are as follows: 1H NMR (500 MHz, DMSO-d6) δ 7.70–7.64 (m,2H), 7.63 (d, J = 2.1 Hz, 1H), 7.31–7.20 (m, 3H), 7.18 (s, 1H), 5.97 (s, 2H),2.78–2.69 (m, 2H), 1.22 (t, J = 7.2 Hz, 3H).HRMS: [M + H] + 267.1016.

[0113] Example 6: Preparation of Compound 6

[0114]

[0115] Its synthesis method is the same as the general synthesis method, and the synthetic route includes the following:

[0116]

[0117] The structural characterization results of the obtained product are as follows: 1 H NMR (500 MHz, DMSO-d6) δ 7.67 (ddd, J= 9.3, 2.1, 1.2 Hz, 1H), 7.63 (d, J = 2.1 Hz, 1H), 7.54 (td, J = 2.1, 1.0 Hz,1H), 7.28 (dd, J = 9.3, 7.8 Hz, 1H), 7.24–7.17 (m, 3H), 5.97 (s, 1H), 2.65–2.59 (m, 1H), 1.62 (dtd, J = 13.8, 7.5, 6.3 Hz, 2H), 0.94 (t, J = 7.5 Hz,3H).HRMS: [M + H] + 281.1172.

[0118] Example 7 Preparation of Compound 7

[0119]

[0120] Its synthesis method is the same as the general synthesis method, and the synthetic route includes the following:

[0121]

[0122] The structural characterization results of the obtained product are as follows: 1H NMR (500 MHz, DMSO-d6) δ 7.73 (td, J =2.1, 0.9 Hz, 1H), 7.69 (ddd, J = 9.0, 2.2, 1.5 Hz, 1H), 7.63 (d, J = 2.1 Hz,1H), 7.29 (dd, J = 8.8, 7.9 Hz, 1H), 7.27–7.20 (m, 2H), 7.18 (s, 1H), 5.97(s, 2H), 3.09–2.97 (m, 1H), 1.29 (d, J = 6.6 Hz, 6H).HRMS: [M + H] + 281.1172.

[0123] Example 8: Preparation of Compound 8

[0124]

[0125] Its synthesis method is the same as the general synthesis method, and the synthetic route includes the following:

[0126]

[0127] The structural characterization results of the obtained product are as follows: 1 H NMR (500 MHz, DMSO-d6) δ 7.63 (d, J =2.1 Hz, 1H), 7.54 (ddd, J = 9.1, 2.1, 1.1 Hz, 1H), 7.32 (dd, J = 9.2, 7.9 Hz, 1H), 7.24–7.17 (m, 3H), 6.92 (ddd, J = 8.1, 2.2, 1.3 Hz, 1H), 5.97 (s, 1H), 3.81 (s, 2H).HRMS: [M + H] + 269.0808.

[0128] Example 9: Preparation of Compound 9

[0129]

[0130] Its synthesis method is the same as the general synthesis method, and the synthetic route includes the following:

[0131]

[0132] The structural characterization results of the obtained product are as follows: 1H NMR (500 MHz, DMSO-d6) δ 7.63 (d, J =2.1 Hz, 1H), 7.54 (ddd, J = 9.1, 2.2, 1.1 Hz, 1H), 7.30 (dd, J = 9.2, 7.9 Hz, 1H), 7.24–7.17 (m, 3H), 6.89 (ddd, J = 7.9, 2.2, 1.3 Hz, 1H), 5.97 (s, 2H), 4.08 (q, J = 6.8 Hz, 2H), 1.35 (t, J = 6.7 Hz, 3H).HRMS: [M + H] + 283.0965.

[0133] Example 10: Preparation of Compound 10

[0134]

[0135] Its synthesis method is the same as the general synthesis method, and the synthetic route includes the following:

[0136]

[0137] The structural characterization results of the obtained product are as follows: 1 H NMR (500 MHz, DMSO-d6) δ 7.63 (d, J =2.1 Hz, 1H), 7.54 (ddd, J = 9.1, 2.2, 1.1 Hz, 1H), 7.45 (dq, J = 7.3, 0.8 Hz, 2H), 7.40–7.33 (m, 2H), 7.33–7.26 (m, 2H), 7.22 (q, J = 2.2 Hz, 2H), 7.18 (s,1H), 6.91 (ddd, J = 7.9, 2.1, 1.1 Hz, 1H), 5.97 (s, 2H), 5.07 (t, J = 0.9 Hz,2H).HRMS: [M + H] + 345.1121.

[0138] Example 11 Preparation of Compound 11

[0139]

[0140] Its synthesis method is the same as the general synthesis method, and the synthetic route includes the following:

[0141]

[0142] The structural characterization results of the obtained product are as follows: 1 H NMR (500 MHz, DMSO-d6) δ 8.52 (t, J =2.2 Hz, 2H), 8.24 (ddd, J = 7.9, 2.2, 1.2 Hz, 2H), 8.03 (ddd, J = 9.3, 2.2,1.2 Hz, 2H), 7.75 (dd, J = 9.3, 7.8 Hz, 2H), 7.63 (d, J = 2.1 Hz, 2H), 7.24(d, J = 2.1 Hz, 2H), 7.18 (s, 2H), 5.97 (s, 3H).HRMS: [M + H] + 284.0553.

[0143] Example 12 Preparation of Compound 12

[0144]

[0145] Its synthesis method is the same as the general synthesis method, and the synthetic route includes the following:

[0146]

[0147] The structural characterization results of the obtained product are as follows: 1 H NMR (500 MHz, DMSO-d6) δ 7.98 (t, J =2.1 Hz, 1H), 7.83 (ddd, J = 8.2, 2.1, 1.2 Hz, 1H), 7.68–7.61 (m, 2H), 7.49(dd, J = 8.3, 6.5 Hz, 1H), 7.25 (d, J = 2.1 Hz, 1H), 7.18 (s, 1H), 5.97 (s,2H).HRMS: [M + H] + 264.0655.

[0148] Example 13 Preparation of Compound 13

[0149]

[0150] Its synthesis method is the same as the general synthesis method, and the synthetic route includes the following:

[0151]

[0152] The structural characterization results of the obtained product are as follows: 1H NMR (500 MHz, DMSO-d6) δ 7.83 (tq, J= 1.8, 0.8 Hz, 1H), 7.71 (ddd, J = 9.0, 2.2, 1.5 Hz, 1H), 7.63 (d, J = 2.1Hz, 1H), 7.59 (dd, J = 9.8, 8.8 Hz, 1H), 7.54 (dtt, J = 9.8, 2.1, 1.0 Hz,1H), 7.22 (d, J = 2.2 Hz, 1H), 7.18 (s, 1H), 6.67 (d, J = 0.8 Hz, 1H), 5.97(s, 2H).HRMS: [M + H] + 289.0671.

[0153] Example 14 Preparation of Compound 14

[0154]

[0155] Its synthesis method is the same as the general synthesis method, and the synthetic route includes the following:

[0156]

[0157] The structural characterization results of the obtained product are as follows: 1 H NMR (500 MHz, DMSO-d6) δ 8.11 (q, J =1.6 Hz, 1H), 7.78–7.70 (m, 1H), 7.68 (dd, J = 5.6, 1.0 Hz, 2H), 7.63 (d, J =2.1 Hz, 1H), 7.24–7.17 (m, 2H), 5.97 (s, 2H).HRMS: [M + H] + 307.0577.

[0158] Example 15 Preparation of Compound 15

[0159]

[0160] Its synthesis method is the same as the general synthesis method, and the synthetic route includes the following:

[0161]

[0162] The structural characterization results of the obtained product are as follows: 1H NMR (500 MHz, DMSO-d6) δ 8.23 ​​(t, J =2.2 Hz, 1H), 7.75 (ddd, J = 9.1, 2.2, 1.3 Hz, 1H), 7.63 (d, J = 1.2 Hz, 1H),7.64–7.55 (m, 3H), 7.52–7.42 (m, 3H), 7.45–7.37 (m, 1H), 7.23 (d, J = 2.2 Hz,1H), 7.18 (s, 1H), 5.97 (s, 1H).

[0163] HRMS: [M + H] + 315.1016

[0164] Example 16 Preparation of Compound 16

[0165]

[0166] Its synthesis method is the same as the general synthesis method, and the synthetic route includes the following:

[0167]

[0168] The structural characterization results of the obtained product are as follows: 1 H NMR (500 MHz, DMSO-d6) δ 7.63 (d, J =2.1 Hz, 1H), 7.54 (ddd, J = 9.1, 2.2, 1.1 Hz, 1H), 7.43 (t, J = 2.1 Hz, 1H), 7.35 (dd, J = 9.1, 7.1 Hz, 1H), 7.25–7.17 (m, 3H), 5.97 (s, 2H).HRMS: [M + H] + 323.0526.

[0169] Example 17 Preparation of Compound 17

[0170]

[0171] Its synthesis method is the same as the general synthesis method, and the synthetic route includes the following:

[0172]

[0173] The structural characterization results of the obtained product are as follows: 1H NMR (500 MHz, DMSO-d6) δ 7.49 (dd, J =9.5, 2.2 Hz, 2H), 7.21–7.17 (m, 1H), 6.98 (td, J = 2.3, 0.8 Hz, 1H), 5.97 (s,1H).HRMS: [M + H] + 267.1016.

[0174] Example 18 Preparation of Compound 18

[0175]

[0176] Its synthesis method is the same as the general synthesis method, and the synthetic route includes the following:

[0177]

[0178]

[0179] The structural characterization results of the obtained product are as follows: 1 H NMR (500 MHz, DMSO-d6) δ 7.52 (d, J =2.1 Hz, 2H), 7.48 (d, J = 2.1 Hz, 1H), 7.24–7.17 (m, 2H), 7.10 (ddq, J = 3.1,1.9, 0.9 Hz, 1H), 5.97 (s, 2H), 2.69–2.61 (m, 2H), 2.65 (s, 1H), 1.23 (t, J =7.2 Hz, 6H).HRMS: [M + H] + 295.1329.

[0180] Example 19 Preparation of Compound 19

[0181]

[0182] Its synthesis method is the same as the general synthesis method, and the synthetic route includes the following:

[0183]

[0184] The structural characterization results of the obtained product are as follows: 1H NMR (500 MHz, DMSO-d6) δ 7.67 (d, J =2.2 Hz, 1H), 7.48 (d, J = 2.1 Hz, 1H), 7.24–7.16 (m, 2H), 5.97 (s, 1H), 3.03–2.91 (m, 1H), 1.23 (d, J = 6.6 Hz, 7H).HRMS: [M + H] + 323.1642.

[0185] Example 20 Preparation of Compound 20

[0186]

[0187] Its synthesis method is the same as the general synthesis method, and the synthetic route includes the following:

[0188]

[0189]

[0190] The structural characterization results of the obtained product are as follows: 1 H NMR (500 MHz, DMSO-d6) δ 7.95 (d, J =2.2 Hz, 2H), 7.64 (d, J = 1.3 Hz, 2H), 7.65–7.56 (m, 4H), 7.52–7.37 (m, 8H),7.23 (d, J = 2.1 Hz, 1H), 7.19 (s, 1H), 5.97 (s, 2H).HRMS: [M + H] + 391.1329.

[0191] Example 21 Preparation of Compound 21

[0192]

[0193] Its synthesis method is the same as the general synthesis method, and the synthetic route includes the following:

[0194]

[0195]

[0196] The structural characterization results of the obtained product are as follows: 1H NMR (500 MHz, DMSO-d6) δ 7.48 (d, J =2.2 Hz, 0H), 7.21–7.17 (m, 1H), 6.96 (d, J = 2.2 Hz, 1H), 6.46 (t, J = 2.2Hz, 1H), 5.97 (s, 1H), 4.08 (q, J = 6.7 Hz, 2H), 1.35 (t, J = 6.7 Hz, 3H).HRMS: [M + H] + 327.1227.

[0197] Example 22 Preparation of Compound 22

[0198]

[0199] Its synthesis method is the same as the general synthesis method, and the synthetic route includes the following:

[0200]

[0201]

[0202] The structural characterization results of the obtained product are as follows: 1 H NMR (500 MHz, DMSO-d6) δ 8.88 (t, J =2.1 Hz, 1H), 8.80 (d, J = 2.2 Hz, 2H), 7.48 (d, J = 2.1 Hz, 1H), 7.26 (d, J =2.1 Hz, 1H), 7.19 (s, 1H), 5.97 (s, 2H).HRMS: [M + H] + 329.0404.

[0203] Example 23 Preparation of Compound 23

[0204]

[0205] Its synthesis method is the same as the general synthesis method, and the synthetic route includes the following:

[0206]

[0207]

[0208] The structural characterization results of the obtained product are as follows: 1H NMR (500 MHz, DMSO-d6) δ 8.15 (dd, J =15.9, 2.2 Hz, 3H), 7.48 (d, J = 2.1 Hz, 1H), 7.28 (d, J = 2.1 Hz, 1H), 7.19(s, 1H), 5.97 (s, 2H).HRMS: [M + H] + 289.0608.

[0209] Example 24 Preparation of Compound 24

[0210]

[0211] Its synthesis method is the same as the general synthesis method, and the synthetic route includes the following:

[0212]

[0213]

[0214] The structural characterization results of the obtained product are as follows: 1 H NMR (500 MHz, DMSO-d6) δ 7.48 (d, J =2.2 Hz, 1H), 7.23–7.17 (m, 4H), 6.96 (t, J = 2.2 Hz, 1H), 5.97 (s, 2H).HRMS: [M + H] + 407.0349.

[0215] Example 25 Preparation of Compound 25

[0216]

[0217] Its synthesis method is the same as the general synthesis method, and the synthetic route includes the following:

[0218]

[0219]

[0220] The structural characterization results of the obtained product are as follows: 1H NMR (500 MHz, DMSO-d6) δ 7.54–7.46 (m,2H), 7.22–7.17 (m, 1H), 7.01 (dt, J = 3.6, 1.2 Hz, 1H), 5.97 (s, 1H), 2.66–2.58 (m, 1H), 2.23 (s, 2H), 1.23 (t, J = 7.2 Hz, 2H).HRMS: [M + H] + 281.1172.

[0221] Example 26 Preparation of Compound 26

[0222]

[0223] Its synthesis method is the same as the general synthesis method, and the synthetic route includes the following:

[0224]

[0225]

[0226] The structural characterization results of the obtained product are as follows: 1 H NMR (500 MHz, DMSO-d6) δ 7.63 (td, J =2.2, 0.9 Hz, 1H), 7.54 (t, J = 2.4 Hz, 1H), 7.48 (d, J = 2.2 Hz, 1H), 7.23–7.17 (m, 2H), 7.12–7.08 (m, 1H), 5.97 (s, 2H), 3.04–2.93 (m, 1H), 2.23 (s, 3H), 1.23 (d, J = 6.6 Hz, 7H).

[0227] HRMS: [M + H] + 295.1329.

[0228] Example 27 Preparation of Compound 27

[0229]

[0230] Its synthesis method is the same as the general synthesis method, and the synthetic route includes the following:

[0231]

[0232]

[0233] The structural characterization results of the obtained product are as follows: 1H NMR (500 MHz, DMSO-d6) δ 7.71 (t, J =2.1 Hz, 1H), 7.55 (t, J = 2.4 Hz, 1H), 7.48 (d, J = 2.1 Hz, 1H), 7.22–7.13(m, 2H), 5.97 (s, 1H), 1.32 (s, 5H).HRMS: [M + H] + 309.1485.

[0234] Example 28 Preparation of Compound 28

[0235]

[0236] Its synthesis method is the same as the general synthesis method, and the synthetic route includes the following:

[0237]

[0238]

[0239] The structural characterization results of the obtained product are as follows: 1 H NMR (500 MHz, DMSO-d6) δ 7.48 (t, J =2.2 Hz, 2H), 7.42 (tt, J = 2.0, 0.8 Hz, 1H), 7.22–7.17 (m, 2H), 7.03–6.98 (m,1H), 5.97 (s, 2H), 2.56 (dd, J = 7.0, 0.9 Hz, 2H), 2.23 (s, 3H), 1.90 (dp, J= 13.9, 6.9 Hz, 1H), 0.86 (d, J = 6.9 Hz, 6H).HRMS: [M + H] + 309.1485.

[0240] Example 29 Preparation of Compound 29

[0241]

[0242] Its synthesis method is the same as the general synthesis method, and the synthetic route includes the following:

[0243]

[0244]

[0245] The structural characterization results of the obtained product are as follows: 1H NMR (500 MHz, DMSO-d6) δ 7.89 (t, J =2.2 Hz, 1H), 7.66–7.60 (m, 3H), 7.56 (td, J = 2.1, 1.1 Hz, 1H), 7.52–7.44 (m,3H), 7.44–7.37 (m, 1H), 7.24–7.17 (m, 2H), 5.97 (s, 2H), 2.75–2.67 (m, 2H), 1.23 (t, J = 7.2 Hz, 3H).

[0246] HRMS: [M + H] + 343.1329.

[0247] Example 30 Preparation of Compound 30

[0248]

[0249] Its synthesis method is the same as the general synthesis method, and the synthetic route includes the following:

[0250]

[0251]

[0252] The structural characterization results of the obtained product are as follows: 1 H NMR (500 MHz, DMSO-d6) δ 7.76 (t, J =2.2 Hz, 1H), 7.66–7.60 (m, 2H), 7.52–7.45 (m, 3H), 7.45–7.37 (m, 2H), 7.29(dt, J = 12.1, 2.2 Hz, 1H), 7.24 (d, J = 2.1 Hz, 1H), 7.19 (s, 1H), 5.97 (s,1H).HRMS: [M + H] + 333.0921.

[0253] Example 31 Preparation of compound 31

[0254]

[0255] Its synthesis method is the same as the general synthesis method, and the synthetic route includes the following:

[0256]

[0257]

[0258] The structural characterization results of the obtained product are as follows: 1 H NMR (500 MHz, DMSO-d6) δ 7.53 (t, J =2.2 Hz, 1H), 7.48 (d, J = 2.2 Hz, 1H), 7.27 (dt, J = 12.1, 2.2 Hz, 1H), 7.24–7.17 (m, 2H), 6.95 (dt, J = 12.1, 2.5 Hz, 1H), 5.97 (s, 2H).HRMS: [M + H] + 271.0765.

[0259] Example 32 Preparation of compound 32

[0260]

[0261] Its synthesis method is the same as the general synthesis method, and the synthetic route includes the following:

[0262]

[0263]

[0264] The structural characterization results of the obtained product are as follows: 1 H NMR (500 MHz, DMSO-d6) δ 7.62 (t, J =2.1 Hz, 1H), 7.57 (t, J = 2.2 Hz, 1H), 7.48 (d, J = 2.1 Hz, 1H), 7.27 (t, J =2.5 Hz, 1H), 7.24–7.17 (m, 2H), 5.97 (s, 2H).HRMS: [M + H] + 287.0469.

[0265] Example 33 Preparation of compound 33

[0266]

[0267] Its synthesis method is the same as the general synthesis method, and the synthetic route includes the following:

[0268]

[0269]

[0270] The structural characterization results of the obtained product are as follows: 1H NMR (500 MHz, DMSO-d6) δ 7.73 (t, J =2.2 Hz, 1H), 7.57 (t, J = 2.4 Hz, 1H), 7.48 (d, J = 2.1 Hz, 1H), 7.39 (t, J =2.3 Hz, 1H), 7.24–7.17 (m, 2H), 5.97 (s, 2H).HRMS: [M + H] + 330.9964.

[0271] Example 34 Preparation of compound 34

[0272]

[0273] Its synthesis method is the same as the general synthesis method, and the synthetic route includes the following:

[0274]

[0275]

[0276] The structural characterization results of the obtained product are as follows: 1 H NMR (500 MHz, DMSO-d6) δ 7.97 (t, J =2.2 Hz, 1H), 7.61–7.56 (m, 1H), 7.56–7.52 (m, 1H), 7.48 (d, J = 2.2 Hz, 1H), 7.22–7.17 (m, 2H), 5.97 (s, 2H).HRMS: [M + H] + 321.0733.

[0277] Example 35 Preparation of compound 35

[0278]

[0279] Its synthesis method is the same as the general synthesis method, and the synthetic route includes the following:

[0280]

[0281]

[0282] The structural characterization results of the obtained product are as follows: 1H NMR (500 MHz, DMSO-d6) δ 7.48 (d, J =2.1 Hz, 1H), 7.35 (t, J = 2.5 Hz, 1H), 7.23–7.17 (m, 2H), 7.15 (t, J = 2.2Hz, 1H), 6.77 (t, J = 2.0 Hz, 1H), 5.97 (s, 2H).HRMS: [M + H] + 283.0965.

[0283] Example 36 Preparation of Compound 36

[0284]

[0285] Its synthesis method is the same as the general synthesis method, and the synthetic route includes the following:

[0286]

[0287]

[0288] The structural characterization results of the obtained product are as follows: 1 H NMR (500 MHz, DMSO-d6) δ 7.48 (d, J =2.1 Hz, 1H), 7.37–7.33 (m, 1H), 7.23–7.17 (m, 2H), 7.09 (t, J = 2.2 Hz, 1H), 6.76 (t, J = 2.0 Hz, 1H), 5.97 (s, 2H), 4.09 (q, J = 6.7 Hz, 2H), 1.35 (t, J= 6.7 Hz, 3H).HRMS: [M + H] + 297.1121.

[0289] Example 37 Preparation of Compound 37

[0290]

[0291] Its synthesis method is the same as the general synthesis method, and the synthetic route includes the following:

[0292]

[0293]

[0294] The structural characterization results of the obtained product are as follows: 1H NMR (500 MHz, DMSO-d6) δ 7.50–7.42 (m,2H), 7.40–7.33 (m, 2H), 7.33–7.26 (m, 1H), 7.23–7.17 (m, 1H), 7.12 (t, J =2.2 Hz, 1H), 6.82–6.78 (m, 1H), 5.97 (s, 1H), 4.97 (d, J = 1.0 Hz, 1H).HRMS: [M + H] + 359.1278.

[0295] Example 38 Preparation of compound 38

[0296]

[0297] Its synthesis method is the same as the general synthesis method, and the synthetic route includes the following:

[0298]

[0299]

[0300] The structural characterization results of the obtained product are as follows: 1 H NMR (500 MHz, DMSO-d6) δ 7.48 (d, J =2.1 Hz, 1H), 7.29–7.17 (m, 2H), 7.09 (t, J = 2.1 Hz, 1H), 6.70 (dt, J = 12.1,2.1 Hz, 1H), 5.97 (s, 1H), 3.80 (s, 2H).HRMS: [M + H] + 287.0714.

[0301] Example 39 Preparation of compound 39

[0302]

[0303] Its synthesis method is the same as the general synthesis method, and the synthetic route includes the following:

[0304]

[0305]

[0306] The structural characterization results of the obtained product are as follows: 1H NMR (500 MHz, DMSO-d6) δ 7.53–7.46 (m,1H), 7.24–7.15 (m, 2H), 6.97 (t, J = 2.2 Hz, 1H), 5.97 (s, 1H), 3.80 (s, 2H).HRMS: [M + H] + 303.0419.

[0307] Example 40: Preparation of Compound 40

[0308]

[0309] Its synthesis method is the same as the general synthesis method, and the synthetic route includes the following:

[0310]

[0311]

[0312] The structural characterization results of the obtained product are as follows: 1 H NMR (500 MHz, DMSO-d6) δ 7.59 (t, J =2.1 Hz, 1H), 7.48 (d, J = 2.1 Hz, 1H), 7.26–7.13 (m, 3H), 5.97 (s, 1H), 3.80 (s, 2H).HRMS: [M + H] + 346.9913.

[0313] Example 41 Preparation of Compound 41

[0314]

[0315] Its synthesis method is the same as the general synthesis method, and the synthetic route includes the following:

[0316]

[0317]

[0318] The structural characterization results of the obtained product are as follows: 1 H NMR (500 MHz, DMSO-d6) δ 7.84 (t, J =2.2 Hz, 1H), 7.48 (d, J = 2.1 Hz, 1H), 7.27–7.17 (m, 4H), 5.97 (s, 2H).HRMS: [M + H] + 337.0682.

[0319] Example 42 Preparation of compound 42

[0320]

[0321] Its synthesis method is the same as the general synthesis method, and the synthetic route includes the following:

[0322]

[0323]

[0324] The structural characterization results of the obtained product are as follows: 1 H NMR (500 MHz, DMSO-d6) δ 7.94 (t, J =2.1 Hz, 0H), 7.66–7.60 (m, 1H), 7.52–7.37 (m, 2H), 7.28–7.17 (m, 2H), 5.97(s, 1H), 3.79 (s, 1H).HRMS: [M + H] + 345.1121.

[0325] Example 43 Preparation of compound 43

[0326]

[0327] Its synthesis method is the same as the general synthesis method, and the synthetic route includes the following:

[0328]

[0329]

[0330] The structural characterization results of the obtained product are as follows: 1 H NMR (500 MHz, DMSO-d6) δ 7.55 (t, J =2.1 Hz, 1H), 7.48 (d, J = 2.1 Hz, 1H), 7.36 (ddt, J = 12.1, 8.8, 2.2 Hz, 2H),7.24–7.17 (m, 2H), 5.97 (s, 2H).HRMS: [M + H] + 291.0219.

[0331] Example 44 Preparation of compound 44

[0332]

[0333] Its synthesis method is the same as the general synthesis method, and the synthetic route includes the following:

[0334]

[0335]

[0336] The structural characterization results of the obtained product are as follows: 1 H NMR (500 MHz, DMSO-d6) δ 7.67 (t, J =2.2 Hz, 1H), 7.50–7.38 (m, 3H), 7.25 (d, J = 2.1 Hz, 1H), 7.19 (s, 1H), 5.97(s, 2H).

[0337] HRMS: [M + H] + 334.9714.

[0338] Example 45 Preparation of compound 45

[0339]

[0340] Its synthesis method is the same as the general synthesis method, and the synthetic route includes the following:

[0341]

[0342]

[0343] The structural characterization results of the obtained product are as follows: 1 H NMR (500 MHz, DMSO-d6) δ 7.92 (t, J =2.1 Hz, 1H), 7.55–7.46 (m, 2H), 7.40 (dt, J = 12.1, 2.0 Hz, 1H), 7.23 (d, J =2.1 Hz, 1H), 7.19 (s, 1H), 5.97 (s, 2H).HRMS: [M + H] + 325.0482.

[0344] Example 46 Preparation of Compound 46

[0345]

[0346] Its synthesis method is the same as the general synthesis method, and the synthetic route includes the following:

[0347]

[0348] The structural characterization results of the obtained product are as follows: 1H NMR (500 MHz, DMSO-d6) δ 7.80 (t, J =2.2 Hz, 1H), 7.70 (t, J = 2.2 Hz, 1H), 7.65 (t, J = 2.2 Hz, 1H), 7.48 (d, J =2.1 Hz, 1H), 7.25 (d, J = 2.1 Hz, 1H), 7.19 (s, 1H), 5.97 (s, 2H).

[0349] HRMS: [M + H] + 350.9418

[0350] Example 47 Preparation of Compound 47

[0351]

[0352] Its synthesis method is the same as the general synthesis method, and the synthetic route includes the following:

[0353]

[0354]

[0355] The structural characterization results of the obtained product are as follows: 1 H NMR (500 MHz, DMSO-d6) δ 8.01 (t, J =2.2 Hz, 1H), 7.86 (t, J = 2.2 Hz, 1H), 7.64 (t, J = 2.1 Hz, 1H), 7.48 (d, J =2.1 Hz, 1H), 7.23 (d, J = 2.1 Hz, 1H), 7.19 (s, 1H), 5.97 (s, 2H).HRMS: [M +H] + 341.0187.

[0356] Example 48 Preparation of Compound 48

[0357]

[0358] Its synthesis method is the same as the general synthesis method, and the synthetic route includes the following:

[0359]

[0360] The structural characterization results of the obtained product are as follows: 1H NMR (500 MHz, DMSO-d6) δ 8.06 (t, J =2.2 Hz, 1H), 7.87 (dt, J = 16.7, 2.2 Hz, 2H), 7.48 (d, J = 2.1 Hz, 1H), 7.25 (d, J = 2.1 Hz, 1H), 7.19 (s, 1H), 5.97 (s, 2H).HRMS: [M + H] + 384.9682.

[0361] Example 49 Preparation of Compound 49

[0362]

[0363] Its synthesis method is the same as the general synthesis method, and the synthetic route includes the following:

[0364]

[0365]

[0366] The structural characterization results of the obtained product are as follows: 1 H NMR (500 MHz, DMSO-d6) δ 7.58 (t, J =2.2 Hz, 1H), 7.48 (d, J = 2.1 Hz, 1H), 7.24–7.17 (m, 2H), 6.97 (t, J = 2.2Hz, 1H), 5.97 (s, 1H), 1.33 (s, 5H).HRMS: [M + H] + 325.1434.

[0367] Example 50: Preparation of Compound 50

[0368]

[0369] Its synthesis method is the same as the general synthesis method, and the synthetic route includes the following:

[0370]

[0371]

[0372] The structural characterization results of the obtained product are as follows: 1H NMR (500 MHz, DMSO-d6) δ 8.11 (t, J =2.2 Hz, 1H), 7.70–7.62 (m, 2H), 7.66–7.59 (m, 3H), 7.52–7.37 (m, 5H), 7.24(d, J = 2.1 Hz, 1H), 7.19 (s, 1H), 5.97 (s, 2H).HRMS: [M + H] + 349.0626.

[0373] Example 1: Screening for in vitro BMP-2 upregulation activity

[0374] The instruments and reagents used in this example were selected from the following:

[0375] Cell line: Mouse cranial cells MC3T3-E1, provided by the Key Laboratory of Pharmaceutical Biotechnology.

[0376] Plasmid: Recombinant plasmid PMB, provided by the Key Laboratory of Pharmaceutical Biotechnology.

[0377] Culture medium: High glucose DMEM medium, purchased from MREDA, catalog number M212860.

[0378] Main reagents: D-Hank's solution for cell digestion and passage, used as a buffer (1L) in cell culture: NaCl 8.0g; KCl 0.4g; NaHPO4·H2O 0.06g; KH2PO4 0.06g; NaHCO3 0.35g; Phenol red 0.02g; dissolved in 1L deionized water, sterilized by filtration through a 0.2μm filter membrane, and stored at 4°C.

[0379] The plasmid transfection kit LipofectAMINE™ 2000 Reagent was purchased from Thermofisher, catalog number 11668500.

[0380] Instruments: Fluorescence multifunction detector: POLARSTAR Galaxy (BMG GmbH, Germany); CO2 incubator: REVCO3000 (GS Laboratory); Clean bench: SANYO MCV-B161Sb(T).

[0381] The recombinant plasmid PMB was prepared as follows: The luciferase inhibitor plasmid PYJ2, containing the mouse bmp2 promoter region, was digested with SacI and XhoI enzymes to obtain the promoter region fragment from -3365 to -1658 bp. The pGL4.17(Luc2 / Neo) vector containing the mammalian resistance gene neo was also prepared; its structural diagram is shown below. Figure 1 As shown, after double digestion with SacІ and XhoІ, the plasmid was ligated to the promoter region fragment at a molar ratio of 3:1 to obtain the recombinant plasmid PMB.

[0382] The preparation method of plasmid PYJ2 is as described in Example 2 of CN101543502 B, which describes model construction and is also mentioned in this application. Figure 1 As stated above.

[0383] Cell Culture: Mouse cranial cells (MC3T3-E1) were adherent cells, passaged approximately every 48 hours. Once cells reached 90% confluence, the old culture medium was discarded. After rinsing the cells with an appropriate amount of D-Hank's solution, the liquid was aspirated, and an appropriate amount of digestion solution (0.25% trypsin and 0.02% EDTA, volume ratio 1:1) was added. Digestion was performed at room temperature while observing the cells under an inverted microscope. After approximately 3 minutes, when the intercellular spaces gradually opened, the digestion solution was discarded, and high-glucose-DMEM medium containing 10% FBS was immediately added to inhibit trypsin activity. The cells were gently and repeatedly pipetted against the flask wall using a bent pipette to completely detach the cells and disperse them into a single-cell suspension. The cell suspension was then seeded into new cell culture flasks at a 1:4 ratio, and an appropriate amount of serum-containing high-glucose-DMEM medium (without any antibiotics) was added. The flasks were then incubated for further culture. Culture conditions: 37ºC, 5% CO2.

[0384] The anti-osteoporosis effects of derivatives were evaluated by measuring the upregulation rate of BMP-2 in vitro, with a total of 50 derivatives evaluated. The model works by using the BMP-2 promoter as the drug target and the luciferase gene as the reporter gene, screening for BMP-2 upregulation activity of test substances by measuring changes in model fluorescence intensity. Lovastatin was used as a positive control, and 0.1% DMSO was used as a blank control (i.e., 100%). The concentration of all test samples was 4 μM.

[0385] Filtering method:

[0386] 1. Mouse calcaneal cells MC3T3-E1 were prepared into a single-cell suspension using culture medium, and the cell concentration was adjusted to 5 × 10⁻⁶ cells / cells. 5 Cells / ml, seeded at 100μL per well into a 96-well plate and incubated in an incubator for 8 hours.

[0387] 2. Dilute recombinant plasmid PMB to 0.2 μg / well in 25 μl / well serum-free and antibiotic-free DMEM medium in a sterile centrifuge tube; dilute LF2000 Reagent to 0.5 μl / well in 25 μl / well medium in another sterile centrifuge tube; combine the two tubes and incubate for 30 min to obtain the transfection complex.

[0388] 3. Add the prepared transfection complex to each well containing 50 μl of the culture medium and place it back into the incubator for 6 hours. After 6 hours of incubation, discard the original culture medium in the well and replace it with serum-free culture medium for further incubation.

[0389] 4. After changing the medium, add 1 μl of screening sample to each well. The sample solvent is DMSO, and the final concentration of the sample is 4 μM. Continue to culture for 24 hours. After the culture is completed, discard the culture medium and wash the cells with PBS solution.

[0390] 5. After washing, add 20 μl of cell lysis buffer to each well and incubate at 37°C for 15 min to allow the cells to fully lyse.

[0391] 6. Add 70 μl of luciferase assay reagent to each well of cell lysis buffer, and use a fluorescence multifunction detector to measure the fluorescence value of each well.

[0392] The upregulation rate is calculated using the following formula: Upregulation rate = Sample emission count / DMSO emission count × 100%

[0393] The upregulation results are shown in Table 1, which demonstrates that the compound provided by this invention has a good ability to upregulate the expression level of bone morphogenetic protein-2.

[0394] Table 1. Upregulation rate of BMP-2 for each compound

[0395]

[0396] Example 2: Active screening test method for zebrafish model

[0397] The culture medium used in this example is E3 culture medium;

[0398] Wild-type AB zebrafish juveniles that had developed to 3dpf were randomly distributed in 6-well plates, with 30 fish per well. The plates included a normal control group, a prednisone model group, a test compound group (1.25 μM), and an etidronate disodium positive control group (80 μM). The culture medium volume per well was 3 ml.

[0399] Except for the normal control group, all other groups were treated with prednisolone at a final concentration of 15 μmol / L to construct an osteoporosis model. After construction, etidronate disodium aqueous solution at a final concentration of 80 μM was added at a ratio of 0.1% (v / v) with an equal volume of DMSO or a DMSO solution of the test compound with an equal volume of water. The model group and the normal control group were treated with equal volumes of solvent. The culture was incubated at 28℃, and the drug-containing culture medium was replaced every 24 h for 3 consecutive days. After treatment, calcein staining was performed. Ten normally developed zebrafish were randomly selected from each group, anesthetized, fixed, and fluorescence images of the vertebral region were taken under uniform fluorescence parameters. The fluorescence intensity of the vertebral region was analyzed using NIS Elements D 3.20 software.

[0400] The results are as follows Figure 2 As shown in Table 2, compared with the model group, the dorsal fluorescence of zebrafish in each compound group was significantly increased, with some slightly better than etidronate disodium and close to the blank control group that was not damaged by prednisone.

[0401] Table 2 Quantitative results of fluorescence intensity of each compound

[0402]

[0403] Example 3: Pharmacodynamic Evaluation Methods for Osteoporosis in Aged C57 Mice

[0404] C57BL / 6 mice were purchased from Jiangsu Airingfei Biotechnology Co., Ltd. All experimental procedures were performed in accordance with the operating guidelines approved by the Animal Protection and Use Committee (IACUC) of the Institute of Medical Biotechnology, Chinese Academy of Medical Sciences, and were approved by the Animal Ethics Committee of the Institute of Medical Biotechnology, Chinese Academy of Medical Sciences. All mice were housed in a specific pathogen-free room with 12 hours of light and 12 hours of darkness. Temperature (23±2℃) and humidity (55%) were maintained during animal housing. All animals had free access to food and water; the mice included 16-month-old male C57 mice and 3-month-old male adult C57 mice.

[0405] After one week of acclimatization, the mice were divided into an administration group, an aged control group, and a young control group, with eight mice in each group. The administration group consisted of 16-month-old male C57 mice, which were administered the drug by gavage. The solvent was distilled water (dosage of 30 mg / kg / day). The 16-month-old male C57 mice served as the aged control group and were administered distilled water by gavage. The 3-month-old male C57 mice served as the young control group and were also administered distilled water by gavage.

[0406] Mice were administered the drug continuously for 4 months, with a 1-day rest period every 6 days. After 4 months of continuous administration, mice were anesthetized with isoflurane (RWD Life Sciences Ltd.), and bone mineral density (BMD) was measured using a Quantum FX micro-CT (PerkinElmer, Germany). Specimens were placed in a 140 mm diameter cylindrical scaffold with the long axis of the bone tissue perpendicular to the X-ray beam, at a spatial resolution of 73 mm for the whole body or 40 mm for the femur (55 kV, 114 mA, scan time 4.5 min). For the whole body, the region of interest (ROI) was selected from the thoracic vertebrae to the ischium. For the femur, the ROI was selected from the proximal to the distal end of the femur. The data were analyzed using the rodent analysis software (Analyze 2.0) of the device. Mouse skeletons were filled into the software environment, and bone mineral density in these regions was calculated.

[0407] CT images of animals with bone volume fractions closest to the mean in each group were selected for illustration, as shown in Figure 3. Observational analysis revealed that the femoral scan area of ​​the young group mice showed significantly denser bone structure, with more and longer trabeculae compared to the aged control group. The bone structure in the compound 6 group was even denser than that in the aged control group, with thicker and more numerous trabeculae and smaller gaps. The cortical bone of the aged control group was thinner compared to the compound 6 group. Quantitative results are as follows: Figure 4 As shown. The detection indicators are explained below: Bone Volume (BV) (mm²) 3 Bone volume within the region of interest (ROI); Bone volume fraction (BV / TV) (%): the ratio of bone volume to ROI volume; Trabecular thickness (Tb.Th) (mm): the average thickness of trabeculae within the ROI; Trabecular number (Tb.N) (1 / mm): the number of trabeculae per millimeter within the ROI; Trabecular separation (Tb.Sp) (mm): the average width between trabeculae within the ROI; Structural model index (SMI): reflects the lamellar or rod-like structure of trabeculae. In osteoporosis, trabeculae transform from a lamellar to a rod-like structure, and the SMI value increases; Bone mineral density (BMD) (g / cm³). 3 The results showed that compound 6 provided by this invention has a good therapeutic effect on osteoporosis.

[0408] Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. An alfalfa furan analog, its pharmaceutically acceptable salt, or its stereoisomer, characterized in that: The structure of the compound is shown in Formula I: ； Formula I R1 and R2 are independently selected from hydrogen, C1-C8 alkyl, C1-C8 alkoxy, halogen, nitro, cyano, 1-3 halogen-substituted C1-C8 alkyl, 1-3 halogen-substituted C1-C8 alkoxy, phenyl, benzyl, and benzyloxy.

2. The alfalfa furan analogue, its pharmaceutically acceptable salt, or its stereoisomer according to claim 1, characterized in that: R1 and R2 are independently selected from hydrogen, C1-C5 alkyl, C1-C5 alkoxy, halogen, nitro, cyano, C1-C5 alkyl with 1-3 halogen substituted, C1-C5 alkoxy with 1-3 halogen substituted, phenyl, benzyl, and benzyloxy.

3. An alfalfa furan analogue, its pharmaceutically acceptable salt, or its stereoisomer according to claim 2, characterized in that: The C1-C5 alkyl group is selected from one of the following structures: —CH3, —CH2CH3, —CH(CH3)2, —(CH2)2CH3, —(CH2)3CH3, —CH2CH(CH3)2, —C(CH3)3, —CH(CH3)CH2CH3, —(CH2)4CH3, —CH(CH3)(CH2)2CH3, —CH(CH2CH3)2, —CH2C(CH3)3, —CH2CH(CH3)CH2CH3, —C(CH3)2CH2CH3, —CH(CH3)CH(CH3)2, —(CH2)2CH(CH3)2; The C1-C5 alkoxy group is selected from one of the following structures: —OCH3, —OCH2CH3, —OCH(CH3)2, —O(CH2)2CH3, —O(CH2)3CH3, —OCH2CH(CH3)2, —OC(CH3)3, —OCH(CH3)CH2CH3, —O(CH2)4CH3, —OCH(CH3)(CH2)2CH3, —OCH(CH2CH3)2, —OCH2C(CH3)3, —OCH2CH(CH3)CH2CH3, —OC(CH3)2CH2CH3, —OCH(CH3)CH(CH3)2, —O(CH2)2CH(CH3)2; The halogen is selected from F, Cl, Br or I; The 1-3 halogen-substituted C1-C5 alkyl groups are selected from -CH2F, -CHF2, -CF3, -CH2CF3, -CH2CH2CF3, -CH2CH2CH2CF3, and -CH2CH2CH2CH2CF3; The 1-3 halogen-substituted C1-C5 alkoxy groups are selected from -OCF3, -OCH2CF3, -OCH2CH2CF3, -OCH2CH2CH2CF3, and -OCH2CH2CH2CH2CF3.

4. An alfalfa furan analogue, its pharmaceutically acceptable salt, or its stereoisomer according to claim 1, characterized in that, R1 and R2 are selected from one of the following structures: H, nitro, cyano, phenyl, benzyl, benzyloxy, -F, -Cl, -Br, -CH3, -CH2CH3, -CH(CH3)2, -OCH2CH3, -C(CH3)3, -CH2CH2CH3, -OCH3, -CHF2, -CF3, -OCF3, -CH2CH(CH3)2.

5. An alfalfa furan analogue, its pharmaceutically acceptable salt, or its stereoisomer according to claim 1, characterized in that, The compound is selected from one of the following structures: 、 、 、 Compound 1 Compound 2 Compound 3 、 、 、 Compound 4, Compound 5, Compound 6 、 、 、 Compound 7, Compound 8, Compound 9 、 、 、 Compound 10, Compound 11, Compound 12 、 、 、 Compound 13, Compound 14, Compound 15 、 、 、 Compound 16, Compound 17, Compound 18 、 、 、 Compound 19, Compound 20, Compound 21 、 、 、 Compound 22, Compound 23, Compound 24 、 、 、 Compound 25, Compound 26, Compound 27 、 、 、 Compound 28, Compound 29, Compound 30 、 、 、 Compound 31, Compound 32, Compound 33 、 、 、 Compound 34, Compound 35, Compound 36 、 、 、 Compound 37, Compound 38, Compound 39 、 、 、 Compound 40, Compound 41, Compound 42 、 、 、 Compound 43, Compound 44, Compound 45 、 、 、 Compound 46, Compound 47, Compound 48 、 ； Compound 49 Compound 50.

6. A method for preparing the compound according to any one of claims 1-5, characterized in that, Prepared via the following route: S1： ； S2： ； S3： 。 7. A pharmaceutical composition, characterized in that, Includes the alfalfa furan analogue as described in any one of claims 1-5, its pharmaceutically acceptable salt, or its stereoisomer.

8. The pharmaceutical composition according to claim 7, characterized in that, It also includes one or more pharmaceutically acceptable carriers or excipients.

9. The use of the alfalfa furan analogue, its pharmaceutically acceptable salt, or its stereoisomer as described in any one of claims 1-5 in the preparation of a medicament for treating osteoporosis.

10. The use of any one of the alfalfa furan analogues, pharmaceutically acceptable salts thereof, or stereoisomers thereof, in the preparation of a drug for upregulating bone morphogenetic protein-2 expression levels.

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