Screening and verification method of small molecule inhibitor targeting specific domain of sclerostin

Abstract

The present application relates to the technical field of biological medicine, and discloses a screening and verification method of a small-molecule inhibitor targeting a specific domain of sclerostin. The compound provided by the present application is obtained by structural modification, skeleton derivation design and molecular docking screening of a lead small-molecule compound tetrahydrofolic acid targeting the loop3 domain of sclerostin. The compound can specifically target and combine with the loop3 domain of sclerostin, can re-activate the Wnt signal inhibited by sclerostin, and restore the effect of Wnt signal in promoting bone formation. The sclerostin inhibitor containing the compound can inhibit various muscle-skeletal system diseases mediated by sclerostin activation, has wide application range and strong targeting property. Based on the characteristics that the compound targets and combines with the loop3 domain of sclerostin but not the loop2 domain, when the compound is used for preparing a drug for preventing and treating diseases mediated by sclerostin, the risk of cardiovascular diseases caused in the process of using the drug can be reduced.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a method for screening and validating small molecule inhibitors that target specific domains of sclerosstatin. Background Technology

[0002] The Wnt signaling pathway plays a crucial role in bone formation, growth, and development. The Wnt / β-catenin signaling pathway is a typical Wnt signaling pathway and a key regulator of bone formation and metabolism. In this pathway, Wnt proteins bind to low-density lipoprotein receptor-associated protein 5 / 6 (LRP5 / 6) to promote the further expression of osteoblast-related genes.

[0003] Sclerosstatin is a protein secreted by osteocytes, containing 189 amino acids in total. It includes a disordered N-terminus (amino acids 1-56) and C-terminus (amino acids 145-189), and three loop domains surrounding a cysteine ​​motif: loop 1 (amino acids 57-80), loop 2 (amino acids 86-109), and loop 3 (amino acids 111-140). Sclerosstatin binds to LRP5 / 6, preventing their interaction with Wnt, thereby inhibiting the formation of the WNT-Frizzled-LRP5 / 6 ternary complex. This, in turn, suppresses the classical Wnt signaling pathway, ultimately leading to musculoskeletal diseases mediated by sclerosstatin, such as osteoporosis, osteogenesis imperfecta, hypophosphatemic rickets, and sarcopenia.

[0004] Currently, the only marketed drug targeting sclerosstatin is the Romosozumab monoclonal antibody, which is mainly used to treat postmenopausal osteoporosis. However, the drug received a black box warning from the FDA in April 2019: treatment may pose a potential cardiovascular risk, and patients who have had a heart attack or stroke within one year will be prohibited from using the drug.

[0005] Therefore, developing drugs that target sclerosstatin with low risk of inducing cardiovascular disease is of great significance for the prevention and treatment of musculoskeletal diseases. Summary of the Invention

[0006] The present invention aims to at least solve one of the aforementioned technical problems existing in the prior art. Therefore, one object of the present invention is to provide a compound.

[0007] A second objective of this invention is to provide a method for screening such compounds.

[0008] A third objective of this invention is to provide a method for preparing this compound.

[0009] The fourth objective of this invention is to provide a sclerosstatin inhibitor.

[0010] The fifth objective of this invention is to provide the application of this sclerosstatin inhibitor.

[0011] The sixth objective of this invention is to provide a drug that targets sclerosingin.

[0012] The inventive concept of this invention is as follows: The loop 3 domain of sclerosstatin is a β-sheet short peptide composed of 29 amino acids, containing a large number of positively charged lysine and arginine, and forms a disulfide bond with the loop 1 domain to stabilize the core structure of the protein. Its structure is simple and has few interactions. Existing technology shows that the loop 2 domain of sclerosstatin participates in cardiovascular protection, while the loop 3 domain does not. That is, after specifically targeting and binding to the loop 3 domain of sclerosstatin, the antagonistic effect of sclerosstatin on the Wnt signaling pathway is inhibited, but its cardiovascular protective effect remains unaffected. Studies have shown that tetrahydrofolate can specifically target and bind to the loop 3 domain of sclerosstatin, inhibiting various musculoskeletal system diseases mediated by sclerosstatin activation. Therefore, this invention develops an inhibitor based on the tetrahydrofolate core molecular framework that targets the loop 3 domain of sclerosstatin but does not bind to the loop 2 domain, thereby reducing the inhibition of the Wnt signaling pathway by sclerosstatin while maintaining its cardiovascular protective effect and reducing the risk of cardiovascular disease.

[0013] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0014] A first aspect of the present invention provides a compound having the structural formula shown in formula (A):

[0015]

[0016] Wherein, R1 is H or a halogen; R2 is H or an alkyl group.

[0017] In some embodiments of the present invention, in the compound of formula (A), the combination of R1 and R2 is selected from any of the following: R1 is H and R2 is alkyl or R1 is halogen and R2 is H.

[0018] In some embodiments of the present invention, the compound is as shown in formula (I) or formula (II):

[0019]

[0020] A second aspect of the present invention provides a method for screening the compounds described in the first aspect of the present invention, comprising the following steps:

[0021] Simplify the structure of tetrahydrofolate to obtain As a structural framework, a framework derivation was performed at the Z site to obtain candidate molecules; the candidate molecules were molecularly docked with the loop3 domain of sclerosstatin protein, and the compounds were screened based on the binding mode and binding energy of the candidate molecules in the loop3 binding pocket.

[0022] In some embodiments of the present invention, the skeleton derivation is performed using Libinvent, one of the core modules of Reinvent4.

[0023] In some embodiments of the invention, the molecular docking is performed using Maestro's Glide module.

[0024] In some embodiments of the present invention, the compound is a candidate molecule having the optimal binding mode and the highest binding energy in the loop 3 binding pocket.

[0025] In this invention, the binding mode of tetrahydrofolate to the loop 3 domain of osteosclerosingin was analyzed in detail. It was found that the negatively charged carboxyl group at the terminal of tetrahydrofolate can form hydrogen bonds and salt bridges with the positively charged residues Arg115 and His59 in the loop 3 binding pocket. These interactions are beneficial to the stable binding of tetrahydrofolate to osteosclerosingin, thus confirming... As a core group, the large and complex structure of tetrahydrofolate is simplified by truncating unnecessary groups, and the derivation site Z is determined to be a specific substitution position of the aromatic ring.

[0026] A third aspect of the present invention provides a method for preparing the compound described in the first aspect of the present invention, comprising the following steps:

[0027] S1. React 2-bromo-4-nitrophenol with 4-methoxycarbonylphenylboronic acid to give intermediate (a).

[0028] S2, Intermediate (a) is obtained from intermediate (b) via nitro reduction reaction.

[0029] S3, Intermediate (b) and Compound (x) The reaction yields intermediate (c).

[0030] S4. Intermediate (c) is obtained as intermediate (d) through esterification and hydrolysis.

[0031] S5. Intermediate (d) reacts with glycine methyl ester hydrochloride to give intermediate (e).

[0032] S6. The intermediate (e) is obtained by esterification and hydrolysis reaction to yield the compound;

[0033] R1 and R2 are defined as described in the first aspect of this invention.

[0034] In some embodiments of the present invention, the mass ratio of 2-bromo-4-nitrophenol to 4-methoxycarbonylphenylboronic acid is (0.8-1.5):1.

[0035] In some embodiments of the present invention, the reaction conditions for 2-bromo-4-nitrophenol and 4-methoxycarbonylphenylboronic acid include at least one of the following:

[0036] 1) The reaction is carried out under a protective atmosphere;

[0037] 2) The reaction temperature is 90-110℃;

[0038] 3) The reaction time is 10-13 hours.

[0039] In some embodiments of the present invention, step S1 further includes the use of a catalyst, an alkaline medium, and a solvent.

[0040] In some specific embodiments of the present invention, the mass ratio of the catalyst to 2-bromo-4-nitrophenol is 1:(2-4); the catalyst comprises 1,1'-bis(diphenylphosphine)ferrocenepalladium dichloride (Pd(Cl)2dppf).

[0041] In some specific embodiments of the present invention, the mass ratio of the alkaline medium to 2-bromo-4-nitrophenol is (1-2):1; the alkaline medium includes potassium carbonate (K2CO3).

[0042] In some specific embodiments of the present invention, the liquid-to-solid ratio of the solvent to 2-bromo-4-nitrophenol is 100 mL:(8-10) g; the solvent includes N,N-dimethylformamide (DMF).

[0043] In some embodiments of the present invention, step S1, after the reaction is completed, further includes the steps of extracting the reaction solution, drying the organic phase, removing the solvent by rotary evaporation under reduced pressure, and purifying by rapid column chromatography.

[0044] In some embodiments of the present invention, the reaction conditions for the nitro reduction reaction of the intermediate (a) include at least one of the following:

[0045] 1) The reaction temperature is 70-90℃;

[0046] 2) The reaction time is 3-5 hours.

[0047] In some embodiments of the present invention, step S2 further includes using a catalyst, a reducing agent, and a solvent.

[0048] In some specific embodiments of the present invention, the mass ratio of the catalyst to the intermediate (a) is 1:(1-2); the catalyst comprises ammonium chloride (NH4Cl).

[0049] In some specific embodiments of the present invention, the mass ratio of the reducing agent to the intermediate (a) is 1:(1-2); the reducing agent includes iron powder (Fe).

[0050] In some specific embodiments of the present invention, the liquid-to-solid ratio of the solvent to the intermediate (a) is (9-12) mL:1 g; the solvent includes anhydrous ethanol and water (5:1, v / v).

[0051] In some embodiments of the present invention, step S2, after the reaction is completed, further includes the steps of solid-liquid separation to remove the reducing agent, vacuum rotary evaporation to remove the solvent, extraction of the reaction solution, drying of the organic phase, vacuum rotary evaporation to remove the solvent, and rapid column chromatography purification.

[0052] In some embodiments of the present invention, the mass ratio of the intermediate (b) to the compound (x) is 1:(1-1.5).

[0053] In some embodiments of the present invention, the reaction conditions between the intermediate (b) and the compound (x) include at least one of the following:

[0054] 1) The reaction temperature is 20-30℃;

[0055] 2) The reaction time is 12-13 hours.

[0056] In some specific embodiments of the present invention, the compound (x) includes

[0057] In some embodiments of the present invention, step S3 further includes using a catalyst, a condensing agent, and a solvent.

[0058] In some specific embodiments of the present invention, the mass ratio of the catalyst to the intermediate (b) is (1-2):1; the catalyst comprises N,N-diisopropylethylamine (DIPEA).

[0059] In some specific embodiments of the present invention, the mass ratio of the condensing agent to intermediate (b) is (1-2):1; the condensing agent includes N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (HATU).

[0060] In some specific embodiments of the present invention, the liquid-to-solid ratio of the solvent to the intermediate (b) is (9-12) mL:1 g; the solvent includes N,N-dimethylformamide.

[0061] In some embodiments of the present invention, step S3, after the reaction is completed, further includes the steps of extracting the reaction solution, drying the organic phase, removing the solvent by rotary evaporation under reduced pressure, and purifying by rapid column chromatography.

[0062] In some embodiments of the present invention, the reaction conditions for the esterification and hydrolysis reaction of the intermediate (c) include at least one of the following:

[0063] 1) The reaction temperature is 20-30℃;

[0064] 2) The reaction time is 10-13 hours.

[0065] In some embodiments of the present invention, step S4 further includes using a catalyst and a solvent.

[0066] In some specific embodiments of the present invention, the mass ratio of the catalyst to the intermediate (c) is 1:(5-6); the catalyst comprises lithium hydroxide (LiOH).

[0067] In some specific embodiments of the present invention, the liquid-to-solid ratio of the solvent to the intermediate (c) is (7-10) mL:1 g; the solvent comprises anhydrous methanol and water (5:1, v / v).

[0068] In some embodiments of the present invention, step S4, after the reaction is completed, further includes steps of removing the solvent by rotary evaporation under reduced pressure, adjusting the pH to 3-4, collecting the solid phase by solid-liquid separation, washing, and drying.

[0069] In some embodiments of the present invention, the mass ratio of the intermediate (d) to glycine methyl ester hydrochloride is (1-2):1.

[0070] In some embodiments of the present invention, the reaction conditions of the intermediate (d) with glycine methyl ester hydrochloride include at least one of the following:

[0071] 1) The reaction temperature is 20-30℃;

[0072] 2) The reaction time is 12-13 hours.

[0073] In some specific embodiments of the present invention, the glycine methyl ester hydrochloride includes

[0074] In some embodiments of the present invention, step S5 further includes using a catalyst, a condensing agent, and a solvent.

[0075] In some specific embodiments of the present invention, the mass ratio of the catalyst to the intermediate (d) is (1-2):1; the catalyst comprises N,N-diisopropylethylamine.

[0076] In some specific embodiments of the present invention, the mass ratio of the condensing agent to intermediate (b) is (1-2):1; the condensing agent includes N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea.

[0077] In some specific embodiments of the present invention, the liquid-to-solid ratio of the solvent to the intermediate (d) is (6-8) mL:1 g; the solvent includes N,N-dimethylformamide.

[0078] In some embodiments of the present invention, step S5, after the reaction is completed, further includes the steps of extracting the reaction solution, drying the organic phase, removing the solvent by rotary evaporation under reduced pressure, and purifying by rapid column chromatography.

[0079] In some embodiments of the present invention, the reaction conditions for the esterification and hydrolysis reaction of the intermediate (e) include at least one of the following:

[0080] 1) The reaction temperature is 20-30℃;

[0081] 2) The reaction time is 11-13 hours.

[0082] In some embodiments of the present invention, step S6 further includes using a catalyst and a solvent.

[0083] In some specific embodiments of the present invention, the mass ratio of the catalyst to the intermediate (c) is 1:(6-7); the catalyst comprises lithium hydroxide.

[0084] In some specific embodiments of the present invention, the liquid-to-solid ratio of the solvent to the intermediate (c) is (6-8) mL:1 g; the solvent comprises anhydrous methanol and water (5:1, v / v).

[0085] In some embodiments of the present invention, step S6, after the reaction is completed, further includes steps of removing solvent by rotary evaporation under reduced pressure, adjusting pH to 3-4, solid-liquid separation to collect the solid phase, washing, and drying.

[0086] A fourth aspect of the invention provides a sclerosstatin inhibitor comprising the compound described in the first aspect of the invention or a pharmaceutically acceptable salt thereof.

[0087] In some embodiments of the present invention, the pharmaceutically acceptable salt includes various salts obtained by reacting the acidic groups of the compound with organic or inorganic bases.

[0088] In some specific embodiments of the invention, the pharmaceutically acceptable salt includes calcium, sodium, magnesium, glucosamine, or arginine salts of the compound.

[0089] In some embodiments of the present invention, the sclerosstatin inhibitor targets and binds to the loop 3 domain of sclerosstatin, but does not bind to the loop 2 domain.

[0090] In some embodiments of the present invention, the binding target sites of the sclerosstatin inhibitor are located at the Arg115 and Arg118 sites of the loop 3 domain and the His59 and Thr76 sites of the loop 1 domain in the sclerosstatin protein.

[0091] In some embodiments of the present invention, the efficacy of the sclerostatin inhibitor is verified by the following methods:

[0092] If the addition of the sclerosstatin inhibitor to osteocytes or animal bone tissue enhances Wnt pathway signaling, it indicates that the sclerosstatin inhibitor can target specific domains of sclerosstatin.

[0093] The fifth aspect of the present invention provides the use of the sclerosstatin inhibitor described in the fourth aspect of the present invention in the preparation of a medicament for the prevention and treatment of sclerosstatin-mediated diseases.

[0094] In some embodiments of the present invention, the diseases mediated by sclerosstatin include musculoskeletal system diseases.

[0095] In some embodiments of the present invention, the musculoskeletal system diseases include osteoporosis, osteopenia, osteomalacia, osteogenesis imperfecta, ischemic osteonecrosis, rheumatoid arthritis, fractures, osteoarthritis, or myeloma.

[0096] A sixth aspect of the present invention provides a sclerosstatin-targeting drug, including the sclerosstatin inhibitors described in the fourth aspect of the present invention.

[0097] In some embodiments of the present invention, the medicament further includes pharmaceutically acceptable excipients.

[0098] In some specific embodiments of the present invention, the pharmaceutically acceptable excipients include excipients, diluents, humectants, binders, disintegrants, or lubricants.

[0099] In some embodiments of the present invention, the dosage form of the drug includes tablets, capsules, powders, granules, pills, or solutions.

[0100] Compared with the prior art, the beneficial effects of the present invention are:

[0101] 1) The compound provided by this invention is obtained by structural modification, skeleton derivation design and molecular docking screening of tetrahydrofolate, a lead small molecule compound that targets the loop 3 domain of osteosarcoma. It has been verified that the compound can specifically target and bind to the loop 3 domain of osteosarcoma, which can reactivate the Wnt signal that is inhibited by osteosarcoma and restore the bone formation-promoting effect of Wnt signal. Moreover, it does not bind to the loop 2 domain of osteosarcoma and can maintain the cardiovascular protective effect of the loop 2 domain.

[0102] 2) The sclerosatin inhibitor containing the compound or its pharmaceutically acceptable salt provided by the present invention can inhibit various musculoskeletal system diseases mediated by sclerosatin activation, such as osteoporosis, osteopenia, osteomalacia, osteogenesis imperfecta, ischemic osteonecrosis, rheumatoid arthritis, fractures, osteoarthritis, and myeloma. It has a wide range of applications and strong targeting.

[0103] 3) The sclerosstatin-targeting drug containing a sclerosstatin inhibitor or its pharmaceutically acceptable salt provided by the present invention, based on the characteristic that the sclerosstatin inhibitor or its pharmaceutically acceptable salt can target and bind to the loop 3 domain of sclerosstatin without binding to the loop 2 domain, can reduce the risk of cardiovascular disease during drug use when used for the prevention and treatment of sclerosstatin-mediated diseases, and has higher safety, thus meeting the market demand for sclerosstatin-targeting drugs. Attached Figure Description

[0104] Figure 1 The flowchart shows the simplified structure and skeletal derivation design of tetrahydrofolate in the examples;

[0105] Figure 2 This is a flowchart illustrating the screening process for molecules generated by Reinvent4 in this embodiment.

[0106] Figure 3 A schematic diagram of the three-dimensional binding modes of compounds 669(a) and 190(b) to the target region loop 3 of sclerosstatin protein;

[0107] Figure 4 This is a statistical graph showing the effect of compounds 190 and 669 prepared in Examples 2 and 3 on the Wnt signal. Detailed Implementation

[0108] The present invention will be further described in detail below through specific embodiments. Unless otherwise specified, the raw materials, reagents, or apparatus used in the embodiments can be obtained from conventional commercial sources or by existing technical methods. Unless otherwise specified, the experimental or testing methods are conventional methods in the art.

[0109] Example 1

[0110] In this embodiment, by extracting the core framework of tetrahydrofolate, performing framework derivatization, and molecular docking, compounds 190 and 669 with the potential to target the loop 3 domain of osteosclerosingin were screened.

[0111] Figure 1 This is a flowchart illustrating the simplified structure and skeletal derivation design of tetrahydrofolic acid in the embodiments. Figure 2 This is a flowchart illustrating the screening process for molecules generated by Reinvent4 in this embodiment. Figure 1 and Figure 2 It can be seen that the screening steps for compounds 190 and 669 are as follows:

[0112] 1) Structural simplification of tetrahydrofolate and extraction of its core framework. Furthermore, by utilizing Libinvent, one of the core modules of Reinvent4, the Z site of the core backbone was used for backbone modification and derivation, resulting in a total of 936 ligand molecules.

[0113] 2) First, use Python's RDKit library to convert these ligand molecules from SMILES format to 3D format for preprocessing. Use the LigPrep module of the Schrodinger Maestro package to add polar hydrogen and Gasteiger charge to the ligand molecules so that the ligand molecules can dock with the receptor molecules. Download the sclerosstatin protein structure (PDBID: 2K8P) from the RCSB protein database. Use Maestro's LigPrep module to prepare the initial structure and parameters for docking, add charge and hydrogen atoms, and fuse nonpolar hydrogen atoms to the sclerosstatin protein structure to complete the protein preprocessing.

[0114] 3) Molecular docking was performed using Maestro's Glide module. The docking site was selected as the target region loop 3 of sclerosstatin protein, with center coordinates of (16.68, 14.43, -11.62), and the docking precision was XP. For each input small molecule ligand, the output conformation with the highest binding energy was selected. This conformation and binding energy were used for subsequent analysis. Finally, compounds 190 and 669 were selected as candidate compounds from 936 molecules for subsequent activity testing.

[0115] Figure 3 This diagram illustrates the three-dimensional binding modes of compounds 669(a) and 190(b) to the target region loop 3 of sclerosingin protein. Green bars represent the molecular structures of compounds 669 and 190, yellow dashed lines represent hydrogen bonds, black dashed lines represent cation-π interactions, and purple dashed lines represent electrostatic interactions. Figure 3It is known that compounds 190 and 669 have the optimal binding mode to the target region loop 3 of sclerosstatin protein. The carboxyl end of the ligand forms more hydrogen bonds and ionic interactions with Arg115 of loop 3 and His59 of loop 1. The structure of an aromatic ring at the other end of the ligand forms a cation-π interaction with the side chain of Arg118 of loop 3, and forms hydrogen bond interactions with its main chain.

[0116] Table 1. Docking scores of compounds 190 and 669 in Example 1

[0117]

[0118] Example 2

[0119] In this embodiment, compound 190 was synthesized. The steps and synthetic route are illustrated below:

[0120] S11. Weigh 2.18 g of 2-bromo-4-nitrophenol, 2.16 g of 4-methoxycarbonylphenylboronic acid, 0.73 g of 1,1'-bis(diphenylphosphine)ferrocene palladium dichloride, and 2.76 g of potassium carbonate into a 100 mL flask. Under nitrogen protection, add 25 mL of ultra-dry N,N-dimethylformamide to the flask, and then place the flask at 100 °C for 12 h. After the reaction is complete, extract the reaction solution three times with ethyl acetate, dry the organic phase with anhydrous sodium sulfate, remove the solvent by rotary evaporation under reduced pressure, and purify by rapid column chromatography (petroleum ether / ethyl acetate) to obtain intermediate (a) with a yield of 62%.

[0121]

[0122] S21. Weigh 2.73 g of intermediate (a), 2.12 g of ammonium chloride, and 2.20 g of iron powder and add them to a 100 mL flask. Add 25 mL of anhydrous ethanol and 5 mL of water to the flask, and then place the flask at 80 °C for 4 h. After the reaction is complete, filter the reaction system to remove the remaining iron powder, remove the ethanol by rotary evaporation under reduced pressure, extract the reaction solution three times with ethyl acetate, dry the organic phase with anhydrous sodium sulfate, remove the solvent by rotary evaporation under reduced pressure, and purify the intermediate (b) by rapid column chromatography (petroleum ether / ethyl acetate) with a yield of 81%.

[0123]

[0124] S31. Weigh 2.43 g of intermediate (b), 2.80 g of m-fluorobenzoic acid, and 3.90 g of N,N-diisopropylethylamine (DIPEA) and add them to a 100 mL flask. Add 25 mL of ultra-dry N,N-dimethylformamide to the flask and stir at 25 °C for 30 min. Then add 3.80 g of N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (HATU) to the flask and continue to react at 25 °C for 12 h. After the reaction is complete, extract the reaction solution three times with ethyl acetate, dry the organic phase with anhydrous sodium sulfate, remove the solvent by rotary evaporation under reduced pressure, and purify by rapid column chromatography (petroleum ether / ethyl acetate) to obtain intermediate (c) with a yield of 67%.

[0125]

[0126] S41. Weigh 3.65 g of intermediate (c) and 0.69 g of lithium hydroxide into a 100 mL flask. Add 25 mL of anhydrous methanol and 5 mL of water to the flask. Then place the flask at 25 °C and react for 12 h. After the reaction is complete, remove methanol by rotary evaporation under reduced pressure. Adjust the pH value to 3-4 with 1 mol / L hydrochloric acid. Filter the reactants. The resulting filter cake is washed with water and dried under reduced pressure to obtain intermediate (d) with a yield of 86%.

[0127]

[0128] S51. Weigh 3.51 g of intermediate (d), 2.51 g of glycine methyl ester hydrochloride, and 3.90 g of N,N-diisopropylethylamine (DIPEA) and add them to a 100 mL flask. Add 25 mL of ultra-dry N,N-dimethylformamide to the flask and stir at 25 °C for 30 min. Then add 3.80 g of N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (HATU) to the flask and continue to react at 25 °C for 12 h. After the reaction is complete, extract the reaction solution three times with ethyl acetate, dry the organic phase with anhydrous sodium sulfate, remove the solvent by rotary evaporation under reduced pressure, and purify by rapid column chromatography (petroleum ether / ethyl acetate) to obtain intermediate (e) with a yield of 53%.

[0129]

[0130] S61. Weigh 4.22 g of intermediate (e) and 0.69 g of lithium hydroxide into a 100 mL flask. Add 25 mL of anhydrous methanol and 5 mL of water to the flask, and then place the flask at 25 °C for 12 h. After the reaction is complete, remove methanol by rotary evaporation under reduced pressure. Adjust the pH to 3-4 with 1 mol / L hydrochloric acid. Filter the reactants. The resulting filter cake is washed with water and dried under reduced pressure to obtain compound 190 as a white solid with a yield of 32%.

[0131]

[0132] Compound 190 in Example 2 was analyzed using nuclear magnetic resonance. 1 HNMR and 13 C NMR characterization, the results are as follows:

[0133] 1 H NMR(500MHz,DMSO)δ12.62(s,1H),10.20(s,1H),9.63(s,1H),8.87(d,J=4.4H z,1H),7.96–7.56(m,9H),7.45–7.42(m,1H),6.99–6.95(m,1H),3.97(s,2H).;

[0134] 13 C NMR (125MHz, DMSO) 171.86, 166.79, 164.06, 163.39, 161.45, 151.42, 141.91, 137.80 (d, J = 6.75Hz), 131.96 (d, J = 122.21Hz), 131.02 (d, J = 7.90Hz), 129.33, 127.48, 126.90, 124.20 (d, J = 1.61Hz), 123.36, 122.28, 118.75 (d, J = 21.32Hz), 116.55, 114.79 (d, J = 22.88Hz), 41.71.

[0135] Example 3

[0136] In this embodiment, compound 669 was synthesized. The steps and synthetic route are illustrated below:

[0137] S11. Weigh 2.18 g of 2-bromo-4-nitrophenol, 2.16 g of 4-methoxycarbonylphenylboronic acid, 0.73 g of 1,1'-bis(diphenylphosphine)ferrocene palladium dichloride, and 2.76 g of potassium carbonate into a 100 mL flask. Under nitrogen protection, add 25 mL of ultra-dry N,N-dimethylformamide to the flask, and then place the flask at 100 °C for 12 h. After the reaction is complete, extract the reaction solution three times with ethyl acetate, dry the organic phase with anhydrous sodium sulfate, remove the solvent by rotary evaporation under reduced pressure, and purify by rapid column chromatography (petroleum ether / ethyl acetate) to obtain intermediate (a) with a yield of 62%.

[0138]

[0139] S21. Weigh 2.73 g of intermediate (a), 2.12 g of ammonium chloride, and 2.20 g of iron powder and add them to a 100 mL flask. Add 25 mL of anhydrous ethanol and 5 mL of water to the flask, and then place the flask at 80 °C for 4 h. After the reaction is complete, filter the reaction system to remove the remaining iron powder, remove the ethanol by rotary evaporation under reduced pressure, extract the reaction solution three times with ethyl acetate, dry the organic phase with anhydrous sodium sulfate, remove the solvent by rotary evaporation under reduced pressure, and purify the intermediate (b) by rapid column chromatography (petroleum ether / ethyl acetate) with a yield of 81%.

[0140]

[0141] S31. Weigh 2.43 g of intermediate (b), 2.72 g of p-methylbenzoic acid, and 3.90 g of N,N-diisopropylethylamine (DIPEA) and add them to a 100 mL flask. Add 25 mL of ultra-dry N,N-dimethylformamide to the flask and stir at 25 °C for 30 min. Then add 3.80 g of N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (HATU) to the flask and continue to react at 25 °C for 12 h. After the reaction is complete, extract the reaction solution three times with ethyl acetate, dry the organic phase with anhydrous sodium sulfate, remove the solvent by rotary evaporation under reduced pressure, and purify by rapid column chromatography (petroleum ether / ethyl acetate) to obtain intermediate (c) with a yield of 79%.

[0142]

[0143] S41. Weigh 3.61 g of intermediate (c) and 0.69 g of lithium hydroxide into a 100 mL flask. Add 25 mL of anhydrous methanol and 5 mL of water to the flask. Then place the flask at 25 °C and react for 12 h. After the reaction is complete, remove methanol by rotary evaporation under reduced pressure. Adjust the pH value to 3-4 with 1 mol / L hydrochloric acid. Filter the reactants. The resulting filter cake is washed with water and dried under reduced pressure to obtain intermediate (d) with a yield of 82%.

[0144]

[0145] S51. Weigh 3.47 g of intermediate (d), 2.51 g of glycine methyl ester hydrochloride, and 3.90 g of N,N-diisopropylethylamine (DIPEA) and add them to a 100 mL flask. Add 25 mL of ultra-dry N,N-dimethylformamide to the flask and stir at 25 °C for 30 min. Then add 3.80 g of N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (HATU) to the flask and continue to react at 25 °C for 12 h. After the reaction is complete, extract the reaction solution three times with ethyl acetate, dry the organic phase with anhydrous sodium sulfate, remove the solvent by rotary evaporation under reduced pressure, and purify by rapid column chromatography (petroleum ether / ethyl acetate) to obtain intermediate (e) with a yield of 57%.

[0146]

[0147] S61. Weigh 4.18 g of intermediate (e) and 0.69 g of lithium hydroxide into a 100 mL flask. Add 25 mL of anhydrous methanol and 5 mL of water to the flask, and then place the flask at 25 °C for 12 h. After the reaction is complete, remove methanol by rotary evaporation under reduced pressure. Adjust the pH to 3-4 with 1 mol / L hydrochloric acid. Filter the reactants. The resulting filter cake is washed with water and dried under reduced pressure to obtain compound 669 as a white solid with a yield of 43%.

[0148]

[0149] Compound 669 in Example 3 was analyzed using nuclear magnetic resonance. 1 H NMR and 13 C NMR characterization, the results are as follows:

[0150] 1 H NMR(500MHz,DMSO)δ12.62(s,1H),10.03(s,1H),9.57(s,1H),8.85(t,J=5.8 3Hz,1H),7.94(s,1H),7.92(s,1H),7.89(s,1H),7.87(s,1H),7.73(d,J=2.30 Hz,1H),7.69(s,1H),7.67(s,1H),7.62(dd,J=8.70,2.34Hz,1H),7.34(s,1H ),7.32(s,1H),6.95(d,J=8.71Hz,1H),3.96(d,J=5.81Hz,2H),2.39(s,3H).;

[0151] 13C NMR(125MHz,DMSO)δ171.87,166.80,165.31,151.14,142.00,141.79,132.62,132.40,1 31.86,129.35,129.33,128.01,127.46,126.82,123.28,122.21,116.50,41.74,21.47..

[0152] Example 4

[0153] This embodiment verifies the targeting effect of compound 190 prepared in Example 2 and compound 669 prepared in Example 3 on specific domains of sclerosstatin:

[0154] Experimental materials:

[0155] DMEM complete medium: DMEM medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin;

[0156] MEM-α complete medium: MEM-α medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin;

[0157] Cell signaling pathways: HEK293 cells (human embryonic kidney cells 293) were transfected with Wnt1 protein and full-length human recombinant sclerosstatin protein (FLhSOST), respectively.

[0158] Experimental methods:

[0159] HEK293 cells were cultured at 37°C under humid atmospheric conditions in DMEM medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. The cells were sporulated at a rate of 5 × 10⁶ cells / year. 4 Cells were seeded in 100 μL of DMEM complete medium in 96-well plates at a density of 100 μL and cultured for 24 h. The experiment was divided into 3 groups:

[0160] Wnt1+Veh group: Wnt1 protein particles (25 ng per well) were co-transfected into cells using Lipofectamine 2000 (0.35 μL per well). During this process, each 25 ng plasmid and 0.35 μL of Lipofectamine 2000 were first mixed in 30 μL of OptiMem medium and incubated for 20 min. Then, 70 μL of serum- and antibiotic-free DMEM medium was added and gently mixed to obtain the transfection mixture. Next, the old medium was removed, and the transfection mixture was added to the wells containing cells to transfect the Wnt1 protein particles. After 12 h of transfection, the transfection mixture was removed to obtain transfected cells, and 100 μL of DMEM complete medium was added to treat the cells for 12 h.

[0161] Wnt1+FL hSOST+Veh group: Wnt1 protein particle (25 ng per well) and FL hSOST plasmid (25 ng per well) were co-transfected into cells using Lipofectamine 2000 (0.35 μL per well). During this process, 25 ng of Wnt1 protein particle, 25 ng of FL hSOST plasmid and 0.35 μL of Lipofectamine 2000 were first mixed in 30 μL of OptiMem medium and incubated for 20 min. Then, 70 μL of serum- and antibiotic-free DMEM medium was added and gently mixed to obtain the transfection mixture. Next, the old medium was removed, and the transfection mixture was added to the wells containing cells to transfect Wnt1 protein particle and FL hSOST plasmid. After 12 h of transfection, the transfection mixture was removed to obtain transfected cells, and 100 μL of DMEM complete medium was added to treat the cells for 12 h.

[0162] Wnt1+FLhSOST+190 / 669 group: Wnt1 protein particle (25 ng per well) and FLhSOST plasmid (25 ng per well) were co-transfected into cells using Lipofectamine 2000 (0.35 μL per well). During this process, 25 ng of Wnt1 protein particle, 25 ng of FLhSOST plasmid, and 0.35 μL of Lipofectamine 2000 were added before 30 μL of the other two. Mix and incubate in OptiMem medium for 20 min, then add 70 μL of serum- and antibiotic-free DMEM medium and mix gently to obtain a transfection mixture; then remove the old medium and add the transfection mixture to wells containing cells to transfect Wnt1 protein and FLhSOST plasmids. After 12 h of transfection, remove the transfection mixture to obtain transfected cells, and treat the cells with 100 μL of DMEM complete medium containing 5 μmol / L compound 190 / 669 (dissolved in 1x concentration of phosphate buffer containing 5% DMSO) for 12 h.

[0163] After the above grouping treatment, the culture medium was removed, and the cells were lysed in 100 μL of 1× inactive lysis buffer (PLB) by shaking in a room temperature vibrating incubator for 15 min. Then, 15 μL of lysis buffer was transferred to black 96-well microplates, and the parameters were set according to the manufacturer's instructions for the Dual-Luciferase Reporter Assay System. Luciferase activity and Wnt signal expression were measured using the MD SpectraMaxi3X Multi-Mode Microplate Reader.

[0164] Figure 4 This is a statistical graph showing the effect of compounds 190 and 669 prepared in Examples 2 and 3 on the Wnt signal. Figure 4 It was found that the Wnt signal in cells treated with compounds 190 and 669 was significantly higher than that in cells not treated with compounds 190 or 669. Wnt signal plays a key role in bone development and bone homeostasis. A decrease in this signal may lead to reduced bone density, osteoporosis, and an increased risk of fracture. Therefore, the higher Wnt signal indicates that compounds 190 and 669 are beneficial in restoring Wnt signal and promoting bone formation.

Claims

1. A compound, characterized in that, Its structural formula is shown in formula (I) or formula (II):

2. The method for screening compounds according to claim 1, characterized in that, Includes the following steps: Simplify the structure of tetrahydrofolate to obtain As a structural framework, a framework derivation was performed at the Z site to obtain candidate molecules; the candidate molecules were molecularly docked with the loop3 domain of sclerosstatin protein, and the compounds were screened based on the binding mode and binding energy of the candidate molecules in the loop3 binding pocket.

3. The method for preparing the compound according to claim 1, characterized in that, Includes the following steps: S1. React 2-bromo-4-nitrophenol with 4-methoxycarbonylphenylboronic acid to give intermediate (a). ; S2, Intermediate (a) is obtained from intermediate (b) via nitro reduction reaction. ; S3, Intermediate (b) and Compound (x) The reaction yields intermediate (c). ; S4. Intermediate (c) is obtained as intermediate (d) through esterification and hydrolysis. ; S5. Intermediate (d) reacts with glycine methyl ester hydrochloride to give intermediate (e). ; S6. The intermediate (e) is obtained by esterification and hydrolysis reaction to yield the compound; Where R1 is H and R2 is -CH3, or R1 is F and R2 is H.

4. The preparation method according to claim 3, characterized in that, The mass ratio of 2-bromo-4-nitrophenol to 4-methoxycarbonylphenylboronic acid is (0.8-1.5):

1.

5. The preparation method according to claim 3, characterized in that, The mass ratio of the intermediate (b) to the compound (x) is 1:(1-1.5).

6. The preparation method according to claim 3, characterized in that, The mass ratio of the intermediate (d) to glycine methyl ester hydrochloride is 1:(1-2).

7. A sclerosstatin inhibitor, characterized in that, Includes the compound of claim 1 or a pharmaceutically acceptable salt thereof.

8. The sclerosstatin inhibitor according to claim 7, characterized in that, The sclerosstatin inhibitor targets and binds to the loop 3 domain of sclerosstatin, but does not bind to the loop 2 domain.

9. A drug targeting sclerosstatin, characterized in that, Including the osteostatin inhibitor as described in claim 7 or 8.

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