CN120004758A - Screening and validation method for a class of small molecule inhibitors targeting a specific structural domain of sclerostin - Google Patents
Screening and validation method for a class of small molecule inhibitors targeting a specific structural domain of sclerostin Download PDFInfo
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Abstract
The invention relates to the technical field of biological medicines, and discloses a screening and verifying method of a targeting sclerostin specific structural domain small molecule inhibitor. The compound is obtained by carrying out structural transformation, skeleton derivative design and molecular docking screening on a lead small molecular compound tetrahydrofolic acid of a targeting sclerostin loop3 structural domain, can specifically target and bind with the sclerostin loop3 structural domain, can reactivate Wnt signals inhibited by the sclerostin and restore the effect of promoting bone formation by the Wnt signals, can inhibit various musculoskeletal system diseases mediated by the activation of the sclerostin, has wide application range and strong targeting, and can reduce the risk of causing cardiovascular diseases in the use process of the medicine based on the characteristics that the compound targets and binds with the loop3 structural domain of the sclerostin but does not bind with the loop2 structural domain.
Description
Technical Field
The invention relates to the technical field of biological medicines, in particular to a screening and verifying method of a targeting sclerostin specific structural domain small molecule inhibitor.
Background
The Wnt signaling pathway plays a vital role in bone formation, growth and development. The Wnt/β -catenin signaling pathway is a typical Wnt signaling pathway, a key regulator of bone formation and metabolism, in which Wnt proteins bind to low density lipoprotein receptor-related protein 5/6 (LRP 5/6) to promote further expression of osteoblast-related genes.
Sclerostin is a protein secreted by bone cells and contains 189 amino acids throughout its length, including the unordered N-terminus (amino acids 1-56) and C-terminus (amino acids 145-189), and three loop domains formed around the cysteine knot motif, the loop1 domain (amino acids 57-80), the loop2 domain (amino acids 86-109) and the loop3 domain (amino acids 111-140), respectively. The sclerostin can be combined with low-density lipoprotein receptor related protein 5/6 to prevent interaction between the sclerostin and Wnt, so that the WNT-Frizzled-LRP5/6 ternary complex is prevented from being established, and then classical Wnt signal paths are inhibited, and finally diseases of musculoskeletal system mediated by the sclerostin, such as osteoporosis, osteogenesis imperfecta, low-phosphorus rickets, sarcopenia and the like are caused.
Currently, there is Romosozumab monoclonal antibody targeting sclerostin as a marketed drug, mainly for the treatment of postmenopausal osteoporosis, but this drug has been given black frame warning by the FDA in month 4 of 2019, treatment is at risk of potential cardiovascular disease and patients with heart attacks or strokes will be disabled for one year.
Therefore, the development of the drug targeting the sclerostin with small cardiovascular disease induction risk has great significance for preventing and treating musculoskeletal system diseases.
Disclosure of Invention
The present invention aims to solve at least one of the above technical problems in the prior art. To this end, one of the objects of the present invention is to provide a compound.
It is a second object of the present invention to provide a method for screening such compounds.
It is a further object of the present invention to provide a process for the preparation of such compounds.
The fourth object of the present invention is to provide a sclerostin inhibitor.
The fifth object of the present invention is to provide the use of such a sclerostin inhibitor.
The invention aims at providing a targeting sclerostin drug.
The invention is characterized in that a loop3 structural domain in the sclerostin is a beta-sheet short peptide consisting of 29 amino acids, contains more positively charged lysine and arginine, forms a core structure of disulfide stabilized protein with the loop1 structural domain, and has simple structure and less interaction. The prior art shows that the loop2 domain in the sclerostin participates in cardiovascular protection, while the loop3 domain does not participate, namely after the loop3 domain of the sclerostin is specifically targeted and combined, the antagonism of the sclerostin on the Wnt signal path can be inhibited, but the cardiovascular protection is not affected. Studies show that tetrahydrofolate can specifically target and bind with a sclerostin loop3 structural domain, and can inhibit various musculoskeletal system diseases mediated by activation of sclerostin, so that the invention develops an inhibitor which targets the sclerostin loop3 structural domain but does not bind with a loop2 structural domain based on a tetrahydrofolate core molecular skeleton, thereby reducing inhibition of the sclerostin on a Wnt signal path, maintaining cardiovascular protection effect of the sclerostin and reducing cardiovascular disease risk.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
in a first aspect, the present invention provides a compound having the structural formula (a):
Wherein R 1 is H or halogen, and R 2 is H or alkyl.
In some embodiments of the invention, the combination of R 1 and R 2 in the compound of formula (A) is selected from any one of R 1 is H and R 2 is alkyl or R 1 is halogen and R 2 is H.
In some embodiments of the invention, the compound is represented by formula (I) or formula (II):
In a second aspect, the present invention provides a method for screening a compound according to the first aspect of the present invention, comprising the steps of:
simplified tetrahydrofolic acid structure, and acquisition And carrying out molecular docking on the candidate molecule and a loop3 structural domain of the sclerostin protein, and screening out the compound according to the combination mode and the combination energy of the candidate molecule in a loop3 combination pocket.
In some embodiments of the invention, the backbone derivatization is performed using one of the core modules Libinvent of Reinvent 4.
In some embodiments of the invention, the molecular docking is performed using a slide module of Maestro.
In some embodiments of the invention, the compound is the candidate molecule in the loop3 binding pocket with the optimal binding mode and highest binding energy.
In the invention, the binding mode of the tetrahydrofolic acid and the sclerostin loop3 domain is deeply analyzed, and the fact that the carboxyl with the negative charge at the tail end of the tetrahydrofolic acid can form hydrogen bonds and salt bridges with positive residues Arg115 and His59 in a loop3 binding pocket is found, and the interaction is favorable for the stable binding of the tetrahydrofolic acid and the sclerostin is confirmedAs core groups, the large and complex structure of tetrahydrofolate is simplified by cleaving unnecessary groups and the derivatization site Z is determined to be a specific substitution position of the aromatic ring.
A third aspect of the present invention provides a process for the preparation of a compound according to the first aspect of the present invention, comprising the steps of:
S1, reacting 2-bromo-4-nitrophenol with 4-methoxycarbonylphenylboronic acid to obtain an intermediate (a)
S2, obtaining an intermediate (b) from the intermediate (a) through nitroreduction reaction
S3, intermediate (b) and compound (x)The reaction gives an intermediate (c)
S4, obtaining an intermediate (d) from the intermediate (c) through esterification hydrolysis reaction
S5, reacting the intermediate (d) with glycine methyl ester hydrochloride to obtain an intermediate (e)
S6, obtaining the compound through esterification hydrolysis reaction of the intermediate (e);
wherein R 1 and R 2 are as defined in the first aspect of the invention.
In some embodiments of the invention, the mass ratio of 2-bromo-4-nitrophenol to 4-methoxycarbonylphenylboronic acid is in the range of (0.8-1.5): 1.
In some embodiments of the invention, the reaction conditions of the 2-bromo-4-nitrophenol and 4-methoxycarbonylphenylboronic acid comprise at least one of:
1) The reaction is carried out under a protective atmosphere;
2) The temperature of the reaction is 90-110 ℃;
3) The reaction time is 10-13h.
In some embodiments of the invention, step S1 further comprises using a catalyst, an alkaline medium, and a solvent.
In some embodiments of the invention, the mass ratio of the catalyst to 2-bromo-4-nitrophenol is 1 (2-4), and the catalyst comprises 1,1' -bis-diphenylphosphino ferrocene palladium dichloride (Pd (Cl) 2 dppf).
In some embodiments of the invention, the mass ratio of the base medium to the 2-bromo-4-nitrophenol is (1-2): 1, and the base medium comprises potassium carbonate (K 2CO3).
In some embodiments of the invention, the solvent to 2-bromo-4-nitrophenol ratio is 100mL (8-10) g, and the solvent comprises N, N-Dimethylformamide (DMF).
In some embodiments of the invention, after the reaction is completed in the step S1, the method further comprises the steps of extracting the reaction solution, drying the organic phase, removing the solvent by rotary evaporation under reduced pressure, and purifying by flash column chromatography.
In some embodiments of the invention, the reaction conditions of the intermediate (a) nitroreduction reaction include at least one of:
1) The temperature of the reaction is 70-90 ℃;
2) The reaction time is 3-5h.
In some embodiments of the invention, step S2 further comprises using a catalyst, a reducing agent, and a solvent.
In some embodiments of the invention, the mass ratio of the catalyst to the intermediate (a) is 1 (1-2), and the catalyst comprises ammonium chloride (NH 4 Cl).
In some embodiments of the invention, the mass ratio of the reducing agent to the intermediate (a) is 1 (1-2), and the reducing agent comprises iron powder (Fe).
In some embodiments of the invention, the liquid to solid ratio of the solvent to intermediate (a) is (9-12) mL:1g, and the solvent comprises absolute ethanol and water (5:1, v/v).
In some embodiments of the present invention, after the reaction is completed in the step S2, the method further includes a step of removing the reducing agent by solid-liquid separation, removing the solvent by reduced pressure rotary evaporation, extracting the reaction solution, drying the organic phase, removing the solvent by reduced pressure rotary evaporation, and purifying by flash column chromatography.
In some embodiments of the invention, the mass ratio of intermediate (b) to compound (x) is 1 (1-1.5).
In some embodiments of the invention, the reaction conditions of intermediate (b) with compound (x) include at least one of:
1) The temperature of the reaction is 20-30 ℃;
2) The reaction time is 12-13h.
In some embodiments of the invention, the compound (x) comprises
In some embodiments of the invention, the step S3 further comprises using a catalyst, a condensing agent, and a solvent.
In some embodiments of the invention, the mass ratio of the catalyst to intermediate (b) is (1-2): 1, and the catalyst comprises N, N-Diisopropylethylamine (DIPEA).
In some embodiments of the invention, the mass ratio of condensing agent to intermediate (b) is (1-2): 1, and the condensing agent comprises urea N, N, N ', N' -tetramethyl-O- (7-azabenzotriazol-1-yl) Hexafluorophosphate (HATU).
In some embodiments of the invention, the solvent to intermediate (b) has a liquid to solid ratio of (9-12) mL 1g, and the solvent comprises N, N-dimethylformamide.
In some embodiments of the present invention, after the reaction is completed in the step S3, the method further includes steps of extracting a reaction solution, drying an organic phase, removing a solvent by spin evaporation under reduced pressure, and purifying by flash column chromatography.
In some embodiments of the invention, the reaction conditions of the intermediate (c) esterification hydrolysis reaction include at least one of:
1) The temperature of the reaction is 20-30 ℃;
2) The reaction time is 10-13h.
In some embodiments of the invention, step S4 further comprises using a catalyst and a solvent.
In some embodiments of the invention, the mass ratio of the catalyst to the intermediate (c) is 1 (5-6), and the catalyst comprises lithium hydroxide (LiOH).
In some embodiments of the invention, the liquid to solid ratio of the solvent to intermediate (c) is (7-10) mL:1g, and the solvent comprises anhydrous methanol and water (5:1, v/v).
In some embodiments of the present invention, after the reaction is completed in the step S4, the method further comprises the steps of removing the solvent by spin evaporation under reduced pressure, adjusting the pH to 3-4, collecting the solid phase by solid-liquid separation, washing and drying.
In some embodiments of the invention, the mass ratio of intermediate (d) to glycine methyl ester hydrochloride is (1-2): 1.
In some embodiments of the invention, the reaction conditions of intermediate (d) with glycine methyl ester hydrochloride include at least one of the following:
1) The temperature of the reaction is 20-30 ℃;
2) The reaction time is 12-13h.
In some embodiments of the invention, the glycine methyl ester hydrochloride comprises
In some embodiments of the invention, the step S5 further comprises using a catalyst, a condensing agent, and a solvent.
In some embodiments of the invention, the mass ratio of the catalyst to the intermediate (d) is (1-2): 1, and the catalyst comprises N, N-diisopropylethylamine.
In some embodiments of the invention, the mass ratio of condensing agent to intermediate (b) is (1-2): 1, and the condensing agent comprises urea N, N, N ', N' -tetramethyl-O- (7-azabenzotriazol-1-yl) hexafluorophosphate.
In some embodiments of the invention, the solvent to intermediate (d) has a liquid to solid ratio of (6-8) mL 1g, and the solvent comprises N, N-dimethylformamide.
In some embodiments of the present invention, after the reaction is completed in the step S5, the method further comprises the steps of extracting the reaction solution, drying the organic phase, removing the solvent by rotary evaporation under reduced pressure, and purifying by flash column chromatography.
In some embodiments of the invention, the reaction conditions of the intermediate (e) esterification hydrolysis reaction include at least one of:
1) The temperature of the reaction is 20-30 ℃;
2) The reaction time is 11-13h.
In some embodiments of the invention, step S6 further comprises using a catalyst and a solvent.
In some embodiments of the invention, the mass ratio of the catalyst to the intermediate (c) is 1 (6-7), and the catalyst comprises lithium hydroxide.
In some embodiments of the invention, the liquid to solid ratio of the solvent to intermediate (c) is (6-8) mL:1g, and the solvent comprises anhydrous methanol and water (5:1, v/v).
In some embodiments of the present invention, after the reaction is completed in the step S6, the method further comprises the steps of removing the solvent by spin evaporation under reduced pressure, adjusting the pH to 3-4, collecting the solid phase by solid-liquid separation, washing and drying.
In a fourth aspect, the present invention provides a sclerostin inhibitor comprising a compound according to the first aspect of the invention or a pharmaceutically acceptable salt thereof.
In some embodiments of the invention, the pharmaceutically acceptable salts include various salts obtained by reacting an acidic group of the compound with an organic base and an inorganic base.
In some embodiments of the invention, the pharmaceutically acceptable salt comprises a calcium salt, a sodium salt, a magnesium salt, a glucosamine salt, or an arginine salt of the compound.
In some embodiments of the invention, the sclerostin inhibitor targets the loop3 domain that binds to sclerostin and does not bind to the loop2 domain.
In some embodiments of the invention, the binding target of the sclerostin inhibitor is located at Arg115, arg118 of the loop3 domain and at amino acid positions His59, thr76 of the loop1 domain of the sclerostin protein.
In some embodiments of the invention, the effect of the sclerostin inhibitor is verified by the following method:
And (3) performing hard bone chalone protein dry prognosis on bone cells or animal bone tissues, and adding the hard bone chalone inhibitor, wherein if Wnt channel signals can be enhanced, the hard bone chalone inhibitor can target a hard bone chalone specific structural domain.
In a fifth aspect, the present invention provides the use of a sclerostin inhibitor according to the fourth aspect of the invention in the manufacture of a medicament for the treatment of a sclerostin-mediated disease.
In some embodiments of the invention, the sclerostin-mediated disease comprises a disease of the musculoskeletal system.
In some embodiments of the invention, the musculoskeletal system disease comprises osteoporosis, osteopenia, osteomalacia, osteogenesis imperfecta, ischemic osteonecrosis, rheumatoid arthritis, bone fractures, osteoarthritis, or myeloma.
In a sixth aspect, the invention provides a targeted sclerostin drug comprising a sclerostin inhibitor according to the fourth aspect of the invention.
In some embodiments of the invention, the medicament further comprises a pharmaceutically acceptable excipient.
In some embodiments of the invention, the pharmaceutically acceptable excipients include excipients, diluents, wetting agents, binders, disintegrants or lubricants.
In some embodiments of the invention, the dosage form of the medicament comprises a tablet, capsule, powder, granule, pill, or solution.
Compared with the prior art, the invention has the beneficial effects that:
1) The compound provided by the invention is obtained by carrying out structural transformation, skeleton derivative design and molecular docking screening on a lead small molecular compound tetrahydrofolic acid of a targeting sclerostin loop3 structural domain, and is proved to be capable of specifically targeting and combining with the sclerostin loop3 structural domain, reactivating Wnt signals inhibited by the sclerostin, recovering the effect of promoting bone formation of Wnt signals, not combining with the sclerostin loop2 structural domain, and maintaining the cardiovascular protection effect of the loop2 structural domain.
2) The sclerostin inhibitor containing the compound or the pharmaceutically acceptable salt thereof provided by the invention can inhibit various musculoskeletal system diseases mediated by activation of the sclerostin, such as osteoporosis, osteopenia, osteomalacia, osteogenesis imperfecta, ischemic osteonecrosis, rheumatoid arthritis, fracture, osteoarthritis, myeloma and the like, and has wide application range and strong targeting;
3) The targeting sclerostin drug containing the sclerostin inhibitor or the pharmaceutically acceptable salt thereof provided by the invention can be used for reducing the risk of cardiovascular diseases in the use process of the drug when being used for preventing and treating the sclerostin-mediated diseases based on the characteristic that the sclerostin inhibitor or the pharmaceutically acceptable salt thereof can be targeted to bind with the loop3 domain of the sclerostin but not with the loop2 domain, has higher safety and can meet the market demand of the drug targeting the sclerostin.
Drawings
FIG. 1 is a flow chart of a simplified structure and skeleton derived design of tetrahydrofolate in an embodiment;
FIG. 2 is a flow chart of a screening procedure for Reinvent molecules generated in the examples;
FIG. 3 is a schematic representation of the three-dimensional binding pattern of compound 669 (a) and compound 190 (b) to the target region loop3 of sclerostin protein;
FIG. 4 is a statistical plot of the effect of compounds 190 and 669 prepared in examples 2 and 3 on Wnt signaling.
Detailed Description
The present invention will be described in further detail with reference to specific examples. The starting materials, reagents or apparatus used in the examples are all commercially available from conventional sources or may be obtained by methods known in the art unless otherwise specified. Unless otherwise indicated, assays or testing methods are routine in the art.
Example 1
In the embodiment, through extracting a core skeleton of tetrahydrofolate, skeleton derivative and molecular docking, a compound 190 and a compound 669 with the potential of targeting the loop3 domain of sclerostin are screened out:
Fig. 1 is a flow chart showing the simplified structure and the design of skeleton derivatization of tetrahydrofolate in the example, fig. 2 is a flow chart showing the screening of the molecule produced for Reinvent4 in the example, and as can be seen from fig. 1 and 2, the screening steps of compound 190 and compound 669 are as follows:
1) The tetrahydrofolic acid is structurally simplified, and the core skeleton is extracted And performing framework modification derivatization on a Z site of a core framework by utilizing one Libinvent of the core modules Reinvent to generate 936 ligand molecules in total;
2) Converting the ligand molecules from SMILES format to 3D format by using RDKit library of Python for pretreatment, adding polar hydrogen and Gasteiger charges to the ligand molecules by using LigPrep module of Schrodinger Maestro software package to enable the ligand molecules to dock with receptor molecules, downloading a sclerostin protein structure (PDB ID:2K 8P) from RCSB protein database, preparing initial structure and parameters of docking by using LigPrep module of Maestro, adding charges and hydrogen atoms, and fusing nonpolar hydrogen atoms on the sclerostin protein structure to complete protein pretreatment;
3) Molecular docking was performed using a Maistro Glide module, with a docking site selected for target region loop3 of sclerostin protein, centered at (16.68,14.43, -11.62), and docking precision XP. For each incoming small molecule ligand, the conformation with the highest binding energy of 1 is selected for output, which conformation and binding energy are used for subsequent analysis, and compound 190 and compound 669 are finally screened out of 936 molecules as candidate compounds for subsequent activity testing.
FIG. 3 is a schematic diagram showing three-dimensional binding patterns of compound 669 (a) and compound 190 (b) with a target region loop3 of sclerostin protein, wherein the green bars represent molecular structures of compound 669 and compound 190, the yellow dotted lines represent hydrogen bonds, the black dotted lines represent cation-pi interactions, and the purple dotted lines represent electrostatic interactions. As can be seen from FIG. 3, the compound 190 and the compound 669 have optimal binding modes with the target region loop3 of the sclerostin protein, one end of the ligand with carboxyl forms more hydrogen bonds and ionic interactions with Arg115 of loop3 and His59 of loop1, and the other end of the ligand has a structure of one aromatic ring which forms cation-pi interactions with the side chain of Arg118 of loop3 and forms hydrogen bonds interactions with the main chain of the ligand.
Table 1 docking scoring of compound 190 and compound 669 in example 1
Example 2
The procedure and synthetic route for the synthesis of compound 190 in this example is shown below:
S11, 2.18g of 2-bromo-4-nitrophenol, 2.16g of 4-methoxycarbonyl phenylboronic acid, 0.73g of 1,1' -bis-diphenylphosphine ferrocene palladium dichloride and 2.76g of potassium carbonate are weighed and added into a 100mL flask, under the protection of nitrogen, 25mL of ultra-dry N, N-dimethylformamide is added into the flask, the flask is placed at 100 ℃ for reaction for 12 hours, after the reaction is finished, the reaction liquid is extracted for 3 times by ethyl acetate, the organic phase is dried by anhydrous sodium sulfate, the solvent is removed by reduced pressure rotary evaporation, and the intermediate (a) is obtained by purification through rapid column chromatography (petroleum ether/ethyl acetate) with the yield of 62%;
S21, weighing 2.73g of intermediate (a), 2.12g of ammonium chloride and 2.20g of iron powder, adding 25mL of absolute ethyl alcohol and 5mL of water into a 100mL flask, then placing the flask at 80 ℃ for reaction for 4 hours, filtering a reaction system to remove residual iron powder in the reaction system after the reaction is finished, removing ethanol by reduced pressure rotary evaporation, extracting a reaction solution with ethyl acetate for 3 times, drying an organic phase with anhydrous sodium sulfate, removing a solvent by reduced pressure rotary evaporation, and purifying by flash column chromatography (petroleum ether/ethyl acetate) to obtain the intermediate (b) with the yield of 81%;
S31, weighing 2.43g of intermediate (b), 2.80g of m-fluorobenzoic acid and 3.90g of N, N-Diisopropylethylamine (DIPEA) into a 100mL flask, adding 25mL of ultra-dry N, N-dimethylformamide into the flask, stirring the flask at 25 ℃ for 30min, then adding 3.80g of urea N, N, N ', N' -tetramethyl-O- (7-azabenzotriazole-1-yl) Hexafluorophosphate (HATU) into the flask, continuing to react at 25 ℃ for 12h, extracting the reaction solution with ethyl acetate for 3 times after the reaction is finished, drying the organic phase with anhydrous sodium sulfate, removing the solvent by reduced pressure spin evaporation, and purifying by flash column chromatography (petroleum ether/ethyl acetate) to obtain the intermediate (C) with the yield of 67%;
S41, weighing 3.65g of the intermediate (C) and 0.69g of lithium hydroxide, adding the 3.65g of the intermediate and the 0.69g of lithium hydroxide into a 100mL flask, adding 25mL of anhydrous methanol and 5mL of water into the flask, then placing the flask at 25 ℃ for reaction for 12h, removing the methanol by reduced pressure rotary evaporation after the reaction is finished, adjusting the pH value to 3-4 by using 1mol/L hydrochloric acid, filtering the reactant, and washing and drying the obtained filter cake under reduced pressure to obtain an intermediate (d) with the yield of 86%;
S51, weighing 3.51g of the intermediate (d), 2.51g of glycine methyl ester hydrochloride and 3.90g of N, N-Diisopropylethylamine (DIPEA) into a 100mL flask, adding 25mL of ultra-dry N, N-dimethylformamide into the flask, stirring the flask at 25 ℃ for 30min, then adding 3.80g of urea N, N, N ', N' -tetramethyl-O- (7-azabenzotriazol-1-yl) Hexafluorophosphate (HATU) into the flask, continuing to react at 25 ℃ for 12h, extracting the reaction solution with ethyl acetate for 3 times after the reaction is finished, drying the organic phase with anhydrous sodium sulfate, removing the solvent by reduced pressure spin evaporation, and purifying by flash column chromatography (petroleum ether/ethyl acetate) to obtain the intermediate (e) with the yield of 53%;
S61, weighing 4.22g of the intermediate (e) and 0.69g of lithium hydroxide, adding the mixture into a 100mL flask, adding 25mL of anhydrous methanol and 5mL of water into the flask, then placing the flask at 25 ℃ for reaction for 12 hours, removing the methanol by reduced pressure rotary evaporation after the reaction is finished, adjusting the pH value to 3-4 by using 1mol/L hydrochloric acid, filtering the reactant, washing the obtained filter cake with water and drying the filter cake under reduced pressure to obtain a compound 190 white solid with the yield of 32%.
Compound 190 in example 2 was characterized by 1 HNMR and 13 C NMR using nuclear magnetic resonance, resulting in:
1H NMR(500MHz,DMSO)δ12.62(s,1H),10.20(s,1H),9.63(s,1H),8.87(d,J＝4.4Hz,1H),7.96–7.56(m,9H),7.45–7.42(m,1H),6.99–6.95(m,1H),3.97(s,2H).;
13C 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..
example 3
The synthetic compound 669 of this example is shown below in the steps and synthetic route:
S11, 2.18g of 2-bromo-4-nitrophenol, 2.16g of 4-methoxycarbonyl phenylboronic acid, 0.73g of 1,1' -bis-diphenylphosphine ferrocene palladium dichloride and 2.76g of potassium carbonate are weighed and added into a 100mL flask, under the protection of nitrogen, 25mL of ultra-dry N, N-dimethylformamide is added into the flask, the flask is placed at 100 ℃ for reaction for 12 hours, after the reaction is finished, the reaction liquid is extracted for 3 times by ethyl acetate, the organic phase is dried by anhydrous sodium sulfate, the solvent is removed by reduced pressure rotary evaporation, and the intermediate (a) is obtained by purification through rapid column chromatography (petroleum ether/ethyl acetate) with the yield of 62%;
S21, weighing 2.73g of intermediate (a), 2.12g of ammonium chloride and 2.20g of iron powder, adding 25mL of absolute ethyl alcohol and 5mL of water into a 100mL flask, then placing the flask at 80 ℃ for reaction for 4 hours, filtering a reaction system to remove residual iron powder in the reaction system after the reaction is finished, removing ethanol by reduced pressure rotary evaporation, extracting a reaction solution with ethyl acetate for 3 times, drying an organic phase with anhydrous sodium sulfate, removing a solvent by reduced pressure rotary evaporation, and purifying by flash column chromatography (petroleum ether/ethyl acetate) to obtain the intermediate (b) with the yield of 81%;
s31, weighing 2.43g of intermediate (b), 2.72g of p-methylbenzoic acid and 3.90g of N, N-Diisopropylethylamine (DIPEA) into a 100mL flask, adding 25mL of ultra-dry N, N-dimethylformamide into the flask, stirring the flask at 25 ℃ for 30min, then adding 3.80g of urea N, N, N ', N' -tetramethyl-O- (7-azabenzotriazole-1-yl) Hexafluorophosphate (HATU) into the flask, continuing to react at 25 ℃ for 12h, extracting the reaction solution with ethyl acetate for 3 times after the reaction is finished, drying the organic phase with anhydrous sodium sulfate, removing the solvent by reduced pressure spin evaporation, and purifying by flash column chromatography (petroleum ether/ethyl acetate) to obtain the intermediate (C) with the yield of 79%;
S41, weighing 3.61g of the intermediate (C) and 0.69g of lithium hydroxide, adding the 3.61g of the intermediate and the 0.69g of lithium hydroxide into a 100mL flask, adding 25mL of anhydrous methanol and 5mL of water into the flask, then placing the flask at 25 ℃ for reaction for 12h, removing the methanol by reduced pressure rotary evaporation after the reaction is finished, adjusting the pH value to 3-4 by using 1mol/L hydrochloric acid, filtering the reactant, and washing and drying the obtained filter cake under reduced pressure to obtain an intermediate (d) with the yield of 82%;
S51, weighing 3.47g of intermediate (d), 2.51g of glycine methyl ester hydrochloride and 3.90g of N, N-Diisopropylethylamine (DIPEA) into a 100mL flask, adding 25mL of ultra-dry N, N-dimethylformamide into the flask, stirring the flask at 25 ℃ for 30min, then adding 3.80g of urea N, N, N ', N' -tetramethyl-O- (7-azabenzotriazol-1-yl) Hexafluorophosphate (HATU) into the flask, continuing to react at 25 ℃ for 12h, extracting the reaction solution with ethyl acetate for 3 times after the reaction is finished, drying the organic phase with anhydrous sodium sulfate, removing the solvent by reduced pressure spin evaporation, and purifying by flash column chromatography (petroleum ether/ethyl acetate) to obtain the intermediate (e) with the yield of 57%;
S61, weighing 4.18g of the intermediate (e) and 0.69g of lithium hydroxide, adding the mixture into a 100mL flask, adding 25mL of anhydrous methanol and 5mL of water into the flask, then placing the flask at a temperature of 25 ℃ for reaction for 12h, removing the methanol by reduced pressure rotary evaporation after the reaction is finished, adjusting the pH value to 3-4 by using 1mol/L hydrochloric acid, filtering the reactant, and washing and drying the obtained filter cake under reduced pressure to obtain a white solid of the compound 669, wherein the yield is 43%.
The compound 669 in example 3 was characterized by 1 H NMR and 13 C NMR using nuclear magnetic resonance, resulting in:
1H NMR(500MHz,DMSO)δ12.62(s,1H),10.03(s,1H),9.57(s,1H),8.85(t,J＝5.83Hz,1H),7.94(s,1H),7.92(s,1H),7.89(s,1H),7.87(s,1H),7.73(d,J＝2.30Hz,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).;
13C NMR(125MHz,DMSO)δ171.87,166.80,165.31,151.14,142.00,141.79,132.62,132.40,131.86,129.35,129.33,128.01,127.46,126.82,123.28,122.21,116.50,41.74,21.47..
Example 4
This example demonstrates the targeting of compound 190 prepared in example 2, and compound 669 prepared in example 3 to a specific domain of sclerostin:
Experimental materials:
DMEM complete medium DMEM medium containing 10% Fetal Bovine Serum (FBS) and 1% penicillin-streptomycin;
MEM-alpha complete medium MEM-alpha medium containing 10% Fetal Bovine Serum (FBS) and 1% penicillin-streptomycin;
Cell signaling pathway HEK293 cells (human embryonic kidney cells 293) transfected with Wnt1 protein and full length human recombinant sclerostin protein (FLhSOST), respectively.
The experimental method comprises the following steps:
HEK293 cells were cultured in DMEM medium containing 10% Fetal Bovine Serum (FBS) and 1% penicillin-streptomycin at 37 ℃ under humid atmospheric conditions, cells were seeded in 96-well plates at a density of 5×10 4 cells/well in 100 μl DMEM complete medium, cultured for 24h, the experiments were divided into 3 groups:
Wnt1+Veh group Wnt1 protein plasmid (25 ng per well) was co-transfected into cells using Lipofectamine 2000 (0.35. Mu.L per well), during which each 25ng plasmid and 0.35. Mu.L Lipofectamine 2000 were mixed and incubated for 20min in 30. Mu. L OptiMem medium, then 70. Mu.L of DMEM medium without serum and antibiotics were added, gently mixed to obtain a transfection mixture, then the old medium was removed, the transfection mixture was added to a well plate containing cells, wnt1 protein plasmid was transfected for 12h, the transfection mixture was removed to obtain transfected cells, and 100. Mu.L of DMEM complete medium was added to treat cells for 12h;
Wnt1+ FL hSOST +Veh group Wnt1 protein plasmid (25 ng per well) and FL hSOST plasmid (25 ng per well) were co-transfected into cells using Lipofectamine 2000 (0.35. Mu.L per well), during which each 25ngWnt1 protein plasmid, 25ng FL hSOST plasmid and 0.35. Mu.L Lipofectamine 2000 were mixed and incubated in 30. Mu. L OptiMem medium for 20min, then 70. Mu.L of DMEM medium without serum and antibiotics was added, gently mixed to obtain a transfection mixture, then old medium was removed, the transfection mixture was added to a well plate containing cells, wnt1 protein plasmid and FL hSOST plasmid were transfected for 12h, the transfection mixture was removed to obtain transfected cells, and 100. Mu.L of DMEM complete medium was added to treat cells for 12h;
Wnt1+ FLhSOST +190/669 groups Wnt1 protein plasmid (25 ng per well) and FLhSOST plasmid (25 ng per well), were co-transfected into cells using Lipofectamine 2000 (0.35. Mu.L per well), during which each 25. 25ngWnt1 protein plasmid, 25. 25ng FLhSOST plasmid and 0.35. Mu.L Lipofectamine 2000 were mixed and incubated in 30. Mu. L OptiMem medium for 20min, then 70. Mu.L of DMEM medium without serum and antibiotics was added, gently mixed to obtain a transfection mixture, then the old medium was removed, the transfection mixture was added to a well plate containing cells, after transfection of Wnt1 protein plasmid and FLhSOST plasmid, the transfection mixture was removed to obtain transfected cells, and 100. Mu. LDMEM complete medium containing 5. Mu. Mol/L of compound 190/669 (dissolved in phosphate buffer containing 1-fold concentration of 5%) was added to treat cells for 12h;
after the above-mentioned grouping treatment, the medium was removed, and the cells were lysed in 100. Mu.L of 1 Xinactive lysis buffer (PLB) by shaking in a shaking incubator at room temperature for 15min, respectively. Then, 15. Mu.L of the lysate was transferred to a black 96-well plate, parameters were set according to the Dual Luciferase Dual-Luciferase reporter assay System (Dual-Luciferase ReporterAssay System) manufacturer's protocol, luciferase activity was measured using the SpectraMax i3X multifunctional microplate reader system (MD SpectraMax i3X Multi-Mode Microplate Reader), and Wnt signal expression was measured.
Fig. 4 is a statistical plot of the effect of compounds 190 and 669 prepared in examples 2 and 3 on Wnt signaling, and as can be seen from fig. 4, wnt signaling is significantly higher in both compound 190 and compound 669 treated cells than in cells not treated with either compound 190 or 669, wnt signaling plays a critical role in bone development and bone homeostasis, and this decrease in signaling may lead to decreased bone density, osteoporosis and increased risk of fracture, thus higher Wnt signaling suggests that compound 190 and compound 669 are beneficial in restoring the effects of Wnt signaling in promoting bone formation.
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| US20210179583A1 (en) * | 2017-12-15 | 2021-06-17 | H Lee Mofftt Cancer Center And Research Institute, Inc. | Inhibitors for the b-catenin/b-cell lymphoma 9 (bcl9) protein-protein interaction |
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