Application of compound for interfering interaction of beta3 / Src of integrin

Abstract

The invention relates to application of a compound for interfering interactions of integrin beta3 / Src. Specifically, the invention provides application of a compound as shown in a formula I, or an optical isomer or a raceme thereof, or a solvate thereof, or a pharmaceutically acceptable salt thereof, which is used for preparing a composition or a preparation, and also provides application of the compound as shown in the formula I, or the optical isomer or the raceme thereof, or the solvate thereof, or the pharmaceutically acceptable salt thereof. The composition or the preparation is used for one or more applications selected from the following groups: (1) selectively inhibiting the outward-to-inward signal transduction mediated by interaction of integrin beta3 and Src kinase; (2) preventing and / or treating diseases related to outward-to-inward signal transduction mediated by interaction of integrin beta3 and Src kinase; (3) preventing and / or treating thrombus; (4) preventing and / or treating tumors; (5) preventing and / or treating osteoporosis; and (6) preventing and / or treating endothelial cell mediated vascularization.

Description

[0001] This application is a divisional application with an application date of July 24, 2020, application number 202010724483.5, and invention name “Use of a compound that interferes with integrin β3 / Src interaction”. Technical Field

[0002] The present invention relates to the field of medicine, and in particular to the use of a compound that interferes with the interaction of integrin β3 / Src. Background Art

[0003] At present, cardiovascular and cerebrovascular thrombotic diseases such as myocardial infarction and cerebral infarction have become the main causes of serious threats to human life and health, with high morbidity and mortality. According to statistics from the World Health Organization, about 12 million people die prematurely from cardiovascular and cerebrovascular diseases each year. The mortality rate of cardiovascular and cerebrovascular diseases ranks first among all diseases, and the number of people who die from cardiovascular and cerebrovascular diseases each year accounts for about 1 / 3 of the global death toll. Platelets play a key role in cardiovascular and cerebrovascular thrombosis. When the vascular endothelium is damaged or the atherosclerotic plaque ruptures, platelets are activated and cause pathological thrombosis through a series of functions such as adhesion, extension, and aggregation, causing ischemic necrosis of the heart and brain tissues in the affected blood vessel distribution area, which can seriously endanger the patient's life. Therefore, antiplatelet therapy has become the first choice for the treatment of cardiovascular and cerebrovascular thrombotic diseases. The most direct cause of pathological thrombosis in thrombotic diseases is abnormal activation and aggregation of platelets. The final common pathway of platelet aggregation and thrombosis caused by various platelet agonists in the body is mediated by integrin αIIbβ3 (GPIIb / IIIa). Therefore, integrin αIIbβ3 has become an ideal target for antiplatelet therapy. However, the difficulty of current antithrombotic drugs is that they inhibit normal physiological hemostasis while antithrombotic, often leading to bleeding side effects.

[0004] Integrin αIIbβ3 belongs to the integrin family. Integrins are a class of transmembrane proteins widely present in cells. They mediate the interaction between cells (by binding to cadherins and selectins) and between cells and extracellular matrix (by binding to extracellular matrix such as fibronectin, collagen, and laminin), and participate in a series of physiological and pathological processes such as cell signal transduction, adhesion, extension, tumor migration, hemostasis, and thrombosis. Integrins are heterodimers formed by α subunits and β subunits. Currently, 18 α subunits have been found in mammals, which together with 8 β subunits form 24 different integrins. Among them, integrin β3 can form two integrins with αIIb and αv subunits: αIIbβ3 and αvβ3. Integrin αIIbβ3 is mainly expressed on the surface of platelets and megakaryocytes. It is the main membrane receptor on the surface of platelets and plays an important role in thrombosis and hemostasis. Integrin αvβ3 is widely distributed in the body, such as endothelial cells, osteoclasts, tumor cells, smooth muscle cells, fibroblasts, etc., mediating various pathophysiological processes such as angiogenesis, bone resorption, and tumor metastasis.

[0005] Therefore, there is a need in the art to develop a drug targeting integrin targets, so that the drug does not affect the normal physiological hemostasis function while resisting thrombosis, avoiding the occurrence of bleeding side effects, and at the same time targeting integrin targets enables the drug to have multiple therapeutic effects. Summary of the invention

[0006] The purpose of the present invention is to provide a drug targeting the interaction between integrin and Src kinase, thereby having excellent therapeutic effects on diseases such as thrombosis, tumor, osteoporosis and endothelial cell-mediated angiogenesis.

[0007] In a first aspect, the present invention provides a use of a compound of formula I, or an optical isomer or a racemate thereof, or a solvate thereof, or a pharmaceutically acceptable salt thereof, for preparing a composition or a preparation, wherein the composition or preparation is used for one or more uses selected from the following groups: (1) selectively inhibiting outside-in signal transduction mediated by the interaction between integrin β3 and Src kinase; (2) preventing and / or treating diseases related to outside-in signal transduction mediated by the interaction between integrin β3 and Src kinase; (3) preventing and / or treating thrombus ; (4) prevention and / or treatment of tumors; (5) prevention and / or treatment of osteoporosis; (6) prevention and / or treatment of endothelial cell-mediated angiogenesis;

[0008]

[0009] in,

[0010] R1 is hydrogen, substituted or unsubstituted C1-C12 alkyl, substituted or unsubstituted C3-C12 cycloalkyl, substituted or unsubstituted C6-C16 aryl, substituted or unsubstituted 3-12 membered heteroaryl, -substituted or unsubstituted C1-C12 alkylene-C6-C16 aryl, -substituted or unsubstituted C1-C12 alkylene-3-12 membered heteroaryl;

[0011] R2 is substituted or unsubstituted C1-C12 alkyl, substituted or unsubstituted C3-C12 cycloalkyl, isothiourea, guanidinyl, substituted or unsubstituted C1-C12 alkylene-isothiourea, substituted or unsubstituted C1-C12 alkylene-guanidinyl, substituted or unsubstituted C3-C12 cycloalkylene-isothiourea, or substituted or unsubstituted C3-C12 cycloalkylene-guanidinyl;

[0012] R3 is hydrogen, substituted or unsubstituted C1-C12 alkyl, substituted or unsubstituted C3-C12 cycloalkyl, or substituted or unsubstituted C2-C12 ester, or substituted or unsubstituted C2-C12 acyl;

[0013] R4 is hydrogen, substituted or unsubstituted C1-C12 alkyl, substituted or unsubstituted C3-C12 cycloalkyl, substituted or unsubstituted C2-C12 ester, substituted or unsubstituted C1-C12 alkoxy, substituted or unsubstituted C1-C12 alkylthio, substituted or unsubstituted C3-C12 cycloalkyloxy, or substituted or unsubstituted C3-C12 cycloalkylthio;

[0014] R5 is hydrogen, substituted or unsubstituted C1-C12 alkyl, substituted or unsubstituted C3-C12 cycloalkyl, halogen, substituted or unsubstituted C6-C16 aryl, or substituted or unsubstituted 3-12 membered heteroaryl;

[0015] W and Z are each independently CH, C-R6, or N;

[0016] R6 is substituted or unsubstituted C1-C12 alkyl, substituted or unsubstituted C3-C12 cycloalkyl, halogen, substituted or unsubstituted C6-C16 aryl, or substituted or unsubstituted 3-12 membered heteroaryl;

[0017] Wherein, any "substituted" means that one or more (preferably 1, 2, 3, 4 or 5) hydrogen atoms on the group are replaced by a substituent selected from the group consisting of C1-C8 alkyl, C3-C8 cycloalkyl, C1-C8 haloalkyl, C3-C8 halocycloalkyl, halogen, nitro, -CN, hydroxyl, thiol, amino, C1-C4 carboxyl, C2-C4 ester, C2-C4 amide, C1-C8 alkoxy, C1-C8 alkylthio, C1-C8 haloalkoxy, C1-C8 haloalkylthio, C6-C12 aryl, 5-10 membered heteroaryl, 5-10 membered heterocycloalkyl;

[0018] The heterocyclic ring of the heteroaryl group has 1 to 4 (preferably 1, 2, 3 or 4) heteroatoms selected from N, O and S.

[0019] In another preferred embodiment, at least one of R4 and R5 is not hydrogen.

[0020] In another preferred embodiment, R1 is hydrogen, substituted or unsubstituted C1-C8 alkyl, substituted or unsubstituted C3-C10 cycloalkyl, substituted or unsubstituted C6-C12 aryl, substituted or unsubstituted 3-10 membered heteroaryl, substituted or unsubstituted C1-C8 alkyl-C6-C12 aryl, substituted or unsubstituted C1-C8 alkyl-3-10 membered heteroaryl.

[0021] In another preferred embodiment, R1 is hydrogen, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C3-C8 cycloalkyl, substituted or unsubstituted C6-C12 aryl, substituted or unsubstituted 3-10 membered heteroaryl, -substituted or unsubstituted C1-C6 alkylene-C6-C12 aryl, -substituted or unsubstituted C1-C6 alkylene-3-10 membered heteroaryl.

[0022] In another preferred embodiment, R1 is hydrogen, substituted or unsubstituted C1-C4 alkyl, substituted or unsubstituted C3-C8 cycloalkyl, substituted or unsubstituted C6-C12 aryl, substituted or unsubstituted 3-10 membered heteroaryl, -substituted or unsubstituted C1-C4 alkylene-C6-C12 aryl, -substituted or unsubstituted C1-C4 alkylene-3-10 membered heteroaryl.

[0023] In another preferred embodiment, R1 is methyl or benzyl.

[0024] In another preferred embodiment, R2 is substituted or unsubstituted C1-C8 alkyl, substituted or unsubstituted C3-C8 cycloalkyl, isothiourea, guanidinyl, -substituted or unsubstituted C1-C8 alkylene-isothiourea, -substituted or unsubstituted C1-C8 alkylene-guanidinyl, -substituted or unsubstituted C3-C8 cycloalkylene-isothiourea, or -substituted or unsubstituted C3-C8 cycloalkylene-guanidinyl.

[0025] In another preferred embodiment, R2 is substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C3-C8 cycloalkyl, isothiourea, guanidinyl, -substituted or unsubstituted C1-C6 alkylene-isothiourea, -substituted or unsubstituted C1-C6 alkylene-guanidinyl, -substituted or unsubstituted C3-C8 cycloalkylene-isothiourea, or -substituted or unsubstituted C3-C8 cycloalkylene-guanidinyl.

[0026] In another preferred embodiment, R2 is substituted or unsubstituted C1-C4 alkyl, substituted or unsubstituted C3-C8 cycloalkyl, isothiourea, guanidinyl, -substituted or unsubstituted C1-C4 alkylene-isothiourea, -substituted or unsubstituted C1-C4 alkylene-guanidinyl, -substituted or unsubstituted C3-C8 cycloalkylene-isothiourea, -or substituted or unsubstituted C3-C8 cycloalkylene-guanidinyl.

[0027] In another preferred embodiment, R2 is substituted or unsubstituted methyl-isothiourea, or methyl-guanidinyl.

[0028] In another preferred embodiment, the structural formula of the isothiourea group is as follows:

[0029]

[0030] In another preferred embodiment, the structural formula of the guanidine group is as follows:

[0031]

[0032] In another preferred embodiment, R3 is hydrogen, substituted or unsubstituted C1-C8 alkyl, substituted or unsubstituted C3-C10 cycloalkyl, or substituted or unsubstituted C2-C8 ester, or substituted or unsubstituted C2-C8 acyl.

[0033] In another preferred embodiment, R3 is hydrogen, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C3-C8 cycloalkyl, or substituted or unsubstituted C2-C6 ester, or substituted or unsubstituted C2-C6 acyl.

[0034] In another preferred embodiment, R3 is hydrogen, substituted or unsubstituted C1-C4 alkyl, substituted or unsubstituted C3-C8 cycloalkyl, or substituted or unsubstituted C2-C4 ester, or substituted or unsubstituted C2-C4 acyl.

[0035] In another preferred embodiment, R3 is ethyl ester, propyl ester, or butyl ester.

[0036] In another preferred embodiment, R3 is C1-C8 alkyl-OC(O)- or C1-C8 alkyl-C(O)-O-.

[0037] In another preferred embodiment, R3 is C1-C6 alkyl-OC(O)- or C1-C6 alkyl-C(O)-O-.

[0038] In another preferred embodiment, R3 is C1-C4 alkyl-OC(O)- or C1-C4 alkyl-C(O)-O-.

[0039] In another preferred embodiment, R3 is C1-C3 alkyl-OC(O)- or C1-C3 alkyl-C(O)-O-.

[0040] In another preferred embodiment, R3 is ethyl-OC(O)- or ethyl-C(O)-O-.

[0041] In another preferred embodiment, R4 is hydrogen, substituted or unsubstituted C1-C8 alkyl, substituted or unsubstituted C3-C10 cycloalkyl, substituted or unsubstituted C2-C8 ester, substituted or unsubstituted C1-C8 alkoxy, substituted or unsubstituted C1-C8 alkylthio, substituted or unsubstituted C3-C10 cycloalkyloxy, or substituted or unsubstituted C3-C10 cycloalkylthio.

[0042] In another preferred embodiment, R4 is hydrogen, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C3-C8 cycloalkyl, substituted or unsubstituted C2-C6 ester, substituted or unsubstituted C1-C6 alkoxy, substituted or unsubstituted C1-C6 alkylthio, substituted or unsubstituted C3-C8 cycloalkyloxy, or substituted or unsubstituted C3-C8 cycloalkylthio.

[0043] In another preferred embodiment, R4 is hydrogen, substituted or unsubstituted C1-C4 alkyl, substituted or unsubstituted C3-C8 cycloalkyl, substituted or unsubstituted C2-C4 ester, substituted or unsubstituted C1-C4 alkoxy, substituted or unsubstituted C1-C4 alkylthio, substituted or unsubstituted C3-C8 cycloalkyloxy, or substituted or unsubstituted C3-C8 cycloalkylthio.

[0044] In another preferred embodiment, R4 is hydrogen, substituted or unsubstituted C1-C4 alkyl, substituted or unsubstituted C5-C7 cycloalkyl, substituted or unsubstituted C2-C4 ester, substituted or unsubstituted C1-C4 alkoxy, substituted or unsubstituted C1-C4 alkylthio, substituted or unsubstituted C5-C7 cycloalkyloxy, or substituted or unsubstituted C5-C7 cycloalkylthio.

[0045] In another preferred example, R4 is hydrogen, C1-C6 alkyl-OC(O)-, C1-C6 alkyl-C(O)-O-, C1-C6 alkyl-O-, C1-C6 alkyl-S-, C3-C8 cycloalkyl-O-, or C3-C8 cycloalkyl-S-.

[0046] In another preferred example, R4 is hydrogen, C1-C4 alkyl-OC(O)-, C1-C4 alkyl-C(O)-O-, C1-C4 alkyl-O-, C1-C4 alkyl-S-, C5-C7 cycloalkyl-O-, or C5-C7 cycloalkyl-S-.

[0047] In another preferred embodiment, R4 is hydrogen, ethoxy, propyl, or butyl, methoxy, methylthio, cyclopentyl-O-, cyclopentyl-S-, cyclohexyl-O-, or cyclohexyl-S-.

[0048] In another preferred embodiment, R4 is hydrogen, methyl-OC(O)-, methyl-C(O)-O-, propyl group, or butyl group, methoxy group, methylthio group, cyclopentyl-O-, cyclopentyl-S-, cyclohexyl-O-, or cyclohexyl-S-.

[0049] In another preferred embodiment, R5 is hydrogen, substituted or unsubstituted C1-C8 alkyl, substituted or unsubstituted C3-C10 cycloalkyl, halogen, substituted or unsubstituted C6-C12 aryl, or substituted or unsubstituted 3-10 membered heteroaryl;

[0050] In another preferred embodiment, R5 is hydrogen, halogen, phenyl, naphthyl, quinolyl, isoquinolyl, C1-C4 alkyl-substituted quinolyl, C1-C4 alkyl-substituted isoquinolyl.

[0051] In another preferred embodiment, R5 is hydrogen, chlorine, bromine, phenyl, naphthyl, quinolyl, isoquinolyl, monomethyl-substituted quinolyl, or monomethyl-substituted isoquinolyl.

[0052] In another preferred embodiment, the halogen is fluorine, chlorine, bromine, or iodine.

[0053] In another preferred embodiment, W and Z are each independently CH, C-R6, or N;

[0054] R6 is substituted or unsubstituted C1-C4 alkyl, substituted or unsubstituted C3-C6 cycloalkyl, halogen, substituted or unsubstituted C6-C12 aryl, or substituted or unsubstituted 3-10 membered heteroaryl.

[0055] In another preferred embodiment, W and Z are not -N- at the same time.

[0056] In another preferred embodiment, W and Z are each independently -CH-.

[0057] In another preferred embodiment, the compound of formula I has a structure as shown in the following formula I-1.

[0058]

[0059] in,

[0060] R1, R3, R4 and R5 are each independently as defined above;

[0061] A is S or NH.

[0062] In another preferred embodiment, the compound of formula I is:

[0063]

[0064] In another preferred embodiment, the inhibiting the interaction between integrin β3 and Src kinase comprises inhibiting the interaction between integrin β3 and the SH3 domain of Src kinase.

[0065] In another preferred embodiment, the selective inhibition of outside-in signal transduction mediated by the interaction between integrin β3 and Src kinase refers to the selective inhibition of outside-in signal transduction mediated by the interaction between integrin β3 and Src kinase but without affecting inside-out signal transduction.

[0066] In another preferred embodiment, the interaction between integrin β3 and Src kinase refers to the interaction between integrin β3 and c-Src kinase in platelets.

[0067] In another preferred embodiment, the integrin β3 is β3 of integrin αIIbβ3 and / or integrin αvβ3.

[0068] In another preferred embodiment, the disease associated with outside-in signal transduction mediated by the interaction between integrin β3 and Src kinase is selected from the group consisting of thrombosis, tumor, osteoporosis, endothelial cell-mediated angiogenesis, or a combination thereof.

[0069] In another preferred embodiment, the prevention and / or treatment of thrombosis does not affect or improve bleeding while achieving anti-thrombotic effects.

[0070] In another preferred embodiment, the improvement of bleeding includes inhibiting bleeding, not increasing the risk of bleeding, reducing the risk of bleeding, not causing bleeding side effects and / or not affecting the hemostatic function.

[0071] In another preferred embodiment, the hemostatic function includes platelet hemostatic function.

[0072] In another preferred embodiment, the hemostasis includes physiological hemostasis.

[0073] In another preferred embodiment, the thrombus is a cardiovascular and cerebrovascular disease thrombus, and the thrombus is a cardiovascular and cerebrovascular disease thrombus selected from the following group: myocardial infarction thrombus, cerebral infarction thrombus, ischemic stroke, atherosclerotic thrombus, or a combination thereof.

[0074] In another preferred embodiment, the prevention and / or treatment of thrombosis comprises one or more methods selected from the following group:

[0075] (3-1) Inhibit the stretching function of platelets on solid fibrinogen;

[0076] (3-2) inhibiting platelet aggregation, adhesion and / or extension;

[0077] (3-3) Inhibits the retraction of fibrin clots;

[0078] (3-3) Does not affect the platelet binding to free fibrinogen;

[0079] In another preferred embodiment, the inhibition of platelet aggregation includes inhibiting one-phase aggregation or two-phase aggregation of platelets.

[0080] In another preferred embodiment, the prevention and / or treatment of tumors comprises one or more methods selected from the following group:

[0081] (4-1) Inhibit the growth or proliferation of tumor cells;

[0082] (4-2) Inhibit the formation or growth of tumor blood vessels;

[0083] In another preferred embodiment, the tumor is selected from the following group: solid tumors, hematological tumors, or a combination thereof.

[0084] In another preferred embodiment, the solid tumor is selected from the group consisting of lung cancer, breast cancer, melanoma, gastric cancer, or a combination thereof.

[0085] In another preferred embodiment, the tumor is selected from the following group: lung cancer, breast cancer, melanoma, gastric cancer, or a combination thereof.

[0086] In another preferred embodiment, the tumor is selected from the following group: lung cancer, breast cancer, melanoma, gastric cancer, or a combination thereof.

[0087] In another preferred embodiment, the lung cancer is selected from the group consisting of small cell lung cancer, non-small cell lung cancer, myocardial cancer, or a combination thereof.

[0088] In another preferred embodiment, the prevention and / or treatment of osteoporosis comprises one or more methods selected from the following group:

[0089] (5-1) Inhibit the proliferation and differentiation of osteoclasts.

[0090] In another preferred embodiment, the prevention and / or treatment of endothelial cell-mediated angiogenesis comprises one or more methods selected from the following group:

[0091] (6-1) Inhibit the proliferation of endothelial cells;

[0092] In another preferred embodiment, the blood vessel is selected from the following group: tumor blood vessel, umbilical vein blood vessel, or a combination thereof.

[0093] In another preferred embodiment, the endothelial cells are selected from the following group: tumor vascular endothelial cells, umbilical vein vascular endothelial cells, or a combination thereof.

[0094] In another preferred embodiment, the composition or preparation is a pharmaceutical composition or preparation.

[0095] In another preferred embodiment, the composition or preparation further comprises a pharmaceutically acceptable carrier.

[0096] In another preferred embodiment, the composition or preparation further comprises other antithrombotic drugs, other anti-tumor drugs, other drugs for treating osteoporosis, and / or other anti-angiogenesis drugs.

[0097] In another preferred embodiment, the composition or preparation is in the form of tablets, injections, infusions, ointments, gels, solutions, microspheres or films.

[0098] In a second aspect, the present invention provides a method for (1) selectively inhibiting outside-in signal transduction mediated by the interaction between integrin β3 and Src kinase; (2) preventing and / or treating diseases related to outside-in signal transduction mediated by the interaction between integrin β3 and Src kinase; (3) preventing and / or treating thrombosis; (4) preventing and / or treating tumors; (5) preventing and / or treating osteoporosis; and / or (6) preventing and / or treating endothelial cell-mediated angiogenesis, the method comprising the steps of administering to a subject in need thereof a compound of formula I as described in the first aspect of the present invention, or an optical isomer or a racemate thereof, or a solvate thereof, or a pharmaceutically acceptable salt thereof.

[0099] In another preferred embodiment, the subject is a human or non-human mammal (rodent, rabbit, monkey, livestock, dog, cat, etc.).

[0100] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0101] Figure 1 Shows the screening flow chart for small molecule compounds.

[0102] Figure 2 The results show the inhibitory effect of DCDBS84 on platelet extension. DCDBS84 can inhibit platelet extension in a concentration-dependent manner. DMSO and RGDS are negative and positive controls, respectively.

[0103] Figure 3Surface plasmon resonance (SPR) detection showed that DCDBS84 could bind to c-Src SH3 and its affinity (KD).

[0104] Figure 4 It showed that different concentrations of DCDBS84 did not affect the c-Src tyrosine kinase activity, while PP2 (an inhibitor of the binding of PXXP domain-containing proteins to c-Src) and Dasatinib (a c-Src tyrosine kinase inhibitor) could inhibit the c-Src tyrosine kinase activity.

[0105] Figure 5 It was shown that DCDBS84 did not affect the interaction between RLP1 and c-Src SH3, while both PP2 and Dasatinib were able to disrupt the interaction between RLP1 and c-Src SH3.

[0106] Figure 6 The GST-pull down method was used to detect that DCDBS84 could dissociate the interaction between integrin β3 and c-Src SH3 in a concentration-dependent manner.

[0107] Figure 7 The Co-IP method was used to detect that DCDBS84 could dissociate the interaction between integrin β3 and c-Src.

[0108] Figure 8 The results showed that DCDBS84 could reduce the adhesion of platelets on solid fibrinogen in a concentration-dependent manner, while DMSO and RGDS were used as negative and positive controls, respectively.

[0109] Fig. 9 It was shown that DCDBS84 could reduce ADP-induced platelet aggregation in a concentration-dependent manner.

[0110] Fig.10 It was shown that DCDBS84 could reduce Thrombin-induced platelet aggregation in a concentration-dependent manner.

[0111] Fig.11 Photographic images showing that DCDBS84 is able to inhibit fibrin clot retraction in a concentration-dependent manner.

[0112] Fig.12 Statistical graph showing that DCDBS84 can inhibit fibrin clot retraction in a concentration-dependent manner.

[0113] Fig.13 The results showed that the binding ability of DCDBS84 to soluble fibrinogen was the same as that of DMSO, while that of RGDS was reduced.

[0114] Fig.14The results showed that in the Fecl3-induced carotid artery injury model, DCDBS84 could reduce thrombosis in a concentration-dependent manner, while the clinical dosage of the control drug integrilin was 0.18 mg / kg. When it was used at 16 mg / kg, it had serious side effects and could not be used clinically. The effect of DCDBS84 10 mg / kg was stronger than that of integrilin 16 mg / kg, indicating that DCDBS84 can deeply resist thrombosis.

[0115] Fig.15 The results showed that in the mouse tail-cutting experiment, DCDBS84 10mg / kg did not cause increased bleeding in mice (compared with the DMSO control), while integrilin 0.18mg / kg and 4.2mg / kg could significantly increase the amount of bleeding in mice. Due to the bleeding side effect, integrilin cannot be used at high doses.

[0116] Fig.16 The results showed that in the laser-induced mouse cremaster artery injury model, the DMSO-treated group was able to form very obvious thrombi, DCDBS84 4.2 mg / kg was able to significantly inhibit thrombus formation, but there was still a layer of activated platelets attached to the wound site, and although integrilin 4.2 mg / kg treatment was also able to significantly inhibit thrombus formation, there was no platelet attachment to the wound site.

[0117] Fig.17 Statistical graph showing the results of the laser-induced mouse cremaster artery injury model. CD41 is a platelet-specific antibody (αIIb), and P-selectin (CD 62P) marks activated platelets.

[0118] Fig.18 The fluid technology showed that DCDBS84 could inhibit thrombosis under flow conditions and had platelet adhesion at 1500s-1, while integrilin had no platelet adhesion when inhibiting thrombosis.

[0119] Fig.19 A statistical graph showing the results of fluid technology tests.

[0120] Fig. 20 It was shown that DCDBS84 could inhibit the adhesion of MCF-7 (breast cancer cells) transfected with β3 on fibrinogen. The adhesion of cells was reduced by β3Δ759 (RGT at the C-terminus was deleted, destroying the outside-in function). For MCF-7 without β3 expression, the cells did not adhere.

[0121] Fig.21 DCDBS84 was shown to inhibit the adhesion of MDA-MB-231 (breast cancer cells) expressing β3 on fibrinogen.

[0122] Fig. 22 It was shown that DCDBS84 had little effect on the adhesion of B16 (mouse melanoma cells, expressing β3) cells on fibrinogen.

[0123] Fig.23 It was shown that high concentrations of DCDBS84 could inhibit the growth of B16 cells in the scratch area, suggesting that it could inhibit tumor metastasis.

[0124] Fig.24 It was shown that different concentrations of DCDBS84 could inhibit the growth of MDA-MB-231 in the scratch area, suggesting that it could inhibit tumor metastasis.

[0125] Fig.25 The results showed that MCF-7 cells, which do not express β3, could not grow in the scratched area regardless of whether DCDBS84 was applied or not.

[0126] Fig.26 The results showed that MCF-7 cells were transfected with full-length β3, and DCDBS84 could inhibit the growth of MCF-7-β3 cells in the scratch area in a concentration-dependent manner, suggesting that it could inhibit tumor metastasis.

[0127] Fig. 27 The results showed that when MCF-7 cells were transfected with β3Δ759 (C-terminal RGT truncated), the growth of cells in the scratch area was less, regardless of whether DCDBS84 was applied or not, and its cell growth was between that of MCF-7 and MCF-7-β3.

[0128] Fig.28 The figure shows the comparison of cell growth of MCF-7, MCF-7-β3 and MCF-7-β3Δ759 cells in the scratch area, suggesting the important role of β3 / c-Src interaction in cell growth and adhesion.

[0129] Fig.29 It was shown that different concentrations of DCDBS84 could inhibit the growth of NIH-3T3 (mouse embryonic fibroblast cell line, expressing αvβ3) in the scratch area.

[0130] Fig.30 The results show that the inhibitory effects of different concentrations of DCDBS84 on the proliferation of three cell lines, MCF-7, MCF-7-β3 and MCF-7-β3Δ759, were compared. It had almost no effect on MCF-7, the most obvious effect on MCF-7-β3, and a weaker effect on MCF-7-β3Δ759.

[0131] Fig.31The results showed that different concentrations of DCDBS84 had an inhibitory effect on the proliferation of three cell lines: MCF-7, MDA-MB-231 and B16. It had almost no effect on MCF-7, but had an inhibitory effect on MDA-MB-231 and B16.

[0132] Fig.32 It showed that different concentrations of DCDBS84 had a certain proliferation inhibitory effect on A549 and NCI-H1975 non-small cell lung cancer cell lines.

[0133] Fig.33 The results show that DCDBS84 at different concentrations inhibits osteoclast proliferation and osteoclast differentiation.

[0134] Fig.34 It showed that different concentrations of DCDBS84 could effectively inhibit the expression of osteoclast marker genes.

[0135] Fig.35 The results show that DCDBS84 at different concentrations inhibits the proliferation of Huvec (human umbilical vein endothelial cells expressing β3).

[0136] Fig.36 The results show that DCDBS84 at different concentrations inhibits the proliferation of HL-1 (mouse cardiomyocytes expressing β3).

[0137] Fig.37 The inhibitory effects of DCDBS84 derivatives (Formula I-XII) on platelet extension are shown, with DCDBS84 serving as a control.

[0138] Fig.38 The derivatives of DCDBS84 (Formula II-XV) showed the proliferation inhibitory effect on A549 non-small cell lung cancer.

[0139] Fig.39 The derivatives of DCDBS84 (Formula II-XV) showed the proliferation inhibitory effect on MCF-7 breast cancer cells.

[0140] Fig.40 The results show that the derivatives of DCDBS84 (Formula II-XV) have an inhibitory effect on the proliferation of MCF-7-β3 cells.

[0141] Fig.41 The derivatives of DCDBS84 (Formula II-XV) showed the inhibitory effect on the proliferation of MDA-MB-231 cells.

[0142] Fig.42 The results show that the derivatives of DCDBS84 (Formula II-XV) have an inhibitory effect on the proliferation of Huvec cells.

[0143] Fig.43The derivatives of DCDBS84 (Formula II-XV) showed the inhibitory effect on the proliferation of HL-1 cells. DETAILED DESCRIPTION

[0144] After long-term and in-depth research and extensive screening, the present inventors unexpectedly discovered that a specific class of compounds of formula I that interfere with integrin β3 / Src interaction can (1) selectively inhibit outside-in signal transduction mediated by the interaction between integrin β3 and Src kinase, (2) prevent and / or treat diseases related to outside-in signal transduction mediated by the interaction between integrin β3 and Src kinase, (3) prevent and / or treat thrombosis, (4) prevent and / or treat tumors, (5) prevent and / or treat osteoporosis, and (6) prevent and / or treat endothelial cell-mediated angiogenesis, thereby having excellent therapeutic effects.

[0145] Specifically, the experiments of the present invention show that the compound DCDBS84 can target the SH3 domain of c-Src, specifically dissociate β3 and interact with c-Src, inhibit platelet adhesion, aggregation, and fibrin clot retraction without affecting the binding to soluble fibrinogen. In the FeCl3-induced carotid artery thrombosis model, thrombosis can be deeply inhibited. In the tail-cutting experiment, the bleeding volume of mice was not increased compared with the negative control mice (DMSO treatment), while the bleeding volume of mice in the control drug Integrilin treatment group increased significantly. In the laser-induced mouse cremaster artery thrombosis model, the compound DCDBS84 can significantly inhibit platelet thrombosis, and the activated platelets can lay a layer on the wound to prevent bleeding. DMSO-treated mice can form very obvious platelet thrombi, almost blocking the entire blood vessel. The Integrilin-treated group can also significantly inhibit platelet thrombosis, but there are almost no platelets on the wound, which leads to the cause of bleeding side effects. In addition, fluid technology was used to simulate the platelet thrombus formation in vitro under the flow state of blood. Collagen-coated tubes simulated the exposure of damaged subendothelial collagen. The platelet thrombus formation was displayed by fluorescent CD41 antibody. It was found that at 5000s in the occluded artery, the platelet thrombus formation was significantly reduced. -1 Under the shear force of 1500s, the compound DCDBS84 can significantly inhibit platelet thrombus formation; -1When the shear force (equivalent to the shear force in the peripheral arteriole) is applied, the compound DCDBS84 has no obvious inhibitory effect on platelet emboli, which indicates that the compound DCDBS84 can effectively resist thrombosis while having little effect on the physiological effect of hemostasis. This result is consistent with the result of the in vivo laser-induced mouse cremaster artery thrombosis model. In summary, it can be seen that the compound of formula I of the present invention (especially the compound DCDBS84) can be used as a platelet function inhibitor targeting the SH3 domain of c-Src kinase and selectively inhibiting the regulation of platelet outside-in signal transduction, exerting an inhibitory effect on platelet thrombosis without affecting the physiological hemostasis.

[0146] In addition, the experiments of the present invention also show that the compound DCDBS84 can specifically dissociate the interaction between β3 and c-Src. As an inhibitor of the interaction between integrin β3 and c-Src (β3RGT / c-Src SH3), in addition to affecting the interaction between αIIbβ3 and c-Src in platelets, it also affects the interaction between αvβ3 and c-Src, thereby affecting the function of cells expressing αvβ3. The experimental results of the present invention show that the compound DCDBS84 has a therapeutic effect on tumors, osteoporosis and endothelial cell-mediated related diseases.

[0147] the term

[0148] As used herein, the terms "comprise", "include", and "contain" are used interchangeably and include not only closed definitions, but also semi-closed and open definitions. In other words, the terms include "consisting of", "consisting essentially of".

[0149] As used herein, "Src kinase" is Src kinase, which is a non-receptor tyrosine kinase.

[0150] As used herein, "SH3", "SH3 domain", "SH3 domain protein", and "SH3 protein" are used interchangeably.

[0151] It should be understood that one of ordinary skill in the art can select substituents and substitution patterns on the compounds of the present invention to produce chemically stable compounds that can be synthesized by techniques known in the art and the methods described below. If substituted with more than one (or more) substituent groups, it should be understood that these multiple groups can be on the same carbon or on different carbons, as long as a stable structure is produced.

[0152] As used herein, the term "substituted" or "substituted" means that a hydrogen atom on a group is replaced by a non-hydrogen atom group, but its valence requirements need to be met and the substitution generates a chemically stable compound, that is, a compound that does not spontaneously undergo transformations such as cyclization, elimination, etc.

[0153] As used herein, "R1", "R1" and "R 1 " have the same meaning and are interchangeable. Other similar definitions have the same meaning.

[0154] As used herein, Indicates the attachment site of a group.

[0155] As used herein, the term "alkyl" refers to a straight chain (i.e., unbranched) or branched saturated hydrocarbon group containing only carbon atoms, or a group of a combination of straight and branched chains. When the alkyl group is preceded by a carbon number limit (such as a C1-C10 alkyl group), it means that the alkyl group contains 1-10 carbon atoms. For example, a C1-C4 alkyl group refers to an alkyl group containing 1-4 carbon atoms. Representative examples include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, or similar groups.

[0156] As used herein, the term "alkylene" refers to an alkyl group from which a hydrogen atom is removed.

[0157] In the present invention, the term "halogen" refers to F, Cl, Br or I.

[0158] In the present invention, the term "halo" means substituted with halogen.

[0159] As used herein, the term "haloalkyl" means that one or more (preferably 1, 2, 3 or 4) hydrogen atoms of an alkyl group are substituted by halogen, and the alkyl group and halogen are as defined above. When the number of carbon atoms is limited before the alkyl group (such as C1-C6 haloalkyl), it means that the alkyl group contains 1-6 carbon atoms. For example, C1-C6 haloalkyl refers to a haloalkyl group containing 1-6 carbon atoms. Representative examples include but are not limited to -CF3, -CHF2, monofluoroisopropyl, difluorobutyl, or the like.

[0160] As used herein, the term "cycloalkyl" refers to a saturated or partially saturated unit ring, a bicyclic or polycyclic (condensed ring, bridged ring or spirocyclic) ring system group. When a cycloalkyl group is preceded by a carbon atom number limit (such as C3-C12), it means that the cycloalkyl group has 3-12 ring carbon atoms. In some preferred embodiments, the term "C3-C8 cycloalkyl" refers to a saturated or partially saturated monocyclic or bicyclic alkyl group with 3-8 ring carbon atoms, including cyclopropyl, cyclobutyl, cyclopentyl, cycloheptyl, or similar groups. "Spirocycloalkyl" refers to a bicyclic or polycyclic group in which a carbon atom (called a spiro atom) is shared between monocyclic rings, which may contain one or more double bonds, but none of the rings has a completely conjugated π electron system. "Condensed cycloalkyl" refers to a full carbon bicyclic or polycyclic group in which each ring in the system shares a pair of adjacent carbon atoms with other rings in the system, wherein one or more rings may contain one or more double bonds, but none of the rings has a completely conjugated π electron system. "Bridged cycloalkyl" refers to a full-carbon polycyclic group in which any two rings share two carbon atoms that are not directly connected, which may contain one or more double bonds, but none of the rings has a completely conjugated π electron system. The following are representative examples of cycloalkyl, including but not limited to:

[0161]

[0162] As used herein, the term "cycloalkylene" refers to a group formed by removing a hydrogen atom from a cycloalkyl group.

[0163] As used herein, the term "halocycloalkyl" refers to a cycloalkyl group in which one or more (preferably 1, 2, 3 or 4) hydrogen atoms are replaced by halogens. The cycloalkyl group and halogen are as defined above. When the cycloalkyl group is preceded by a carbon atom number limit (such as C3-C8 haloalkyl), it means that the cycloalkyl group contains 3-8 ring carbon atoms. For example, C3-C8 haloalkyl refers to a halocycloalkyl group containing 3-6 carbon atoms. Representative examples include, but are not limited to, monofluorocyclopropyl, monochlorocyclobutyl, monofluorocyclopentyl, difluorocycloheptyl, or the like.

[0164] The term "alkoxy" refers to a RO-group, where R is an alkyl group, and the alkyl group is as defined herein above, and when the alkoxy group is preceded by a carbon atom number, such as a C1-C8 alkoxy group, the alkyl group in the alkoxy group has 1-8 carbon atoms. Representative examples of alkoxy groups include, but are not limited to, methoxy, ethoxy, n-propoxy, isopropoxy, tert-butoxy, or similar groups.

[0165] As used herein, the term "alkylthio" refers to a RO-group, wherein R is an alkyl group, and the alkyl group is as defined herein above, and when the alkylthio group is preceded by a carbon atom number, such as C1-C8 alkylthio, the alkyl group in the alkylthio group has 1-8 carbon atoms. Representative examples of alkylthio groups include, but are not limited to, methylthio, ethylthio, n-propylthio, isopropylthio, tert-butylthio, or similar groups.

[0166] The term "cycloalkoxy" refers to a RO-group, wherein R is a cycloalkyl group, and the cycloalkyl group is as defined herein above, and when the cycloalkoxy group is preceded by a carbon atom number, such as a C3-C8 cycloalkoxy group, the cycloalkyl group in the cycloalkoxy group has 1-8 carbon atoms. Representative examples of alkoxy groups include, but are not limited to, cyclopropyl-O-, cyclopentyl-O-, cyclohexyl-O-, or similar groups.

[0167] The term "cycloalkylthio" refers to a RO-group, wherein R is a cycloalkyl group, and the cycloalkyl group is as defined herein above, and when the cycloalkylthio group is preceded by a carbon atom number limit, such as C3-C8 cycloalkylthio group means that the cycloalkyl group in the cycloalkylthio group has 1-8 carbon atoms. Representative examples of alkylthio groups include, but are not limited to, cyclopropyl-S-, cyclopentyl-S-, cyclohexyl-S-, or similar groups.

[0168] As used herein, the term "haloalkoxy" refers to a haloalkyl-O- group, wherein the haloalkyl group is as defined above, for example, a C1-C6 haloalkoxy group refers to a haloalkoxy group containing 1 to 6 carbon atoms, representative examples of which include but are not limited to, monofluoromethoxy, monofluoroethoxy, bisfluorobutoxy, or the like.

[0169] As used herein, the term "haloalkylthio" refers to a haloalkyl-S- group, wherein the haloalkyl group is as defined above, for example, a C1-C6 haloalkylthio group refers to a haloalkylthio group containing 1 to 4 carbon atoms, representative examples of which include but are not limited to, monofluoromethylthio, monofluoroethylthio, bisfluorobutylthio, or the like.

[0170] The term "aryl" refers to an all-carbon monocyclic or fused polycyclic (i.e., a ring that shares adjacent pairs of carbon atoms) group with a conjugated π electron system, and is an aromatic cyclic hydrocarbon compound group. When the aryl group is preceded by a carbon atom number limit, such as C6-C12 aryl, it means that the aryl group has 6-12 ring carbon atoms, such as phenyl and naphthyl. The aryl ring can be fused to other cyclic groups (including saturated or unsaturated rings), but cannot contain heteroatoms such as nitrogen, oxygen, or sulfur, and the point of connection to the parent must be on a carbon atom on the ring with a conjugated π electron system. The following are representative examples of aryl groups, including but not limited to:

[0171]

[0172] The term "heteroaryl" refers to an aromatic heterocyclic group having one to multiple (preferably 1, 2, 3 or 4) heteroatoms, which may be a single ring (monocyclic) or a polycyclic (bicyclic, tricyclic or polycyclic) fused together or covalently linked, and each heterocyclic ring containing a heteroatom may carry one or more (such as 1, 2, 3, 4) heteroatoms independently selected from the following group: oxygen, sulfur and nitrogen. When there is a number limit before the heteroaryl, it refers to the number of ring atoms of the heteroaryl. For example, a 5-12-membered heteroaryl refers to a heteroaryl having 5-12 ring atoms. Representative examples include, but are not limited to: pyrrolyl, pyrazolyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, thiadiazolyl, isothiazolyl, furanyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, triazolyl, triazolyl and tetrazolyl.

[0173] As used herein, the term "carboxyl" refers to a -COOH group or an -alkyl-COOH group, wherein the alkyl group is as defined herein above, for example, a "C2-C4 carboxyl" refers to a group of a -C1-C3 alkyl-COOH structure, and representative examples of carboxyl include (but are not limited to): -COOH, -CH2COOH, -C2H4COOH, or similar groups.

[0174] As used herein, the term "ester group" refers to a RC(O)-O- group or a -C(O)-OR group, wherein R is an alkyl group and the alkyl group is as defined herein above. For example, a "C2-C4 ester group" refers to a group with a C1-C3 alkyl-C(O)-O- structure or a group with a -C(O)-O-C1-C3 alkyl structure. Representative examples of ester groups include, but are not limited to: CH3COO-, C2H5COO-, C3H8COO-, (CH3)2CHCOO-, -COOCH3, -COOC2H5, -COOC3H8, or similar groups.

[0175] As used herein, the term "acyl" refers to a group having a RC(O)- structure, wherein R is an alkyl group, and the alkyl group is as defined herein above. For example, a "C2-C4 acyl group" refers to a group having a C1-C3 alkyl-C(O)- structure. Representative examples of ester groups include, but are not limited to, CH3C(O)-, C2H5C(O)-, C3H8C(O)-, (CH3)2CHC(O)-, or the like.

[0176] As used herein, the term "amide" refers to an R-CO-N- group or a -CO-NR group, wherein R is an alkyl group, and the alkyl group is as defined herein above. For example, a "C2-C4 amide" refers to a group with a C1-C3 alkyl-CO-N- structure or a group with a -CO-N-C1-C3 alkyl structure. Representative examples of amide groups include, but are not limited to, CH3CO-N-, C2H5CO-N-, C3H8CO-N-, (CH3)2CHCO-N-, -CO-N-CH3, -CO-N-C2H5, -CO-N-C3H8, or similar groups.

[0177] As used herein, the term "amino" by itself or as part of another substituent means -NH2.

[0178] As used herein, the term "nitro" by itself or as part of another substituent means -NO2.

[0179] As used herein, the term "hydroxy" by itself or as part of another substituent means -OH.

[0180] As used herein, the term "mercapto" by itself or as part of another substituent refers to -SH.

[0181] In this specification, it should be interpreted that all substituents are unsubstituted unless explicitly described as "substituted" in this article. The term "substituted" refers to one or more hydrogen atoms on a specific group being replaced by a specific substituent. The specific substituent is a substituent described above, or a substituent appearing in each embodiment. Preferably, the substitution refers to one or more (preferably 1, 2, 3, 4 or 5) hydrogen atoms on a ring or group being replaced by a substituent selected from the group consisting of C1-C8 alkyl, C3-C8 cycloalkyl, C1-C8 haloalkyl, C3-C8 halocycloalkyl, halogen, nitro, -CN, hydroxyl, sulfhydryl, amino, C1-C4 carboxyl, C2-C4 ester, C2-C4 amide, C1-C8 alkoxy, C1-C8 alkylthio, C1-C8 haloalkoxy, C1-C8 haloalkylthio, C6-C12 aryl, 5-10 membered heteroaryl, 5-10 membered heterocycloalkyl. Unless otherwise specified, an optionally substituted group may have a substituent selected from the specified group at any substitutable position of the group, and the substituent may be the same or different at each position.

[0182] In the present invention, the term "prevention" means a method of preventing the onset of a disease and / or its attendant symptoms or protecting a subject from acquiring a disease. "Prevention" as used herein also includes delaying the onset of a disease and / or its attendant symptoms and reducing the risk of a subject acquiring a disease.

[0183] The "treatment" described in the present invention includes delaying and stopping the progression of the disease, or eliminating the disease, and does not require 100% inhibition, elimination and reversal. In some embodiments, compared with the levels observed in the absence of the composition, medicine box, food box or health product box, active ingredient combination described in the present invention, the composition or pharmaceutical composition described in the present invention reduces, inhibits and / or reverses the related diseases (such as tumors) and their complications by inhibiting the mitochondrial oxidative phosphorylation pathway, for example, by at least about 10%, at least about 30%, at least about 50%, or at least about 80%.

[0184] Integrins and integrin β3 / Src interactions

[0185] Integrin αIIbβ3 is a transmembrane heterodimer composed of two subunits, αIIb and β3, through non-covalent bonds. It is mainly expressed on the surface of platelets and megakaryocytes. It is the main membrane receptor on the surface of platelets and can mediate bidirectional signal transduction of platelets. Therefore, it plays a key role in platelet activation, maintaining normal platelet function and thrombosis. Platelet activators such as thrombin and ADP act on the corresponding receptors to cause the conformational change of integrin αIIbβ3 and increase its affinity with its ligand, i.e., soluble fibrinogen. This process is inside-out signal transduction, and the hallmark events are unstable adhesion of platelets, free fibrinogen binding and reversible aggregation. Integrin αIIbβ3 is activated and binds to the ligand, activating outside-in signal transduction. The hallmark events are stable adhesion, extension, irreversible aggregation, retraction of fibrin clots, etc. of platelets, which ultimately promotes platelets to aggregate and form relatively stable thrombi to complete the physiological or pathological process of hemostasis and thrombosis. The current consensus is that the realization of hemostasis and thrombosis requires the participation of both inside-out and outside-in signal transduction, and the pathological process of thrombosis requires the increase of platelet plugs under conditions of high blood flow impact. Therefore, thrombosis is relatively more dependent on outside-in signal transduction.

[0186] Integrin αIIbβ3 is the final common pathway that mediates platelet activation, aggregation, and thrombosis, and is therefore the main target for antithrombotic drug research. In fact, research on integrin αIIbβ3 as an antithrombotic drug target has made important progress. Currently, it is mainly focused on integrin αIIbβ3 receptor antagonists, which have achieved good clinical efficacy. At present, there are three integrin αIIbβ3 receptor antagonist antiplatelet drugs approved by the US Food and Drug Administration (FDA) for clinical antithrombotic treatment, namely abciximab, eptifibatide, and tirofiban. This type of αIIbβ3 receptor antagonist specifically exerts an antithrombotic effect by interfering with the interaction between integrin αIIbβ3 and its ligand. However, this strategy still has obvious problems. αIIbβ3 receptor antagonists block bidirectional signal transduction by preventing integrin αIIbβ3 from binding to its ligand, that is, while exerting an antithrombotic effect, it affects the normal hemostatic function. Clinical trial review statistics show that about 2% of patients treated with integrin αIIbβ3 antagonists experience severe intracranial hemorrhage, about 15% experience gastrointestinal bleeding, about 5-10% experience peritoneal bleeding, and about 60-80% of the patients experience significant bleeding at the femoral artery puncture site. Like the classic antithrombotic drugs aspirin and clopidogrel, integrin αIIbβ3 antagonists not only play an effective antithrombotic role, but also increase the risk of bleeding in patients, which is the most common and important side effect of antithrombotic drugs at present. Therefore, the choice of antithrombotic drug dosage in clinical practice must also take into account the contribution of its bleeding side effects, making it difficult to achieve better antithrombotic effects by increasing the dosage of antithrombotic drugs. Therefore, by developing a new generation of antithrombotic drugs that do not affect normal hemostasis, it will be possible to obtain stronger antithrombotic efficacy at a low risk, which represents the development direction of antithrombotic drugs.

[0187] Integrin β3 is the main subunit that binds to platelet intracellular signaling molecules and regulates bidirectional signal transduction. The interaction between its intracellular segment and signaling molecules and the regulation of bidirectional signal transduction has attracted wide attention from scholars. In recent years, a large number of research results have suggested that selective inhibition of integrin αIIbβ3 outside-in signal transduction can inhibit pathological thrombosis without causing the risk of spontaneous bleeding. Therefore, in order to achieve a more reasonable new anti-thrombotic strategy, a scientific and reasonable approach should avoid directly antagonizing the αIIbβ3 receptor and selectively regulate the outside-in signal transduction by interfering with the interaction between integrin β3 and platelet intracellular signaling molecules, so as to achieve effective anti-thrombotic effect while retaining physiological hemostatic function as much as possible. Therefore, the key issue currently faced is to involve a method that can achieve clinical translational application. The most feasible one is the small molecule interference strategy, which selectively regulates the outside-in signal transduction of human platelet integrins without basically affecting the inside-out signal transduction of platelet integrins, and truly achieves an anti-thrombotic strategy that effectively resists thrombosis while not affecting the normal hemostatic function.

[0188] Integrin αIIbβ3 bidirectional signal transduction is two interconnected but distinct signal pathways. In recent years, great progress has been made in the study of the regulation of bidirectional signal transduction by the cytoplasmic segment of integrin β3 through interaction with cytoplasmic signaling protein molecules. For example, β3-endonexin, ILK, talin head domain, kindlin-3, etc. are involved in the regulation of inside-out signal transduction; while c-Src, Gα13, Talin, VPS33B, Shc, JAM-A, Dok-1, etc. are involved in the regulation of outside-in signal transduction. Among them, the function and mechanism of the interaction between platelet integrin β3 and c-Src are the most clearly studied. c-Src, which constitutively binds to the RGT sequence at the tail end of integrin β3, modulates kinase activity upon receiving activation signals, and then phosphorylates downstream signaling molecules. It is the most basic regulatory pathway for outside-in signal transduction that has been confirmed. It has been confirmed that defects in Src family genes such as c-Src, Hck, Fgr, Lyn, etc. (especially c-Src) or under the action of Src kinase inhibitors, integrin β3T762A mutation and β3Δ760-762 deletion mutation can inhibit platelet function mediated by outside-in signals. The three amino acids RGT at the intracellular terminal of the β3 subunit can directly interact with c-Src, and the artificially synthesized RGT polypeptide can specifically interfere with the interaction between integrin β3 and c-Src in platelets and selectively inhibit platelet outside-in signal transduction. Therefore, it has become a consensus in the academic community that the β3 / c-Src interaction plays a very important role in the outside-in signal transduction mediated by platelet integrin αIIbβ3. Targeting the β3 / c-Src interaction through artificially synthesized molecules and selectively inhibiting outside-in signal transduction has the potential to develop into a new generation of antithrombotic drugs.

[0189] In the resting state, there are about 80,000 integrins αIIbβ3 on the surface of the platelet membrane. When activated, the platelet α granules release integrin αIIbβ3 and the intracellular duct system opens and transfers to the membrane, increasing the number to enhance the hemostatic function. Integrin αIIbβ3 is composed of αIIb subunit and β3 subunit bound by non-covalent bonds. The αIIb subunit has a molecular weight of 140kD and is connected by a heavy chain (containing 871 amino acids) and a light chain (containing 137 amino acids) by a disulfide bond. The β3 subunit has a molecular weight of 105kD and is a transmembrane polypeptide composed of 762 amino acids. Both αIIb and β3 subunits are composed of a longer extracellular segment, a transmembrane α helix, and a shorter intracellular segment. The extracellular segment of integrin can bind to ligands such as fibrinogen and vWF factor. Its binding ability with ligands is not only regulated by the extracellular segment, but all three parts of the extracellular, transmembrane, and intracellular parts are involved in the regulation. Electron microscopy revealed that the extracellular segments of both subunits have an N-terminal "head" that binds to the ligand, connected to the longer C-terminal "leg", and then connected to the transmembrane segment and the cytoplasmic segment.

[0190] Crystal diffraction further revealed that the "head" of the α subunit is composed of seven blade-shaped folds forming a β-propeller domain (β-propeller), and the "legs" are composed of "thigh", "Calf-1" and "Calf-2" domains, with the knee of the leg formed between the "thigh" and "Calf-1" domains; the "head" of the β subunit is composed of a βA domain and an immunoglobulin-like "Hybrid" domain, and the "legs" are composed of a PSI (plexin-semaphorin-integrin) domain, four EGF (epidermal growth factor) domains and a β tail domain, with the knee of the leg formed between the "EGF-1" and "EGF-2" domains. When integrin is at rest, the legs of both subunits are in a "V"-shaped flexed state, so that the head of the subunit is close to the cell membrane, shielding the binding site with the ligand, and the affinity of integrin αIIbβ3 with the ligand is low. When platelet agonists such as thrombin and adenosine diphosphate act on the corresponding receptors on the platelet membrane, through a series of signal transduction, the head of talin binds to the proximal membrane segment of integrin β3 cytoplasm, destroying the salt bridge between αIIbR995-β3D723 that maintains the resting state of αIIbβ3. The spatial conformation of integrin αIIbβ3 changes from a "flexed" state to an "extended" state. The ligand binding site is exposed, and the affinity for the ligand increases. This process is called "inside-out signaling", which is functionally manifested as platelet binding to soluble fibrinogen, initial platelet adhesion, and phase one aggregation. After the conformational change, the integrin αIIbβ3 receptor clusters, aggregates and binds to kinases and anchoring proteins, leading to a series of signal transductions such as calcium influx, protein tyrosine phosphorylation, and skeleton protein reorganization. This process is called "outside-in" signal transduction, which is functionally manifested as stable adhesion and extension of platelets, two-phase aggregation, and retraction of fibrin clots. Integrin αIIbβ3 completes its role in thrombosis and hemostasis through such bidirectional signal transduction. Inside-out signal transduction completes the initial activation of integrins, initial adhesion and reversible aggregation, and basically achieves hemostasis, while outside-in signal transduction achieves platelet extension, irreversible aggregation, and fibrin clot formation of firm thrombus, playing a greater role in thrombosis.

[0191] The cytoplasmic segment of integrin αIIbβ3 is very short, but it can bind to many intracellular proteins and kinases, playing an important role in regulating the signal transduction of integrin αIIbβ3. The proteins that interact with the cytoplasmic segment of β3 include cytoskeletal proteins such as talin, α-actinin, filamin and myosin, kinases include the Src kinase family, integrin-linked kinase (ILK), focal adhesion kinase (FAK), etc., and regulatory proteins include β3-innexin and cell attachment protein-1. In recent years, the role of Src kinase in the bidirectional signal transduction of integrin αIIbβ3, especially the outside-in signal transduction, has attracted the interest of many scholars. Src kinase is a member of the SFK (Src family kinase protein) family. The SFK family contains 9 members, among which Src, Fyn and Yes are widely present in various tissues in the body and play an important regulatory role in cell growth, development and differentiation. When mice lack these three kinases, they die embryonically due to developmental defects. Src kinase is composed of SH3, SH2, kinase region, and C-terminal region domains from N-terminus to C-terminus. Csk kinase can bind to the SH2 domain and phosphorylate Y527 on the C-terminus of Src kinase, thereby maintaining the closed conformation of Src kinase and making it inactive. Under certain signal stimulation, Csk kinase can leave the SH2 domain of Src kinase, dephosphorylating Y527 and phosphorylating Y416 of Src kinase, thereby making Src have kinase activity and can phosphorylate downstream substrates, participating in cell signal transduction.

[0192] Current studies have shown that the SH3 domain of Src kinase forms a constitutive binding with the three amino acids of RGT at the C-terminus of integrin β3. In the resting state of platelets, β3-Src (SH3)-Src (SH2)-Csk forms a complex, which forms the basis for the outside-in signal transduction of integrin αIIbβ3. When mouse platelets lack Src kinase, the outside-in signal transduction of integrin αIIbβ3 is inhibited, and the outside-in signal transduction of platelets is also significantly inhibited by the action of Src kinase inhibitors. During the outside-in signal transduction after integrin activation, Src kinase interacts with the RGT sequence at the C-terminus of β3, and phosphorylation of Y747 and Y759 of the cytoplasmic segment of β3 occurs, which becomes an important event of outside-in signal transduction. When αIIbβ3Δ759 was transferred into CHO cells, the outside-in signal was significantly inhibited compared with that of αIIbβ3 CHO cells, while the outside-in signal was significantly impaired in mice by knocking out the integrin αIIbβ3-RGT sequence, and the phosphorylation of Y747 and Y759 was inhibited. Ablooglu, AJ et al. established integrin αIIbβ3Δ759 transgenic mice, namely RGT knockout mice. The results showed that compared with control mice, the extension of platelets on solid fibrinogen in RGT knockout mice was inhibited; the retraction of fibrin clots was also inhibited; the phosphorylation of Y747 was impaired; but the binding of free fibrinogen as agonists in PAR4 and glycoprotein VI was not affected. In vivo experiments confirmed that RGT knockout mice can avoid thrombosis caused by FeCl3 stimulation of the carotid artery; in the tail cutting experiment, the bleeding time of some mice was prolonged, but there was no spontaneous bleeding, postoperative bleeding, bloody stools, hematuria, anemia, etc. The authors believe that knocking out the RGT sequence of integrin αIIbβ3 can affect the αIIbβ3 signal transduction involving c-Src kinase, thereby preventing arterial thrombosis; it inhibits the outside-in signal transduction of integrin αIIbβ3, while only partially affecting the inside-out signal transduction.

[0193] Through Myr-RGT, it was explained that interfering with the binding of integrin αIIbβ3RGT sequence and Src kinase SH3 domain can selectively inhibit outside-in signal transduction. This mechanism study laid the foundation for applied research. However, due to the limitation of low efficiency, the analog peptide is difficult to be applied in vivo, which has become a major obstacle to drugization and even clinical application. Therefore, it is urgent to explore and find a batch of new small molecule compounds that can target this binding site and be applied in vivo with sufficient efficiency. Therefore, by targeting the β3 / c-Src interaction through artificial synthesis of small molecule compounds and selectively inhibiting outside-in signal transduction, it basically does not affect inside-out signal transduction, and has the potential to develop into a new generation of anti-thrombotic drugs.

[0194] In summary, the biggest challenge in the development of antithrombotic drugs is that the difficulty in the development of such drugs is to separate the synchronous platelet thrombosis and hemostasis, that is, to distinguish the platelet's outside-in signal transduction and inside-out signal transduction for inhibition. Although some studies have found that some oligopeptides or polypeptides can selectively inhibit platelet outside-in signals (related to thrombosis) and have little effect on inside-out signals (related to hemostasis), such peptide mimics have poor membrane permeability and low utilization efficiency, making them difficult to apply in vivo and poor drugability, which has become a major obstacle to drugization and even clinical application. At present, there are no reports of inhibitors for the interaction between β3 and c-Src protein-protein.

[0195] In addition to αIIbβ3, which is specifically expressed in platelets and megakaryocytes, integrin αvβ3 is widely distributed in the body, such as endothelial cells, osteoclasts, tumor cells, smooth muscle cells, fibroblasts, etc., mediating various pathophysiological processes such as angiogenesis, bone resorption, and tumor metastasis. Since the β3 subunits of αIIbβ3 and αvβ3 are exactly the same, the pathophysiological effects mediated by the β3 / c-Src interaction in other tissues and cells may also be affected by inhibitors of the protein-protein interaction between β3 and c-Src. At present, there are no reports on the effects of inhibitors of the protein-protein interaction between β3 and c-Src on αvβ3-expressing cells such as endothelial cells, osteoclasts, tumor cells, smooth muscle cells, and fibroblasts. Src and c-Src

[0196] Src was first discovered as an oncogene protein in Rous sarcoma retrovirus (retrovirus roussarcoma virus), and then v-Src, which is highly conserved and homologous to Src, was found to be ubiquitous in cells.

[0197] The Src kinase family is a group of proteins with protein tyrosine kinase (PTK) activity, of which c-Src is an important component of the Src kinase family.

[0198] Active ingredients

[0199] As used herein, "compounds of the present invention", "compounds of formula I of the present invention", or "compounds of formula I" are used interchangeably to refer to compounds having the structure of formula I, or optical isomers or racemates thereof, or solvates thereof, or pharmaceutically acceptable salts thereof. It should be understood that the term also includes mixtures of the above components.

[0200]

[0201] Specifically, the compound of formula I is as described above in the first aspect of the present invention.

[0202] The term "pharmaceutically acceptable salt" refers to a salt formed by the compound of the present invention and an acid or base that is suitable for use as a drug. Pharmaceutically acceptable salts include inorganic salts and organic salts. A preferred salt is a salt formed by the compound of the present invention and an acid. Acids suitable for forming salts include (but are not limited to): inorganic acids such as hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid, phosphoric acid, formic acid, acetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, picric acid, methanesulfonic acid, benzenesulfonic acid, benzenesulfonic acid, and organic acids such as aspartic acid and glutamic acid. A preferred salt is a metal salt formed by the compound of the present invention and a base. Bases suitable for forming salts include (but are not limited to): inorganic bases such as sodium hydroxide, potassium hydroxide, sodium carbonate, sodium bicarbonate, sodium phosphate, and organic bases such as ammonia water, triethylamine, and diethylamine.

[0203] The compound of formula I described in the present invention can be converted into its pharmaceutically acceptable salt by conventional methods. For example, a solution of the corresponding acid can be added to a solution of the above compound, and the solvent can be removed after the salt is completely formed to obtain the corresponding salt of the compound described in the present invention.

[0204] Preferred compounds of the present invention are selected from the following group:

[0205]

[0206] use

[0207] The present invention also provides a compound of the present invention for one or more uses selected from the following groups: (1) selectively inhibiting outside-in signal transduction mediated by the interaction between integrin β3 and Src kinase; (2) preventing and / or treating diseases related to outside-in signal transduction mediated by the interaction between integrin β3 and Src kinase; (3) preventing and / or treating thrombosis; (4) preventing and / or treating tumors; (5) preventing and / or treating osteoporosis; and (6) preventing and / or treating endothelial cell-mediated angiogenesis.

[0208] In a preferred embodiment of the present invention, the inhibiting the interaction between integrin β3 and Src kinase comprises inhibiting the interaction between integrin β3 and the SH3 domain of Src kinase.

[0209] In another preferred embodiment, the selective inhibition of outside-in signal transduction mediated by the interaction between integrin β3 and Src kinase refers to the selective inhibition of outside-in signal transduction mediated by the interaction between integrin β3 and Src kinase but without affecting inside-out signal transduction.

[0210] In another preferred embodiment, the interaction between integrin β3 and Src kinase refers to the interaction between integrin β3 and c-Src kinase in platelets.

[0211] In another preferred embodiment, the integrin β3 is β3 of integrin αIIbβ3 and / or integrin αvβ3.

[0212] In another preferred embodiment, the diseases associated with outside-in signal transduction mediated by the interaction between integrin β3 and Src kinase include (but are not limited to): thrombosis, tumor, osteoporosis, endothelial cell-mediated angiogenesis, or a combination thereof.

[0213] In another preferred embodiment, the prevention and / or treatment of thrombosis includes anti-thrombosis and improvement of bleeding.

[0214] In another preferred embodiment, the improvement of bleeding includes inhibiting bleeding, not increasing the risk of bleeding, reducing the risk of bleeding, not causing bleeding side effects and / or not affecting the hemostatic function.

[0215] In another preferred embodiment, the hemostatic function includes platelet hemostatic function.

[0216] In another preferred embodiment, the hemostasis includes physiological hemostasis.

[0217] In another preferred embodiment, the thrombus is a cardiovascular and cerebrovascular disease thrombus, and the thrombus is a cardiovascular and cerebrovascular disease thrombus including (but not limited to): myocardial infarction thrombus, cerebral infarction thrombus, ischemic stroke, atherosclerotic thrombus, or a combination thereof.

[0218] In another preferred embodiment, the prevention and / or treatment of thrombosis comprises one or more methods selected from the following group:

[0219] (3-1) Inhibit the stretching function of platelets on solid fibrinogen;

[0220] (3-2) inhibiting platelet aggregation, adhesion and / or extension;

[0221] (3-3) Inhibits the retraction of fibrin clots;

[0222] (3-3) Does not affect the platelet binding to free fibrinogen;

[0223] In another preferred embodiment, the inhibition of platelet aggregation includes inhibiting one-phase aggregation or two-phase aggregation of platelets.

[0224] In another preferred embodiment, the prevention and / or treatment of tumors comprises one or more methods selected from the following group:

[0225] (4-1) Inhibit the growth or proliferation of tumor cells;

[0226] (4-2) Inhibit the formation or growth of tumor blood vessels;

[0227] In another preferred embodiment, the tumor includes (but is not limited to): solid tumor, hematological tumor, or a combination thereof.

[0228] In another preferred embodiment, the solid tumor includes (but is not limited to): lung cancer, breast cancer, melanoma, gastric cancer, or a combination thereof.

[0229] In another preferred embodiment, the tumor includes (but is not limited to): lung cancer, breast cancer, melanoma, gastric cancer, or a combination thereof.

[0230] In another preferred embodiment, the tumor includes (but is not limited to): lung cancer, breast cancer, melanoma, gastric cancer, or a combination thereof.

[0231] In another preferred embodiment, the lung cancer includes (but is not limited to): small cell lung cancer, non-small cell lung cancer, myocardial cancer, or a combination thereof.

[0232] In another preferred embodiment, the prevention and / or treatment of osteoporosis comprises one or more methods selected from the following group:

[0233] (5-1) Inhibit the proliferation and differentiation of osteoclasts.

[0234] In another preferred embodiment, the prevention and / or treatment of endothelial cell-mediated angiogenesis comprises one or more methods selected from the following group:

[0235] (6-1) Inhibit the proliferation of endothelial cells;

[0236] In another preferred embodiment, the blood vessel is selected from the following group: tumor blood vessel, umbilical vein blood vessel, or a combination thereof.

[0237] In another preferred embodiment, the endothelial cells are selected from the following group: tumor vascular endothelial cells, umbilical vein vascular endothelial cells, or a combination thereof.

[0238] Composition or formulation, combination of active ingredients and kit and method of administration

[0239] The present invention also provides a composition, which comprises the compound of formula I of the present invention.

[0240] The composition of the present invention is preferably a pharmaceutical composition. The composition of the present invention may include a pharmaceutically acceptable carrier.

[0241] As used herein, "pharmaceutically acceptable carrier" refers to one or more compatible solid, semisolid, liquid or gel fillers that are suitable for human or animal use and must have sufficient purity and sufficiently low toxicity. "Compatibility" refers to the components in the pharmaceutical composition and the active ingredients of the drug and their mutual blending without significantly reducing the efficacy of the drug.

[0242] It should be understood that in the present invention, the pharmaceutically acceptable carrier is not particularly limited, and can be selected from materials commonly used in the art, or prepared by conventional methods, or purchased from the market. Some examples of pharmaceutically acceptable carriers include cellulose and its derivatives (such as methylcellulose, ethylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, etc.), gelatin, talcum powder, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerol, mannitol, sorbitol, etc.), emulsifiers (such as Tween), wetting agents (such as sodium lauryl sulfate), buffers, chelating agents, thickeners, pH regulators, transdermal enhancers, colorants, flavoring agents, stabilizers, antioxidants, preservatives, antibacterial agents, pyrogen-free water, etc.

[0243] In the present invention, the dosage forms of the compositions and preparations include but are not limited to oral preparations, injection preparations, and external preparations.

[0244] Typically, the dosage forms of the compounds and preparations include, but are not limited to, tablets, injections, infusions, ointments, gels, solutions, microspheres, and films.

[0245] Typically, the injection is an intratumoral injection.

[0246] The pharmaceutical preparation should match the mode of administration. The preferred modes of administration are oral administration and injection (such as intratumoral injection). When used, a therapeutically effective amount of the drug is administered to the desired subject (such as a human or non-human mammal). As used herein, the term "therapeutically effective amount" refers to an amount that produces a function or activity on humans and / or animals and can be accepted by humans and / or animals. It should be understood by those of ordinary skill in the art that the "therapeutically effective amount" may vary depending on the form of the pharmaceutical composition, the route of administration, the excipients of the drug used, the severity of the disease, and the combination with other drugs.

[0247] In one mode of administration, the safe and effective daily dose of the first active ingredient is usually at least about 0.1 mg, and in most cases does not exceed about 2500 mg. Preferably, the dose is 1 mg-500 mg; the safe and effective amount of the second active ingredient is usually at least about 0.01 mg, and in most cases does not exceed 2500 mg. Preferably, the dose range is 0.1 mg to 2500 mg. Of course, the specific dose should also take into account factors such as the route of administration and the patient's health status, which are all within the skill range of skilled physicians.

[0248] The main advantages of the present invention include:

[0249] 1. The present invention unexpectedly discovered that the compounds of the present invention (such as compound DCDBS84) can effectively dissociate the interaction between integrin β3 and c-Src, inhibit platelet adhesion, aggregation, and fibrin clot retraction, without affecting the binding to soluble fibrinogen, thereby being able to specifically inhibit platelet outside-in signals (related to thrombosis) without substantially affecting platelet inside-out signals (related to hemostasis), thereby being able to achieve deep anti-thrombotic effects without causing bleeding side effects, and being able to become a new generation of effective drugs for preventing and treating thrombosis-related cardiovascular and cerebrovascular diseases.

[0250] 2. The compounds of the present invention (such as compound DCDBS84) can inhibit tumor growth and metastasis, play a therapeutic effect on osteoporosis by inhibiting osteoclasts, and the inhibitory effect on endothelial cells can develop into inhibiting tumor angiogenesis. In addition, the compounds of the present invention also have certain effects on endothelial cells, cardiomyocytes, fibroblasts and other αvβ3 expressing cells.

[0251] 3. The compounds of the present invention are small molecule compounds with advantages such as good drugability and low side effects.

[0252] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples without specifying specific conditions are usually based on conventional conditions or the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight.

[0253] Example

[0254] The structural formula of the compound is as follows:

[0255]

[0256] Symbols represent:

[0257] "RGDS" is an αIIbβ3 antagonist.

[0258] "ADP" is adenosine diphosphate.

[0259] Example 1: Screening model diagram of small molecule compounds targeting β3 / c-Src interaction.

[0260] Based on the crystal structure of the SH3:RGT complex (PDB number: 4HXJ), we used molecular docking to virtually screen SPECS containing 200,000 small molecule compounds. The software used for molecular docking was Glide, and the docking mode was extra precision. From the docking results, the top 1,200 small molecule compounds were selected. After deduplication, a total of 898 candidate small molecules were obtained. Then, cluster analysis was performed on these 898 small molecules. From the clustering results, 124 small molecules were selected to purchase physical compounds. The screening mode is shown in the figure below. Figure 1 .

[0261] Example 2: Effect of small molecule compound DCDBS84 on platelet extension on solid fibrinogen.

[0262] First, add 50 μl of fibrinogen (0.1M, pH 8.3 sodium bicarbonate dilution, 20 μg / ml) to a 96-well plate and coat overnight at 4°C. The next morning, wash three times with PBS and block with bovine serum albumin (20 mg / ml) at 37°C for 60 min. Then take 100 μl of the washed platelet suspension and add each group of small molecule compounds to make the final concentration 250 μM, and incubate at 37°C for 60 min. Take 50 μl of the incubated platelets and add them to a 96-well plate, adhere in a 37°C incubator for 60 min, wash three times with PBS to remove non-adherent platelets, fix the adhered platelets with 4% paraformaldehyde, and wash three times with PBS. Then, the platelet membrane was perforated with 0.5% Triton X-100, and then the platelets were stained with 0.5 μg / ml phalloidin-rhodamine at 37°C for 60 minutes, and washed with PBS three times (10 minutes each time). After washing, the fluorescence color was observed with a fluorescence microscope (Leica), and the drug concentration was reduced to observe the effect of different concentration gradients of small molecule compounds on the extension function of platelets on solid fibrinogen. The results are as follows Figure 2 As shown, from Figure 2 It can be seen that the compound DCDBS84 can inhibit the extension function of platelets on solid fibrinogen to varying degrees at a concentration gradient of 20-80 μM.

[0263] Example 3: In a surface plasmon resonance experiment, the dissociation constant diagram between the compound and the protein SH3 was determined.

[0264] The surface plasmon resonance test was performed on a BIACORE T200 instrument (GE). Fresh purified Src kinase SH3 domain protein (concentration 2 mg / ml) was diluted to 0.1 mg / ml with 10 mM CH3COONa (pH 4.2), and then the SH3 domain protein was coupled to the CM5 chip by the standard amino coupling method. The small molecule compound DCDBS84 was diluted step by step with a buffer solution (20 mM Tris-HCl, pH 8.0, 100 mM NaCl), and then continuously injected for 60 seconds at a flow rate of 20 μl / s and dissociated for 120 seconds. The changes in the response value over time were recorded, and the dissociation constant Kd between the compound and the SH3 protein was obtained by analysis using the BIA Evaluation Software (GE Healthcare) program. Figure 3 As shown, from Figure 3 It can be seen that the dissociation constant of compound DCDBS84 and SH3 protein is 980nM.

[0265] Example 4: Detecting the effect of the small molecule compound DCDBS84 on the activity of c-Src tyrosine kinase and the effect on the classical binding between PXXP domain-containing proteins and c-Src SH3.

[0266] The c-Src tyrosine phosphorylation kit (MBL) was used to detect the effects of different concentrations of DCDBS84, PP2 (c-Src inhibitor) and Dasatinib (tyrosine kinase inhibitor) on c-Src tyrosine kinase activity. Figure 4 As shown, it can be seen that different concentrations of DCDBS84 do not inhibit the tyrosine kinase activity of c-Src, while PP2 and Dasatinib can significantly inhibit the tyrosine kinase activity of c-Src.

[0267] In addition, we used ELISA to detect the effects of DCDBS84, PP2 and Dasatinib on the binding of PXXP domain-containing peptide (RLP1) to c-Src SH3. Ligand protein molecules that interact with Src kinase in cells generally target the Src-SH3 domain through the classic (PXXP) motif. RLP1 peptide (containing the classic (PXXP) motif) is a common (PXXP) motif sequence of multiple ligand protein molecules that interact with Src-SH3 domain in cells. The specific experimental process is as follows: 50 μL of coating solution containing Flag antibody (Sigma) (1 μg / mL, diluted with 0.1M NaHCO3 PH 8.3) was added to each well of the 96-well plate and placed at 4°C overnight. On the second day, the plate was washed 3 times with TBST for 5 minutes each time, blocked with 5% BSA for 2 hours, and then washed 3 times with TBST for use. In the early stage, pFlag-CMV4-Src (WT) and four other Src mutant overexpression vectors (lipo2000) were transfected in 293T cells. After 48 hours, 293T cells were lysed with RIPA for 30 minutes, centrifuged (4°C, 18000rpm) to collect the lysate supernatant, and 50μL of lysate supernatant was added to each well of the 96-well plate coated with Flag antibody, and placed at 4°C overnight. Part of the supernatant of each experimental group was retained for relative quantification by Western blot. On the third day, the cells were washed three times with TBST, and 50μL of biotin-labeled RLP1 and control (RLA) peptides (1μg / mL) were added to each well and incubated at 37°C for 1h. Then, the cells were washed three times with TBST, each time for 5 minutes, and 50μL of HRP-labeled avidin was added to each well, and incubated at 37°C for 1h. After washing with TBST for 3 times, 100 μL of TMB display solution was added to each well, and the well was placed at room temperature for 15 min. Then 100 μL of sulfuric acid (1 M) was added to terminate the reaction, and the absorbance of each well was detected at a wavelength of 450 nm using an enzyme-labeled instrument. Figure 5 As shown, from Figure 5 It can be seen that DCDBS84 does not affect the interaction between RLP1 and c-Src, while PP2 and Dasatinib can significantly inhibit the interaction between RLP1 and c-Src.

[0268] Example 5: Pull-down assay was used to determine the interference of compounds on SH3-RGT interaction in vitro.

[0269] The His-Src-SH3 and GST-β3 proteins were expressed and purified in prokaryotes respectively. The specific methods are as follows:

[0270] The wild-type and mutant plasmids of Src-SH3 were constructed by our laboratory, and the expression vector was pET32A. The N-terminus contained a Trx tag, a His Tag, and a thrombin cleavage site, and a Ni-NTA protein purification column (5mL HisTrap FFcolumn, GE, USA) was used for purification. The wild-type or mutant plasmids of Src-SH3 were transformed into BL21 competent cells, and positive clones were selected on LB (containing 100μg / mL ampicillin) plates. Then the clones were picked and transferred to 10mL LB medium (containing 100μg / mL ampicillin) and cultured overnight on a shaker (37℃, 220rpm). On the second day, 5mL of the bacterial solution was inoculated into 1L LB medium (containing 100μg / mL ampicillin) for expansion culture (37℃, 220rpm). When the optical density of the bacterial solution at a wavelength of 600nm was 0.6-0.8, the temperature of the shaker was adjusted to 16℃, and the culture was continued for 1h (220rpm). Then, 400 μL of isopropylthio-β-D-galactoside (IPTG) (1M) was added to induce the expression of the target protein overnight for 12–14 hs (16°C, 220 rpm). On the third day, the bacterial solution was introduced into a centrifuge tank and the cells were collected by centrifugation (4°C, 5000 rpm, 10 min). The cells were resuspended with pre-cooled buffer A (20 mM Tris-HCl, PH 8.0, 100 mM Nacl), and the cells were ultrasonically disrupted on ice (ultrasonication for 5 s, stop for 5 s, power 40%, 15 min), and then centrifuged at high speed (4°C, 18000 rpm, 40 min) to discard the sediment and keep the supernatant. The collected supernatant was then loaded onto the Ni-NTA protein purification column at a flow rate of 1 mL / min. In the AKTA Pure system (GE, USA), 2% buffer B (20mM Tris-HCl, PH 8.0, 100mM Nacl, 500mM iminazole) was used to elute the impurities. After the protein peak (UV_280) was constant, the buffer B was adjusted to 8% to elute the target protein and collect it. Then, 20% buffer B was used to completely elute the target protein and collect it. Finally, 100% buffer B was used to thoroughly wash the protein on the column. Thrombin (2U / mL) was added to the collected protein eluate and treated at room temperature for 12-16h. On the 4th day, the iminazole was removed by desalting (HiTrap Desalting purification column). Then, the Ni-NTA protein purification column was loaded again at a flow rate of 1mL / min, and the flow-through was the Scr-SH3 protein with the His Tag and Trx-tags cut off. When the flow-through was concentrated to about 1 mL, it was further purified using buffer A through molecular sieve Superdex 75 (24 mL, GE, USA).The purified protein was tested for concentration using NanoDrop 2000, and then glycerol was added to a final concentration of 5% and stored at -80°C. When the His Tag tag needs to be retained in the experiment, thrombin treatment is not performed, and the other operations remain unchanged.

[0271] The GST-β3 intracellular segment plasmid was constructed on the Pgex-6p1 vector and stored in our laboratory. 716 -T 762 ) The initial extraction process is similar to the above, but the purification method is different from that of Src-SH3 protein, and GST affinity chromatography medium (GST Agarose) is used for purification. Resuspend the bacteria with pre-cooled PBS buffer, sonicate on ice, and then centrifuge at high speed (4°C, 18000rpm, 40min) to retain the supernatant, and load the GST affinity chromatography column (GlutathioneSepharose 4Fast Flow) at a flow rate of 0.5mL / min. Then wash with PBS on the AKTK Pure system, and after UV_280 is constant, elute the GST-β3 intracellular segment protein with PBS containing reduced glutathione (GSH, 20mM). After the collected solution is concentrated, it is further purified with molecular sieve Superdex 75 (24mL, GE, USA). The storage method of the GST-β3 intracellular segment protein is the same as above.

[0272] Glutathione-agarose resin was pretreated to form a 50% glutathione-agarose resin homogenate, and purified GST-β3 intracellular segment protein was added, and then purified Src-SH3 protein (wild type or mutant) with His Tag was added and incubated overnight at 4°C. Centrifugation (4°C, 1000g, 1min) was performed on the morning of the second day, and the precipitate was washed 3 times with 200μL buffer H (0.1% NP-40, 50mM KCl, 20% glycerol, 0.007% β-mercaptoethanol, 20mM HEPES, pH 7.7). To analyze the interaction between wild-type Src-SH3 protein and Src-SH3 protein, the pellet was resuspended and DCDBS84 (250 μM) or DMSO control was added and incubated at 4°C for 1 h with rotation, and finally washed again, and then boiled in 1×SDS loading buffer (100 mM Tris-HCl, pH 6.8, 5% β-mercaptoethanol, 4% SDS, 20% glycerol, 0.1% bromophenol blue) (100°C, 10 min). The target protein was detected by Western blot.

[0273] The results are as follows Figure 6As shown, it can be seen that compound DCDBS84 can inhibit the binding of GST-β3 to His-Src-SH3 in a concentration-dependent manner.

[0274] Example 6: Co-immunoprecipitation (Co-IP) was used to determine the interaction of the compound with SH3-RGT in vitro.

[0275] The Co-IP method was used to detect the effect of the small molecule compound DCDBS84 on the interaction between β3 and c-Src. After DCDBS84 was incubated with washed human platelets for 1 hour, the supernatant was removed by centrifugation, and the platelets were lysed on ice with IP buffer (Biyuntian Biotechnology Co., Ltd.) for 30 minutes, and then centrifuged (4℃, 12000rpm, 15min) to retain the supernatant for BCA protein quantification. 50μL of Protein A / G agarose beads pre-washed with IP buffer were added, incubated at 4℃ for 2 hours, centrifuged (4℃, 1000g, 5min), and the supernatant was transferred to a new centrifuge tube. Anti-integrin β3 antibody SZ-21 (10μg) or non-specific mouse IgG (sc-2025, Santa Cruz Biotechnology, 10μg) was added to the protein supernatant, and the antigen-antibody mixture was incubated overnight at 4℃. The next morning, 20 μL of Protein A / G agarose beads pre-washed with IP buffer were added to the mixture, and the mixture was rotated and incubated at 4°C for 2 h. The mixture was then centrifuged (4°C, 1000 g, 5 min) to collect the agarose bead-antigen-antibody complex and washed 3 times with pre-cooled PBS buffer. Finally, the agarose bead-antigen-antibody complex was resuspended with 1× SDS loading buffer and boiled at 100°C for 10 min. The immunoprecipitate was detected by Western blotting.

[0276] The results are as follows Figure 7 As shown, in the control group (no drug added) and DMSO group, the complex contained integrin β3 and c-Src protein; after the platelets were treated with 80μM DCDBS84, only integrin β3 was present in the complex, but no c-Src protein. This indicates that at a concentration of 80μM, DCDBS84 can significantly inhibit the interaction between integrin β3 and c-Src in human platelets.

[0277] Example 7: Effect of the small molecule compound DCDBS84 on the adhesion function of platelets on solid fibrinogen.

[0278] The coating and blocking methods of fibrinogen in the adhesion function experiment were the same as those in the extension experiment. First, 100 μl of the washed platelet suspension was added to each group of reagents to make the final concentrations of DMSO (250 μM), SMDBS84 (20 μM, 40 μM, 80 μM), and RGDS (1 mM) respectively, and incubated at 37°C for 60 min. 50 μl of the incubated platelets were added to a 96-well plate and adhered in a 37°C incubator for 60 min. After adhesion, PBS was washed 5 times to remove unadhered and unstable adhered platelets. Then, 50 μl / well of disodium p-nitrophenol phosphate (PNPP) substrate colorimetric solution was added to the wells where platelets adhered, incubated in a 37°C incubator for 60 min, and an equal amount of 1× sodium hydroxide was added to terminate the reaction. Finally, the OD value was read at a wavelength of 405 nm on an ELISA reader, and the wells without platelets were used as blank controls to calculate the amount of adhered platelets. Three secondary wells were set for each sample, and the results were averaged. Figure 8 As shown, it can be seen that compound DCDBS84 can inhibit the adhesion function of platelets on solid fibrinogen in a concentration-dependent manner.

[0279] Example 8: Effect of small molecule compound DCDBS84 on platelet aggregation.

[0280] First, 10 ml of venous blood (anticoagulated with sodium citrate) from healthy blood donors was centrifuged at 300 × g for 7 min to obtain PRP, and then centrifuged at 500 × g for 10 min to obtain platelet poor plasma (PPP). The platelet concentration in PRP was adjusted to 2 × 10 8 / ml. Then take 200μl PPP to calibrate the zero point of the instrument light transmission aggregation instrument (Chrono-Log). After each group of reagents were incubated with PRP at 37℃ for 60min, 200μl / tube was put on the aggregation instrument (37℃, 1000rpm stirring), and after calibrating the zero point, the inducer ADP (2μM) was added to start aggregation, and the aggregation curve was observed and recorded. When the platelets were aggregated with thrombin as the inducer, the washed platelets were taken and the platelet concentration was adjusted to 2×10 8 / mL, add 0.1U / ml thrombin to start the reaction, and record the aggregation curve. Fig. 9 , Fig.10 As shown, it can be seen that the compound DCDBS84 exhibits concentration-dependent inhibition on platelet aggregation induced by different inducers. For platelet aggregation induced by ADP, DCDBS84 selectively inhibits the two-phase aggregation of platelets without affecting the one-phase aggregation.

[0281] Example 9: Effect of the small molecule compound DCDBS84 on the retraction function of fibrin clot.

[0282] Fibrin clot retraction is an important step in thrombosis mediated by integrin αIIbβ3 outside-in signal transduction. During thrombosis, serum is squeezed out through the fibrin clot to form a firm thrombus. The retraction process is produced by the interaction between integrin and fibrinogen, and integrin and cytoskeleton. In this experiment, the effect of compounds on the retraction function of platelet fibrin clot was observed in a siliconized aggregation tube. In order to observe the effect of compounds on the retraction of platelet fibrin clot, human washed platelets pre-incubated with drugs were added to HEPES buffer containing Fg (2mg / ml), placed in a siliconized aggregation tube, and 1U / mL of thrombin was added to start the reaction, and the formation of fibrin clots was observed. The aggregation tube was incubated in a 37°C incubator, and then the retraction of the fibrin clot was observed and photographed at 15min, 30min, 45min, and 60min after the addition of thrombin. The clot volume was calculated using NIH Image 1.67e software, and the data was processed using Excel. A line graph was drawn with the ratio of clot size to initial volume as the ordinate and each time period as the abscissa to reflect the change in clot volume. Fig.11 It can be seen that compound DCDBS84 inhibits platelet fibrin clot retraction, and the degree of inhibition is dose-dependent. Fig.12 is based on Fig.11 Statistical results obtained from the data.

[0283] Example 10: Effect of the small molecule compound DCDBS84 on the binding of ADP-activated platelets to free fibrinogen.

[0284] The previous experiment observed the effect of the compound on the outside-in signal transduction regulation-related functions of platelet integrin αIIbβ3. In order to detect the effect of the compound on the activation state and ligand binding function of integrin αIIbβ3 regulated in the inside-out signaling pathway of integrin αIIbβ3, a platelet free fibrinogen binding experiment was performed. ADP was used to induce the activation of platelet integrin αIIbβ3 and bind to free fibrinogen. The compound-treated platelets were first stimulated with 20μM ADP, and then 100μg / ml Alexa Fluor 488-labeled fibrinogen was added for co-incubation. The binding amount of Alexa Fluor 488-labeled fibrinogen by platelets in different treatment groups activated by ADP was detected by flow cytometry. The results are shown in the figure. Fig.13As shown in the flow cytometry fluorescence results, it can be seen that when platelets are stimulated by 20 μM ADP, the fluorescence intensity of the control (untreated group) platelets changes, and the amount of bound Alexa Fluor 488-Fg is higher than the negative control (Control), i.e., the free fibrinogen binding amount of platelets not activated by ADP; the fluorescence intensity of the platelets pretreated with the compound, i.e., the fibrinogen binding amount, is similar to that of the platelets in the DMSO-treated group, and the binding of platelets to free fibrinogen is almost completely inhibited under 1 mM RGDS (αIIbβ3 antagonist).

[0285] The free fibrinogen binding function of platelets reflects the activation state of integrin αIIbβ3 and its ability to bind to ligands, and is an important marker of integrin αIIbβ3 inside-out signal transduction. Fig.13 The results showed that the small molecule compound DCDBS84 did not affect the ADP-induced platelet binding to free fibrinogen. This suggests that the small molecule compound DCDBS84 has no significant effect on integrin inside-out signal transduction, and does not affect the conformational change, ligand binding, and receptor activation of integrin αIIbβ3 from low affinity to high affinity. However, RGDS, due to the inhibition of integrin αIIbβ3-mediated inside-out signal transduction, significantly inhibited the ADP-induced platelet binding to free fibrinogen.

[0286] Example 11: The effect of carotid artery occlusion thrombosis on mice was examined after treatment with the small molecule compound DCDBS84.

[0287] First, 6-week-old C57BL / 6 mice were selected as experimental subjects. The drugs were injected by tail vein, and the dosage of DCDBS84 was 2.5μg / g BW, 5μg / g BW, and 10μg / g BW, and the corresponding drug concentrations were 6.25μmol / kg BW, 12.5μmol / kg BW, and 25μmol / kg BW. Integrilin was injected as a positive control drug, and the dosage was 0.18μg / g BW, 4.2μg / g BW, and 16μg / g BW, and the corresponding drug concentrations were 16μmol / kg BW, 50μmol / kg BW, and 192μmol / kg BW. Mice injected with DMSO and saline were used as negative controls for DCDBS84 and integrilin, respectively. According to the experimental method reported in the literature, FeCl3 stimulation of the mouse carotid artery began 15 minutes after drug injection, resulting in endothelial damage and subsequent thrombosis. Blood flow was detected with a Doppler ultrasound probe at the distal end of FeCl3 stimulation. When upstream thrombus formation occurred and blocked the blood vessel, blood flow decreased.

[0288] The results are as follows Fig.14 As shown, from Fig.14 It can be seen that compared with the control group, after the application of compound DCDBS84, the carotid artery thrombosis time of mice under FeCl3 stimulation was significantly prolonged, and it showed time dependence, that is, the effect was most obvious at 10μg / g BW, the thrombosis time was about 12.5min; at 5μg / g BW, it was about 7.5min; and at 2.5μg / g BW, the thrombosis time was about 5min, which was similar to the control group. The thrombosis time of integrilin at 0.18μg / g was about 6min, which was slightly lower than 5μg / g BW DCDBS84; the thrombosis time of integrilin at 4.2μg / g was about 7.5min, which was equivalent to 5μg / g BW DCDBS84; the thrombosis time of integrilin at 16μg / g was about 12.5min, which was equivalent to 10μg / g BW DCDBS84. The results showed that the compound DCDBS84 had a significant inhibitory effect on arterial thrombosis in mice, and this effect was dose-dependent.

[0289] Example 12: The effect of tail snip on bleeding time was detected after mice were treated with the small molecule compound DCDBS84.

[0290] The effect of the small molecule compound DCDBS84 on hemostasis was evaluated. This is the process in which platelets maintain normal hemostasis and block the blood vessel wound after a blood vessel is damaged. First, the drug was injected into the tail vein. After 15 minutes, the mouse tail was quickly cut off with a sharp blade 5 mm away from the tail tip (tail-cut). Then, filter paper was used to dip the blood oozing out of the mouse tail every 15 seconds. Do not touch the mouse tail to avoid causing new damage. The timing standard was when the bleeding stopped and there was no recurrence within 15 seconds.

[0291] The results are as follows Fig.15 As shown, from Fig.15 It can be seen that the control group stopped bleeding at about 160s, and compared with the control group, the bleeding time did not prolong significantly when a higher concentration of compound DCDBS84 (10μg / g BW) was used, which was also about 160s. However, 0.18μg / g and 4.2μg / g of integrilin blocked the binding of integrin αIIbβ3 with the ligand fibrinogen, completely inhibiting bidirectional signal transduction, including the inside-out signal transduction that plays an important role in hemostasis, thereby significantly prolonging the bleeding time (600s). Due to the limitation of prolonged bleeding time, integrilin cannot be used at a sufficient antithrombotic dose of 4.2μg / g or even 16μg / g, but can only be used at a low dose of 0.18μg / g.

[0292] Example 13: Real-time detection of the effect of thrombosis on cremaster artery after treating mice with the small molecule compound DCDBS84.

[0293] In addition, we used a more accurate laser-induced mouse cremaster muscle micro-arteriolar thrombosis model to analyze in real time the effect of DCDBS84 on thrombosis in mice.

[0294] Eight-week-old wild-type C57BL / 6 mice were injected with DCDBS84 (10 μg / g BW) through the tail vein. After 30 minutes of drug action, the mice were anesthetized with 1% pentobarbital intraperitoneally. Anti-CD41-FITC antibody and anti-CD62P red fluorescent antibody were injected through the carotid artery under a stereomicroscope. Then, the mouse cremaster arterioles were isolated and transferred to a laser fluorescence microscope. Laser irradiation was applied to the arteriolar endothelium to cause damage. Thrombosis at the injured site was observed in real time under a fluorescence microscope. Statistical analysis was performed after multiple points of injury. Green light represents CD41 (platelets labeled with Alexa Fluor 488) and red light represents P-selectin (activated platelets labeled with Alexa Fluor 633). The results are shown in Figure 2. Fig.16 As shown in Figure 2, DCDBS84 can significantly inhibit the size of thrombotic plaques in mice. Compared with the DMSO control group, the CD41 fluorescence intensity and the number of activated platelets in the plaques of the DCDBS84-treated group ( Fig.17 ) were significantly reduced. The experimental results of this model also confirmed that DCDBS84 can inhibit thrombosis in mice. However, there are still a small number of activated platelets adhering to the damaged vascular endothelium, which may be the basis for DCDBS84 to basically not affect the hemostatic function. The control drug integrilin can significantly inhibit the size of thrombotic plaques formed in mice ( Fig.16 ), CD41 fluorescence intensity in the plaque and the number of activated platelets in the plaque ( Fig.17 ) were almost undetectable and there was no platelet adhesion under the damaged vascular endothelium, which may be the basis for the bleeding side effects of integrilin.

[0295] Example 14: In vitro fluid technology was used to detect the effect of small molecule compound DCDBS84 on platelet thrombus formation in collagen-coated tubes.

[0296] The in vitro fluid platelet adhesion and aggregation experiment simulates the process of platelet adhesion and aggregation in blood vessels, which is also mediated by integrin αIIbβ3 signal transduction. Integrilin is an FDA-approved antithrombotic drug that inhibits the binding of integrin αIIbβ3 to its ligand.

[0297] Collagen was used to coat the experimental wells of the Bioflux 200 48-well plate overnight. The next day, the wells were washed with PBS and blocked with BSA for 1-2 hours. Fresh peripheral blood from volunteers was collected and anticoagulated with hirudin. DCDBS84 (20μM, 40μM, 80μM) was added and incubated at 37°C for 1-2 hours. DMSO was used as a negative control and Integrilin was used as a positive control. The treated blood samples were added to the experimental wells on the left. The flow shear force (500s -1 , 1500s -1 , 5000s -1 ), samples of different treatment groups will flow from the left hole to the right hole under the set shear force, and real-time video recording will be used under a fluorescent microscope to capture the data of thrombosis in the coated tube, and then statistical analysis will be performed. Fig.18 It shows that the shear force is 5000s -1 Under the condition of simulating pathological stenosis of aorta in vivo, both DCDBS84 and Integrilin treatment groups could inhibit platelet aggregation and adhesion ( Fig.18 ); and when the shear force is 1500s -1 Under the condition of (simulating in vivo peripheral arterioles), the fluorescence signal of the Integrilin-treated group was significantly lower than that of the DMSO group and the DCDBS84 group, indicating that Integrilin significantly inhibited platelet aggregation and adhesion ( Fig.18 ), while DCDBS84 had little effect on platelet aggregation and adhesion under this condition, which is also the mechanism by which DCDBS84 has almost no effect on normal hemostasis function; under shear force of 500s -1 Under the conditions of (simulating in vivo aorta), DCDBS84 can also inhibit platelet aggregation to a certain extent compared with DMSO. The above results show that under the shear stress of 5000s -1 When the shear rate was 1500s, DCDBS84 showed obvious anti-platelet aggregation and adhesion effect, and had the same anti-thrombotic effect as Integrilin. -1 The effect of DCDBS84 is different from that of Integrilin. DCDBS84 has a smaller effect on platelet aggregation and adhesion, thus not increasing the risk of bleeding. The above results show that DCDBS84 can inhibit thrombosis in mice without increasing the risk of bleeding. Fig.19 Based on Fig.18 Statistical results of the data.

[0298] Example 15: Effect of the small molecule compound DCDBS84 on the adhesion of tumor cells on solid fibrinogen.

[0299] In addition to αIIbβ3 specifically expressed on platelets and megakaryocytes, integrin β3 can also form heterodimers with αv subunits to form αvβ3. Since αvβ3 is widely distributed in the body and expressed in a variety of tissues and cells, such as tumor cells, we tested the effect of DCDBS84, a small molecule compound that inhibits β3 / c-Src interaction, on the adhesion of several tumor cells on solid fibrinogen.

[0300] First, add 50μl of fibrinogen (0.1M, pH 8.3 sodium bicarbonate dilution, 20μg / ml) to a 96-well plate and coat overnight at 4℃. First, take various tumor cells, count to ensure the same number of cells in each group, add reagents to each group so that the final concentrations are DMSO (250μM), DCDBS84 (20μM, 40μM, 80μM), and RGDS (1mM) respectively, and incubate at 37℃ for 60min. Take 50μl of incubated tumor cells and add them to a 96-well plate, and adhere in a 37℃ incubator for 60min. After adhesion, wash with PBS 5 times to remove non-adherent and unstable adherent cells. Then add 50μl / well of disodium p-nitrophenol phosphate (PNPP) substrate colorimetric solution to the wells where cells adhere, place in a 37℃ incubator for 60min, and add an equal amount of 1× sodium hydroxide to terminate the reaction. Finally, the OD value was read at a wavelength of 405 nm by an ELISA reader, and the wells without cells were used as blank controls to calculate the number of adhered tumor cells. Three replicate wells were set for each sample, and the results were averaged.

[0301] like Fig. 20 As shown, for MCF-7 breast cancer cells that do not express β3, the cells cannot adhere to solid fibrinogen; when β3 is overexpressed in MCF-7, MCF-7-β3 cells can adhere to solid fibrinogen, and treatment with different concentrations of DCDBS84 can inhibit the adhesion of MCF-7-β3 cells to solid fibrinogen; when β3Δ759 (C-terminal RGT truncation) is transfected into MCF-7, the adhesion of MCF-7-β3Δ759 cells to solid fibrinogen is inhibited, and the effect is equivalent to the effect of MCF-7-β3 treated with DCDBS84.

[0302] In addition, for MDA-MB-231 (human breast cancer cells, Fig.21 ) and B16 (mouse melanoma cells, Fig. 22 ), DCDBS84 could inhibit the adhesion of cells on solid fibrinogen to a certain extent.

[0303] Example 16: Effect of the small molecule compound DCDBS84 on the growth of several cells in the scratched area.

[0304] The cell scratch test can reflect the proliferation and migration ability of cells, and thus can predict the proliferation and metastasis of cells to a certain extent. We tested the effect of the small molecule compound DCDBS84 on the growth of several cells in the scratch area.

[0305] Take various tumor cells, count them to ensure the same number of cells in each group, and plate the cells in a 6-well plate overnight. Add reagents in each group to make the final concentrations of DMSO (250μM), DCDBS84 (20μM, 40μM, 80μM), and RGDS (1mM) respectively, and incubate at 37°C for 60min. Use the pipette tip to forcefully scratch a scratch with a diameter of about 1cm in the middle area of ​​each empty space, and then continue to culture the cells for 12h, 24h, 36h, and 48h, and then take pictures under an inverted microscope to observe the growth of tumor cells in the scratch area.

[0306] Fig.23 It showed that high concentrations of DCDBS84 could inhibit the growth of B16 cells in the scratch area, suggesting that it could inhibit tumor metastasis; Fig.24 It was shown that different concentrations of DCDBS84 could inhibit the growth of MDA-MB-231 in the scratch area; Fig.25 This indicates that MCF-7 cells, because they do not express β3, cannot grow in the scratch area regardless of whether DCDBS84 is applied or not; Fig.26 This indicates that MCF-7 cells were transfected with full-length β3, and DCDBS84 could inhibit the growth of MCF-7-β3 cells in the scratch area in a concentration-dependent manner, suggesting that it could inhibit tumor metastasis; Fig. 27 The results showed that when MCF-7 cells were transfected with β3Δ759 (C-terminal RGT truncated), the growth of cells in the scratch area was less, regardless of whether DCDBS84 was applied or not, and the cell growth was between that of MCF-7 and MCF-7-β3. Fig.28 Comparison of cell growth in the scratched area among MCF-7, MCF-7-β3 and MCF-7-β3Δ759 cells indicates the important role of β3 / c-Src interaction in cell growth and adhesion; Fig.29 It was shown that different concentrations of DCDBS84 could inhibit the growth of NIH-3T3 (mouse embryonic fibroblast cell line, expressing αvβ3) in the scratch area.

[0307] Example 17: Effect of the small molecule compound DCDBS84 on the proliferation of several tumor cells.

[0308] We examined the effect of the small molecule compound DCDBS84 on the proliferation of several tumor cells.

[0309] Take various tumor cells, count to ensure the same number of cells in each group, plate the cells in a 96-well plate overnight, add reagents in each group to make the final concentrations of DMSO (250μM), DCDBS84 (20μM, 40μM, 80μM), RGDS (1mM), and incubate at 37°C for 24h or 48h. Add 1 / 10 volume of CCK-8 for staining, incubate at 37°C for 4h, read the OD value at 405nm wavelength on an enzyme reader, use the wells without cells as blank controls, and calculate the cell proliferation inhibition rate. Set up 3 replicate wells for each sample, and take the average value of the results.

[0310] The results are as follows: Fig.30 The results show the comparison of the inhibitory effects of different concentrations of DCDBS84 on the proliferation of three cell lines: MCF-7, MCF-7-β3, and MCF-7-β3Δ759. It has almost no effect on MCF-7, the most obvious effect on MCF-7-β3, and a weaker effect on MCF-7-β3Δ759. Fig.31 The results show that DCDBS84 at different concentrations has an inhibitory effect on the proliferation of three types of cells, MCF-7, MDA-MB-231 and B16. It has almost no effect on MCF-7, but has inhibitory effects on MDA-MB-231 and B16. The above results suggest that DCDBS84 has almost no inhibitory effect on the proliferation of cells without β3 expression, and for cells expressing β3, it may exert its inhibitory effect on cell proliferation by inhibiting the β3 / c-Src interaction. Fig.32 It showed that different concentrations of DCDBS84 had a certain proliferation inhibitory effect on A549 and NCI-H1975 non-small cell lung cancer cell lines.

[0311] Example 18: Effects of the small molecule compound DCDBS84 on osteoclast proliferation and differentiation.

[0312] Since αvβ3 is also expressed in osteoclasts, we examined the effect of the small molecule compound DCDBS84 on osteoclast proliferation and differentiation.

[0313] Osteoclasts were taken and counted to ensure that the number of cells in each group was consistent. The cells were plated in a 96-well plate overnight, and reagents were added to make the final concentration of DCDBS84 (1μM, 5μM, 10μM, 20μM), and incubated at 37°C for 24h or 48h. 1 / 10 volume of CCK-8 was added for staining, and the cells were incubated at 37°C for 4h. The OD value was read at a wavelength of 405nm on an enzyme reader. The wells without cells were used as blank controls to calculate the cell proliferation inhibition rate. Three replicate wells were set for each sample, and the results were averaged.

[0314] Normal bone metabolism is maintained in balance through osteoblast growth and osteoclast bone resorption. The marker enzyme of osteoclasts is tartrate-resistant acid phosphatase (TRAP). The cell treatment method is the same as above. After the cells are treated with DCDBS84, a colorimetric substrate solution for TRAP enzyme activity staining is added, and then the OD value is read at a wavelength of 405nm using an ELISA reader. The wells without cells are used as blank controls to calculate TRAP activity. The cell treatment method is the same as above. The cells are plated in a 6-well plate, a sterilized cover slip is placed in the 6-well plate in advance, and the cells are plated on the cover slip. After the cells are treated as above, the cells are treated with a colorimetric substrate solution for TRAP enzyme activity staining, and then photographed and observed under a microscope. Fig.33 A shows the inhibitory effect of different concentrations of DCDBS84 on osteoclast proliferation, showing that 20 μM DCDBS84 treatment can significantly inhibit osteoclast proliferation; Fig.33 B Effects of different concentrations of DCDBS84 on osteoclast differentiation. The results showed that DCDBS84 could inhibit osteoclast differentiation in a concentration-dependent manner. Fig.33 C, 33D show that different concentrations of DCDBS84 can inhibit the differentiation of osteoclast precursor cells. Fig.34 The results showed that different concentrations of DCDBS84 could effectively inhibit the number of osteoclasts, suggesting that the small molecule compound DCDBS84 could inhibit the proliferation and differentiation of osteoclasts.

[0315] Example 19: Effect of small molecule compound DCDBS84 on proliferation of endothelial cells and cardiomyocytes

[0316] Since αvβ3 is expressed in endothelial cells and cardiomyocytes, we examined the effect of the small molecule compound DCDBS84 on the proliferation of endothelial cells and cardiomyocytes.

[0317] Take the cardiomyocytes, count them to ensure the same number of cells in each group, and plate the cells in a 96-well plate overnight. Add the reagents in each group to make the final concentrations of DMSO (250μM) and DCDBS84 (0.1μM, 1μM, 10μM, 50μM), respectively, and incubate at 37°C for 24h or 48h. Add 1 / 10 volume of CCK-8 staining, incubate at 37°C for 4h, read the OD value at 405nm wavelength on an enzyme reader, and use the wells without cells as blank controls to calculate the cell proliferation inhibition rate. Set up 3 replicate wells for each sample, and take the average value of the results.

[0318] The results showed that different concentrations of DCDBS84 had an inhibitory effect on the proliferation of Huvec (human umbilical vein endothelial cells, expressing β3) Fig.35 ); Different concentrations of DCDBS84 have an inhibitory effect on the proliferation of HL-1 (mouse cardiomyocytes, expressing β3) ( Fig.36 ).

[0319] Example 20: Effects of structural derivatives of the small molecule compound DCDBS84 (Formula II-XII) on platelet extension on solid fibrinogen.

[0320] In addition to the small molecule compound DCDBS84, we also synthesized its structural derivatives (Formula II-XII) and tested the effects of these derivatives on the extension of platelets on solid fibrinogen.

[0321] The basic process of the platelet extension experiment is as follows: first, add 50 μl of fibrinogen (0.1M, pH 8.3 sodium bicarbonate dilution, 20 μg / ml) to a 96-well plate and coat overnight at 4°C. The next morning, wash with PBS three times and block with bovine serum albumin (20 mg / ml) at 37°C for 60 min. Then, take 100 μl of the washed platelet suspension and add each group of DCDBS84 structural derivatives (Formula II-XII) to make the final concentrations 1 μM, 10 μM, 50 μM, 100 μM, 62.5 μM, 125 μM, 250 μM, respectively, and incubate at 37°C for 60 min. Take 50 μl of the incubated platelets and add them to a 96-well plate, adhere in a 37°C incubator for 60 min, wash with PBS three times to remove non-adherent platelets, fix the stably adhered platelets with 4% paraformaldehyde, and wash with PBS three times. Then, the platelet membrane was perforated with 0.5% Triton X-100, and then the platelets were stained with 0.5 μg / ml phalloidin-rhodamine at 37°C for 60 minutes, and washed with PBS three times (10 minutes each time). After washing, the fluorescence color was observed with a fluorescence microscope (Leica), and the drug concentration was reduced to observe the effect of different concentration gradients of small molecule compounds on the extension function of platelets on solid fibrinogen.

[0322] The results showed that these derivatives could inhibit platelet extension on solid fibrinogen to varying degrees ( Fig.37 ), some of its effects are equivalent to those of DCDBS84, and some of its effects are stronger than those of DCDBS84.

[0323] Example 21: Effects of structural derivatives of the small molecule compound DCDBS84 (Formula II-XII) on the proliferation of several tumor cells.

[0324] Take various tumor cells, count to ensure the same number of cells in each group, and plate the cells in a 96-well plate overnight. Add the structural derivatives of DCDBS84 (Formula II-XII) in each group to make the final concentrations of DMSO (250μM) and DCDBS84 (0.1μM, 1μM, 10μM, 50μM) and incubate at 37°C for 24h or 48h. Add 1 / 10 volume of CCK-8 for staining, incubate at 37°C for 4h, read the OD value at 405nm wavelength on an enzyme reader, and use the wells without cells as blank controls to calculate the cell proliferation inhibition rate. Set up 3 replicate wells for each sample, and take the average value of the results.

[0325] The effects of Formula II-XII on lung cancer ( Fig.38 ), breast cancer( Fig.39 , Fig.40 , Fig.41 ) and found that these derivatives can exert a certain proliferation inhibitory effect on lung cancer and breast cancer.

[0326] Example 22: Effects of structural derivatives of the small molecule compound DCDBS84 (Formula II-XII) on endothelial cell proliferation.

[0327] Take endothelial cells, count to ensure the same number of cells in each group, plate the cells in a 96-well plate overnight, add the structural derivatives of DCDBS84 (Formula II-XII) in each group to make the final concentrations of DMSO (250μM), DCDBS84 (0.1μM, 1μM, 10μM, 50μM), and incubate at 37°C for 24h or 48h. Add 1 / 10 volume of CCK-8 for staining, incubate at 37°C for 4h, read the OD value at 405nm wavelength on an enzyme reader, use the wells without cells as blank controls, and calculate the cell proliferation inhibition rate. Set up 3 replicate wells for each sample, and take the average value of the results.

[0328] The effects of Formula II-XV on Huvec endothelial cells ( Fig.42 ) and found that these derivatives can exert a certain proliferation inhibitory effect on Huvec endothelial cells.

[0329] Example 23: Effects of structural derivatives of the small molecule compound DCDBS84 (Formula II-XII) on cardiomyocyte proliferation.

[0330] Take the cardiomyocytes, count them to ensure the same number of cells in each group, and plate the cells in a 96-well plate overnight. Add the structural derivatives of DCDBS84 (Formula II-XII) in each group to make the final concentrations of DMSO (250μM) and DCDBS84 (0.1μM, 1μM, 10μM, 50μM) respectively, and incubate at 37°C for 24h or 48h. Add 1 / 10 volume of CCK-8 for staining, incubate at 37°C for 4h, read the OD value at 405nm wavelength on an enzyme reader, and use the wells without cells as blank controls to calculate the cell proliferation inhibition rate. Set up 3 replicate wells for each sample, and take the average value of the results.

[0331] The effects of formula II-XV on mouse cardiomyocytes (HL-1) ( Fig.43 ) and found that these derivatives can exert a certain proliferation inhibitory effect on HL-1.

[0332] Summarize:

[0333] Based on the results of the above specific examples, the compounds and derivatives of the present invention can exert an influence on the functions mediated by the outside-in signal of platelets by inhibiting the interaction of integrin β3 / c-Src, while having little effect on the functions mediated by the inside-out signal, thereby effectively inhibiting the platelet functions related to the outside-in signal, such as platelet aggregation, adhesion, extension and fibrin clot retraction, while having little effect on the platelet functions related to the inside-out signal, such as binding to free fibrinogen. Studies in mice have shown that the compounds of the present invention can effectively achieve deep anti-thrombotic effects without substantially affecting the physiological hemostatic function. For the first time, the symmetric regulatory effects of platelet thrombosis and hemostasis have been separately regulated at the compound level, providing a new perspective and prospect for the development of a new generation of anti-thrombotic drugs. In addition to αIIbβ3 specifically expressed on platelets and megakaryocytes, integrin β3 can also form a heterodimer αvβ3 with the αv subunit. Since αvβ3 is widely distributed in the body and expressed in a variety of tissues and cells, such as tumor cells, osteoclasts, endothelial cells, cardiomyocytes, fibroblasts, etc. We also tested the effects of the compounds and derivatives of the present invention on the proliferation of these cells expressing αvβ3, adhesion on solid fibrinogen, cell growth and migration in the scratch area, etc., and found that the compounds and derivatives can inhibit the proliferation, adhesion and migration of tumor cells, osteoclasts, endothelial cells, cardiomyocytes, fibroblasts, etc. to a certain extent by inhibiting the integrin β3 / c-Src interaction, and play a therapeutic role on a variety of malignant tumors; inhibit osteoclast proliferation and differentiation, and play a preventive and therapeutic role on osteoporosis; inhibit the proliferation of endothelial cells, and related functions such as tumor angiogenesis mediated by endothelial cells; and related effects on cardiomyocytes, fibroblasts, etc.

[0334] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A use of a compound of formula I, or an optical isomer thereof, or a racemate thereof, or a solvate thereof, or a pharmaceutically acceptable salt thereof, characterized in that: Used for preparing a composition or preparation, wherein the composition or preparation is used for one or more uses selected from the following groups: (1) selectively inhibiting outside-in signal transduction mediated by the interaction between integrin β3 and Src kinase; (2) preventing and / or treating diseases related to outside-in signal transduction mediated by the interaction between integrin β3 and Src kinase; (3) preventing and / or treating tumors; (4) preventing and / or treating osteoporosis; (5) preventing and / or treating endothelial cell-mediated angiogenesis; in, R1 is hydrogen, substituted or unsubstituted C1-C12 alkyl, substituted or unsubstituted C3-C12 cycloalkyl, substituted or unsubstituted C6-C16 aryl, substituted or unsubstituted 3-12 membered heteroaryl, -substituted or unsubstituted C1-C12 alkylene-C6-C16 aryl, -substituted or unsubstituted C1-C12 alkylene-3-12 membered heteroaryl; R2 is substituted or unsubstituted C1-C12 alkyl, substituted or unsubstituted C3-C12 cycloalkyl, isothiourea, guanidinyl, substituted or unsubstituted C1-C12 alkylene-isothiourea, substituted or unsubstituted C1-C12 alkylene-guanidinyl, substituted or unsubstituted C3-C12 cycloalkylene-isothiourea, or substituted or unsubstituted C3-C12 cycloalkylene-guanidinyl; R3 is hydrogen, substituted or unsubstituted C1-C12 alkyl, substituted or unsubstituted C3-C12 cycloalkyl, or substituted or unsubstituted C2-C12 ester, or substituted or unsubstituted C2-C12 acyl; R4 is hydrogen, substituted or unsubstituted C1-C12 alkyl, substituted or unsubstituted C3-C12 cycloalkyl, substituted or unsubstituted C2-C12 ester, substituted or unsubstituted C1-C12 alkoxy, substituted or unsubstituted C1-C12 alkylthio, substituted or unsubstituted C3-C12 cycloalkyloxy, or substituted or unsubstituted C3-C12 cycloalkylthio; R5 is hydrogen, substituted or unsubstituted C1-C12 alkyl, substituted or unsubstituted C3-C12 cycloalkyl, halogen, substituted or unsubstituted C6-C16 aryl, or substituted or unsubstituted 3-12 membered heteroaryl; W and Z are each independently CH, C-R6, or N; R6 is substituted or unsubstituted C1-C12 alkyl, substituted or unsubstituted C3-C12 cycloalkyl, halogen, substituted or unsubstituted C6-C16 aryl, or substituted or unsubstituted 3-12 membered heteroaryl; Wherein, any "substituted" means that one or more (preferably 1, 2, 3, 4 or 5) hydrogen atoms on the group are replaced by a substituent selected from the group consisting of C1-C8 alkyl, C3-C8 cycloalkyl, C1-C8 haloalkyl, C3-C8 halocycloalkyl, halogen, nitro, -CN, hydroxyl, thiol, amino, C1-C4 carboxyl, C2-C4 ester, C2-C4 amide, C1-C8 alkoxy, C1-C8 alkylthio, C1-C8 haloalkoxy, C1-C8 haloalkylthio, C6-C12 aryl, 5-10 membered heteroaryl, 5-10 membered heterocycloalkyl; The heterocyclic ring of the heteroaryl group has 1 to 4 (preferably 1, 2, 3 or 4) heteroatoms selected from N, O and S.

2. The use according to claim 1, characterized in that The compound of formula I is:

3. The use according to claim 1, characterized in that The disease associated with outside-in signal transduction mediated by the interaction between integrin β3 and Src kinase is selected from the following group: tumor, osteoporosis, endothelial cell-mediated angiogenesis, or a combination thereof.

4. The use according to claim 1, characterized in that The tumor is selected from the group consisting of lung cancer, breast cancer, melanoma, gastric cancer, or a combination thereof.

5. The use according to claim 4, characterized in that The lung cancer is selected from the group consisting of small cell lung cancer, non-small cell lung cancer, myocardial cancer, or a combination thereof.

6. The use according to claim 1, characterized in that The composition or preparation also includes other anti-tumor drugs, other drugs for treating osteoporosis, and / or other anti-angiogenesis drugs.

7. A method for (1) selectively inhibiting outside-in signal transduction mediated by the interaction between integrin β3 and Src kinase; (2) preventing and / or treating diseases related to outside-in signal transduction mediated by the interaction between integrin β3 and Src kinase; (3) preventing and / or treating tumors; (4) preventing and / or treating osteoporosis; and / or (5) preventing and / or treating endothelial cell-mediated angiogenesis, characterized in that: The compound of formula I as claimed in claim 1, or its optical isomer or racemate, or its solvate, or its pharmaceutically acceptable salt is administered to a subject in need thereof.

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