CN102603610B - 1,3-dihydro-1-oxo-2H-isoindole compound, its preparation method and use - Google Patents

Abstract

The present invention discloses 1,3-dihydro-1-oxo-2H-isoindole compounds (I) and pharmaceutically acceptable salts thereof, as well as methods for preparing the compounds and their use in preparing drugs for preventing and/or treating various symptoms _or diseases _of tumor metastasis, tumor growth, solid tumor growth, angiogenesis, retinopathy, macular degeneration, osteoporosis, arthritis, smooth muscle cell migration, and atherosclerosis mediated by integrin α v β 3. .

Description

1,3-dihydro-1-oxo-2H-isoindole compound, its preparation method and use

technical field

The invention belongs to the field of medicinal chemistry, and relates to a class of 1,3-dihydro-1-oxo-2H-isoindole compounds (I) and pharmaceutically acceptable salts thereof, their preparation methods, pharmaceutical compositions and preparation methods. Prevention or/and treatment of various symptoms or diseases mediated by integrin α _v β ₃ , including but not limited to tumor metastasis, tumor growth, solid tumor growth, angiogenesis, retinopathy, macular degeneration, Osteoporosis, arthritis, smooth muscle cell migration, and atherosclerosis, etc.

Background technique

Angiogenesis refers to the process of generating a new vascular system from an existing vascular bed. Under normal physiological conditions, angiogenesis is strictly controlled by certain transient, specific physiological processes, such as reproduction, developmental processes, and wounds. Healing process, etc., while persistent angiogenesis is characteristic of certain diseases, such as: malignant growth of tumor tissue, retinopathy, macular degeneration, arthritis, osteoporosis, etc. Studies have shown that tumor-induced angiogenesis is closely related to the proliferation, invasion and metastasis of tumor cells. New blood vessels not only provide sufficient nutrients for the malignant growth of tumor cells, but also provide a pathway for distant metastasis of tumor cells. Therefore, , inhibit tumor-induced angiogenesis, reduce or block the blood supply of tumor tissue and "starve" tumor cells to death, which is an effective way to prevent and treat tumor metastasis. Compared with traditional cytotoxic antitumor drugs, this starvation therapy works because antiangiogenesis drugs prolong the proliferation cycle of tumor cells and promote tumor cell necrosis by reducing or blocking the blood supply of tumor tissues, rather than directly Kill tumor cells, therefore, this type of drug has no obvious toxic and side effects on normal cells, and shows better tumor targeting; and this therapy can overcome the problem of drug resistance in the treatment process of traditional chemotherapy drugs, and drug resistance is a common problem in current chemotherapy. One of the important reasons for treatment failure. the

Recent studies have shown that tumor-induced angiogenesis and tumor metastasis are closely related to the function of integrins. Integrins belong to the family of cell surface adhesion molecules, which are heterodimeric transmembrane glycoproteins composed of α and β subunits connected by non-covalent bonds. So far, more than 20 integrin subtypes have been discovered, and different subtypes have different cell characteristics and adhesion characteristics. They mediate cell-cell, cell-extracellular matrix (ECM) adhesion and Participate in cell signal transduction, and play an important regulatory role in cell adhesion, proliferation, metastasis, apoptosis, etc. Among them, integrin α _v β ₃ plays an important role in tumor growth, local invasion, metastasis, especially tumor-induced angiogenesis, etc. important role in the process.

Integrin α _v β ₃ is composed of α _v and β ₃ subunits, which are divided into three parts: extracellular region, transmembrane region and intracellular region, among which α _v subunit has 1018 amino acid residues and a molecular weight of 125kD. The β ₃ subunit has 762 amino acid residues and a molecular weight of 105kD; the study on the crystal structure of the extracellular region of integrin α _v β ₃ and its complex with the ligand shows that the extracellular region consists of 12 domains, of which α Assembled with the N-terminal part of the β subunit to form an oval "head", from which two almost parallel "tails"extend; the binding of the ligand occurs in the contact area between the α and β subunits, and the ligand The binding region mainly includes the β-propeller domain of the α subunit and the β _A domain of the β subunit, and requires the participation of divalent cations. The cations not only interact with the acidic groups of the ligands to mediate ligand binding. Binding to α _v β ₃ can also stabilize the ligand-binding surface of α _v β ₃ . The endogenous ligands of integrin α _v β ₃ are extracellular matrix components (such as: fibronectin, laminin, vitaminectin, thrombospondin, etc.) Contains the sequence of arginyl-glycyl-aspartyl (Arg-Gly-Asp, RGD, 1) tripeptide, and is selective for the recognition of extracellular matrix components, when α _v β ₃ and matrix components After the combination of RGD, vascular endothelial cells proliferate, differentiate and migrate through the mitogen-activated protein kinase pathway, and promote the formation of tube lumen to become new blood vessels. Experiments have shown that the use of exogenous RGD-containing sequence peptides or analogs can inhibit the adhesion and migration of tumor cells, induce tumor cell apoptosis and inhibit tumor angiogenesis, and finally lead to inhibition of tumor growth and even tumor regression. Since integrin α _v β ₃ is rarely expressed in normal vascular endothelial cells and most normal organ systems, only activated vascular endothelial cells, activated osteoclasts and Invasive tumor cells are highly expressed; and neovascular endothelial cells are genetically stable non-malignant cells, and the α _v β ₃ receptors on their surface have not undergone mutations, and there will be no drug resistance problems in the process of interacting with inhibitors; In addition, the α _v β ₃ receptor is located on the cell surface, directly in contact with the blood, and the drug is easy to reach the target; therefore, integrin α _v β ₃ is ideal for the design of highly efficient and selective anti-angiogenesis and anti-tumor metastasis drugs target.

Finding integrin α _v β ₃ inhibitors is of great significance for the discovery of anti-angiogenesis and anti-tumor metastasis drugs, and can also be used to treat tumor growth, solid tumor growth, retinopathy, macular degeneration, Common conditions such as osteoporosis, arthritis, smooth muscle cell migration, and atherosclerosis.

At present, anti-angiogenesis and anti-metastasis drugs targeting integrin α _v β ₃ are still in clinical or preclinical trials. These inhibitors can be divided into three categories, namely: monoclonal antibodies, peptides and cyclic peptides And its mimics, non-peptide small molecules. Vitaxin and Vitaxin II are α _v β ₃ monoclonal antibodies developed by Eli Lilly, which can specifically bind to integrin α _v β ₃ , inhibit tumor growth and angiogenesis, and are currently undergoing phase II clinical trials for the treatment of prostate carcinoma, metastatic melanoma, advanced colon cancer, and rheumatoid arthritis. Studies have found that the use of exogenous linear peptides or polymeric peptides containing RGD sequences, such as: RGDS, GRGDS, poly-RGDS, etc., can prevent angiogenesis induced by bFGF, TNF-α and various human tumor fragments, while the existing Blood vessels are unaffected. However, these peptide molecules are flexible, resulting in poor selectivity and affinity for integrin α _v β ₃ receptors. Cyclic peptides have better selectivity for different integrins due to their conformational constraints. The cyclic peptide cyclo(RGDfV) (Cyclo(-Arg-Gly-Asp-D-Phe-Val-), 2) is a highly active and selective integrin α _v β ₃ inhibitor, which inhibits α _v The IC ₅₀ of _β3 binding to its ligand protein is 2.5nmol/L; on this basis, Merck researchers methylated the peptide chain backbone of cyclo(RGDfV) to obtain a more active polypeptide mimic cyclo(RGDf-N( Me)V)(3, common name Cilengitide), with an IC ₅₀ of 0.58nmol/L, can antagonize the interaction between vascular endothelial cells and extracellular matrix mediated by integrin α _v β ₃ , inhibit the growth of melanoma and angiogenesis Neonatal and capable of inducing apoptosis in brain tumor cells grown on vitronectin, Clengitide is currently undergoing Phase II/III clinical trials for the treatment of lung, pancreatic, breast, melanoma, kidney and colon cancers. Due to the shortcomings of antibodies and peptide drugs such as in vivo degradation, short half-life, low bioavailability requiring intravenous administration, and difficulty in large-scale preparation, it is necessary to find and discover non-peptide integrin α _v with high efficiency and high selectivity that can be administered orally β ₃ inhibitors are one of the important fields of anti-angiogenesis and anti-tumor metastasis drug research at home and abroad.

Contents of the invention

The purpose of the present invention is to disclose a class of integrin α _v β ₃ inhibitors with 1,3-dihydro-1-oxo-2H-isoindole chemical structure (I) or pharmaceutically acceptable salts thereof.

Another object of the present invention is to disclose the preparation method of the 1,3-dihydro-1-oxo-2H-isoindole compound (I). the

Another object of the present invention is to disclose a pharmaceutical composition comprising 1,3-dihydro-1-oxo-2H-isoindole compound (I) or a pharmaceutically acceptable salt thereof. the

Another object of the present invention is to disclose 1,3-dihydro-1-oxo-2H-isoindole compounds (I) or pharmaceutically acceptable salts thereof in the preparation of prevention or/and treatment of various factors integrin α _v β ₃ mediates the use of drugs in causing symptoms or diseases, including but not limited to tumor metastasis, tumor growth, solid tumor growth, angiogenesis, retinopathy, macular degeneration, osteoporosis, arthritis, smooth muscle cell migration, and atherosclerosis etc.

1,3-dihydro-1-oxygen-2H-isoindole compound (I) provided by the invention has a general chemical structure formula:

或 

R₃、R₄、R₅各自独立地表示H、C₁～C₆烷基、C₁～C₆烷氧基、羟基、卤素、氨 基、硝基、氰基、羧基、CF₃、NR₇R₈，R₆表示H、C₁～C₆烷基、C₁～C₆烷氧基、卤素、硝基、氰基或CF₃，R₇、R₈各自独立地表示C₁～C₆烷基，R₃、R₄、R₅、R₆可在苯环的任意可能位置，R₃、R₄、R₅可以相同，也可以不同；R₂表示

n表示1-3、m表示0-2，R₉表示C₁～C₆烷基、卤素、羟基或CF₃。  In the _formula : R represents H, phenyl, 2-furyl, 2-thienyl, 3-thienyl, 3-pyridyl, 4-pyridyl, 3,4-methylenedioxyphenyl, 2 ,3-methylenedioxyphenyl,

or

R ₃ , R ₄ , and R ₅ each independently represent H, C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, hydroxyl, halogen, amino, nitro, cyano, carboxyl, CF ₃ , NR ₇ R ₈ and R ₆ represent H, C ₁ to C ₆ alkyl, C ₁ to C ₆ alkoxy, halogen, nitro, cyano or CF ₃ , R ₇ and R ₈ each independently represent C ₁ to C ₆ Alkyl, R ₃ , R ₄ , R ₅ , R ₆ can be in any possible position of the benzene ring, R ₃ , R ₄ , R ₅ can be the same or different; R ₂ means

n represents 1-3, m represents 0-2, R ₉ represents C ₁ -C ₆ alkyl, halogen, hydroxyl or CF ₃ .

When there is a chiral carbon in the molecule of the 1,3-dihydro-1-oxo-2H-isoindole compound (I) disclosed in the present invention, the compound (I) is a racemic body or an optically active body. the

The 1,3-dihydro-1-oxo-2H-isoindole compound (I) disclosed in the present invention can be prepared by the following method:

In the formula: R ₁ and R ₂ are defined as the same as the chemical structure general formula (I);

Using 2-formyl-5-nitrobenzoic acid (4) and β-alanine methyl ester derivatives (5) as starting materials, 3-(6-nitrobenzoic acid) is obtained through reduction and cyclization in a suitable solvent Base-1-oxoisoindol-2-yl) methyl propionate (6), the resulting intermediate (6) uses a reducing agent to reduce the nitro group to amino to obtain 3-(6-amino-1-oxoisoindol Indol-2-yl) methyl propionate (7), and then condensed with guanidine-containing carboxylic acid compound (8) in the presence of a condensing agent to obtain 1,3-dihydro-1-oxo-2H-iso Indole methyl ester compound (9) is hydrolyzed with alkali and acid neutralized to obtain 1,3-dihydro-1-oxo-2H-isoindole compound (I). the

Each reaction step of the above-mentioned method is specifically described as follows:

A) With 2-formyl-5-nitrobenzoic acid (4) and β-alanine methyl ester derivatives (5) as starting materials, 3-(6) is obtained through reduction and cyclization in a suitable solvent -Nitro-1-oxoisoindol-2-yl)methyl propionate (6); wherein, the solvent used for the reaction is: C _1-8 fatty alcohol, N,N-dimethylformamide, ethers ( Such as: ether, isopropyl ether, methyl tert-butyl ether, tetrahydrofuran, ethylene glycol dimethyl ether, etc.), C _1-6 fatty acids, esters of C _1-6 fatty acids and C _1-6 fatty alcohols, halogenated Hydrocarbons (such as dichloromethane, chloroform, 1,2-dichloroethane, etc.), aromatic hydrocarbons or substituted aromatic hydrocarbons (such as: benzene, toluene, xylene, chlorobenzene, o-dichlorobenzene, etc.), or acetonitrile, Preferably methanol, ethanol, acetic acid, ethyl acetate, ether, dichloromethane or tetrahydrofuran; the reducing agent used is: KBH ₄ , NaBH ₄ , LiBH ₄ , NaBH ₃ CN, the preferred reducing agent is NaBH ₄ , KBH ₄ ; 2- The molar feeding ratio of formyl-5-nitrobenzoic acid (4), β-alanine methyl ester derivatives (5) and reducing agent is 1.0: 1.0～5.0: 0.5～5.0, preferably the molar feeding ratio is 1.0 : 1.1 to 3.0: 1.0 to 3.0; the reaction temperature is -20°C to 120°C, preferably 0°C to 100°C; the reaction time is 30 minutes to 72 hours, preferably 1 to 15 hours.

The 3-(6-nitro-1-oxoisoindol-2-yl) methyl propionate (6) obtained in step A) is in a suitable solvent, through chemical reduction or catalytic hydrogenation reduction, the nitro group is reduced to Amino, to get 3-(6-amino-1-oxoisoindol-2-yl) methyl propionate (7); Wherein, when using the chemical reduction method, the solvent used for the reaction is: C ₁ ~ C ₆ fatty alcohol, Tetrahydrofuran, ether, C _1-6 fatty acid, ester of C 1-6 fatty acid and C _1-6 fatty alcohol, benzene, toluene, 1,4 _- dioxane, ethylene glycol dimethyl ether, dichloromethane, Chloroform, 1,2-dichloroethane, hydrochloric acid aqueous solution with a concentration of 1%-36%, ammonium chloride aqueous solution with a concentration of 1%-30%, or a mixture of the above solvents, preferably methanol, ethanol, acetic acid, acetic acid Ethyl ester, ether, dichloromethane or tetrahydrofuran; The reducing agent used is: zinc powder, iron powder or tin dichloride; The molar feed ratio of compound (6) and reducing agent is 1.0: 1.0～10.0, preferably molar feed ratio is 1.0:1.5～5.0; the reaction temperature is 0°C～150°C, preferably the reaction temperature is room temperature～solvent reflux temperature; the reduction reaction time is 0.5～72 hours, preferably the reaction time is 1～12 hours;

When using catalytic hydrogenation reduction, the solvents used in the reaction are: C _1-8 fatty alcohol, diethyl ether, isopropyl ether, methyl tert-butyl ether, tetrahydrofuran, ethylene glycol dimethyl ether, C _1-6 fatty acid, C _1-6 Esters formed by fatty acids and C _1-6 fatty alcohols, benzene, toluene, xylene or acetonitrile, preferably methanol, ethanol, acetic acid or ethyl acetate; the catalysts used are: 1% to 30% Pd-C, 1% to 30 %Pd(OH) ₂ -C, Raney-Ni, the preferred catalyst is: Raney-Ni, 5% to 20% Pd-C, 5% to 20% Pd(OH) ₂ -C; compound (6) and catalyst The mass ratio is 1.0:0.01～1.0; the reaction pressure is normal pressure～10.0MPa, preferably normal pressure～2.0MPa; the reaction temperature is room temperature～150°C, preferably room temperature～80°C; the reaction time is 1～48 hours, preferably 1 to 24 hours.

3-(6-amino-1-oxoisoindol-2-yl) methyl propionate (7) obtained by step B) is in a condensing agent with a guanidine-containing carboxylic acid compound (8) in a suitable solvent Condensation under existing conditions, 1,3-dihydro-1-oxo-2H-isoindole methyl ester compound (9); wherein, the solvent used for the reaction is: pyridine, N, N-dimethylformamide, C _3-8 Fatty ketones, ethers (such as diethyl ether, isopropyl ether, methyl tert-butyl ether, tetrahydrofuran, ethylene glycol dimethyl ether, etc.), esters formed by C _1-6 fatty acids and C _1-6 fatty alcohols , halogenated hydrocarbons (such as: methylene chloride, chloroform, 1,2-dichloroethane, etc.), aromatic hydrocarbons or substituted aromatic hydrocarbons (such as: benzene, toluene, xylene, chlorobenzene, o-dichlorobenzene, etc.) or Acetonitrile, reaction can be carried out in above-mentioned single solvent, also can carry out in above-mentioned mixed solvent, mixed solvent volume ratio is 1: 0.1～10, preferred solvent is pyridine, tetrahydrofuran, N,N-dimethylformamide, acetone, Chlorobenzene or acetonitrile; the condensing agent used is: chloroformic acid C _1-8 fatty alcohol esters (such as: ethyl chloroformate, tert-butyl chloroformate, benzyl chloroformate, etc.), N-ethoxycarbonyl-2- Ethoxy-1,2-dihydroquinoline (EEDQ), carbodiimide compounds (such as: dicyclohexylcarbodiimide (abbreviated as DCC), 1-ethyl-3-(3-dimethyl Aminopropyl) carbodiimide hydrochloride (abbreviated as EDCI)), diethyl cyanophosphate (DEPC), 2-chloro-4,6-dimethoxy-1,3,5-triazine ( CDMT for short), 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM for short), wherein, in DMTMM The anions are chlorine, bromine, perchlorate, fluoroborate, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, camphorsulfonic acid, aminosulfonic acid, and the preferred condensing agent is: ethyl chloroformate , dicyclohexylcarbodiimide (DCC), EDCI, DMTMM; Compound (7): compound (8): the molar feed ratio of condensing agent is 1.0: 1.0～4.5: 1.0～4.5, and preferred molar feed ratio is 1.0: 1.0～2.0:1.0～2.0; the condensation reaction temperature is 0～130°C, preferably the reaction temperature is 0～50°C; the condensation reaction time is 30 minutes～72 hours, preferably the reaction time is 2～48 hours.

The 1,3-dihydro-1-oxo-2H-isoindole methyl ester compound (9) obtained in step C) is subjected to alkali hydrolysis and acid neutralization to obtain 1,3-dihydro-1-oxo-2H - Isoindole compound (I), the obtained (I) is salted with the corresponding acid according to the conventional method to obtain the salt of the compound (I); wherein, the alkali used for hydrolysis is alkali metal or alkaline earth metal hydroxide, alkali Metal or alkaline earth metal carbonate, alkali metal or alkaline earth metal bicarbonate, preferred alkali is: lithium hydroxide, sodium carbonate; The molar feed ratio of alkali and compound (9) is 1.0～5.0: 1.0, preferred molar feed ratio is 1.1～2.5:1.0; the hydrolysis reaction temperature is room temperature～150°C, preferably the reaction temperature is room temperature reaction; the hydrolysis time is 0.5～24 hours, preferably the reaction time is 1～7 hours. the

The 1,3-dihydro-1-oxo-2H-isoindole compound (I) disclosed in the present invention can be combined with any suitable acid to obtain its pharmaceutically acceptable salt through a pharmaceutically conventional salt-forming method, Described acid is hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid, formic acid, trifluoroacetic acid, acetic acid, propionic acid, oxalic acid, benzoic acid, maleic acid, fumaric acid, succinic acid, tartaric acid, mandelic acid, lemon acid, methanesulfonic acid, ethylsulfonic acid, camphorsulfonic acid, benzenesulfonic acid or p-toluenesulfonic acid. the

The starting materials of the present invention—2-formyl-5-nitrobenzoic acid (4) and β-alanine methyl ester derivatives (5) can be prepared by common techniques in the art, including but not limited to Methods disclosed in the following documents: 1.R.L.Shriner, F.J.Wolf.Org.Syn.Coll.Vo1.3 (1955) 737; 2.V.T.Abaev, A.S.Dmitriev, A.V.Gutnov J.Heterocyclic Chem., 43(2006) 1195; 3. C.Y.K. Tana, D.F. Weaver. Tetrahedron 58 (2002) 7449. the

The pharmaceutical composition disclosed in the present invention includes one or more 1,3-dihydro-1-oxo-2H-isoindole compounds (I) or their pharmaceutically acceptable salts in a therapeutically effective amount. The composition may further contain one or more pharmaceutically acceptable carriers or excipients. The "therapeutically effective amount" refers to the amount of the drug or agent that causes the biological or medical response of the tissue, system or animal targeted by the researcher or doctor; the "composition" refers to the combination of more than one substance or The mixed product; the "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable substance, composition or carrier, such as: liquid or solid filler, diluent, excipient, solvent or encapsulation Substances that carry or transport a chemical substance. the

The 1,3-dihydro-1-oxo-2H-isoindole compound (I) or pharmaceutically acceptable salt thereof proposed by the present invention has been screened for the following biological activity:

(1) Solid-phase receptor screening

Screening was performed in 96-well culture plates. Integrin α _v β ₃ protein was prepared with Coating Buffer solution (Tris-HCl 20mM, NaCl 150mM, CaCl ₂ 1mM and NaN ₃ 0.02%) to a concentration of 4 μg/mL, added to a 96-well plate, 100 μL per well, and coated overnight at 4°C , the negative control was coated with 2% BSA in the same way, the supernatant was discarded, and 100 μL of 2% BSA was added to each well, and after blocking for 1 hour at 37 ° C, the Binding Buffer solution (Tris-HCl 50 mM, CaCl ₂ 2 mM, NaN ₃ 0.02% and BSA 1 mg/mL (0.1%)). Dilute the compound to be screened with Binding Buffer solution to different concentrations, take biotin-labeled Bio-Fibronectin solution (8 μg/mL) and mix with different concentrations of the compound to be screened, add to 96-well plate, 100 μL per well, negative control Add the same amount of Binding Buffer solution to the model group and the model group, incubate at 37°C for 3 hours, wash with Binding Buffer solution, add 100 μL of HRP-streptavidin (1:500) to each well, incubate at 37°C for 1 hour, and wash with Binding Buffer solution. Buffer solution was washed three times, and 100 μL of 3,3',5,5'-tetramethylbenzidine stock solution (TMB) was added to each well, incubated at room temperature for 10 minutes, and then 1.0 M sulfuric acid solution (100 μL per well) was added to stop the reaction. Measure the OD value of each hole on a microplate reader (wavelength is 450nm), calculate the binding inhibition rate of the compound to be screened and the integrin α _v β ₃ protein, inhibition rate (%)=[1-(test article OD value-negative Group OD)/(model group OD-negative group OD))×100.

Using the above method, the affinity of the target substance to the integrin α _v β ₃ protein was determined, and the results showed that the 1,3-dihydro-1-oxo-2H-isoindole compound (I) or its pharmaceutical The IC _{50 of the binding inhibition IC 50} of acceptable salts and integrin α _v β ₃ protein is 1.8nM-2000nM, while the IC ₅₀ of the positive control drug cyclo (RGDfV) is 10.0nM.

(2) Determination of cell adhesion inhibition

The 96-well culture plate was coated with 2.0 μg/mL Fibronectin overnight, and the control well was coated with 1% BSA, the supernatant was discarded, 100 μL of 1% BSA was added to each well, and blocked for 1 hour at room temperature. The compound to be screened was made into different concentrations with 0.01M PBS solution, and added to the M21 cell suspension with a concentration of 5×10 ⁵ /ml. The positive control was replaced by cyclo (RGDfV), and the blank control was replaced by 0.01M PBS solution. 37 Incubate at ℃ for 30 minutes, then add the M21 cell suspension to the above-mentioned pre-blocked 96-well culture plate, 100 μL per well, incubate at 37℃ for 1 hour, gently suck out the cell suspension in the well, wash 3 times with pre-warmed PBS , remove non-adherent cells, add 100 μL of 10% glutaraldehyde to each well to fix for 30 minutes, wash with deionized water until the glutaraldehyde is thoroughly washed, dry thoroughly in an oven at 37°C, add 0.1% crystal violet to the cells For staining, shake for 30 minutes, wash the excess crystal violet with distilled water, dry it thoroughly in an oven at 37°C, add 10% acetic acid to extract the crystal violet absorbed by the cells, and measure the OD value on a microplate reader after 1 hour (wavelength is 595nm), calculate the cell adhesion inhibition rate, and then the inhibitory activity of the target substance on integrin α _v β ₃ can be obtained. Using the above method, the adhesion inhibitory activity of some target substances on M21 cells was measured, and the results are shown in Example 10.

(3) Determination of target cytotoxic activity

In order to rule out that the anti-cell adhesion activity of the target substance on M21 cells is caused by cytotoxic factors, we tested the target substance against human liver cancer cell HepG2, human lung adenocarcinoma cell A549, mouse melanoma cell B16, Inhibitory activity of human melanoma cell line M21 and mouse colon cancer cell line C26. Tumor cells were digested and counted separately, and seeded in 96-well plate (5000 cells/well). After 24 hours, they were washed twice with PBS solution of pH 7.4. Drugs with different concentrations were added and cultured for 48 hours. After washing, they were washed with Stain with 0.4% sulforhodamine (SRB) solution at room temperature for 15 minutes, wash 3 times with 1% glacial acetic acid, dry the stained cells in air, dissolve with 10mM Tris-HCl, and measure the absorbance value at 540nm wavelength with Dynatech MR7000 instrument , to calculate the inhibitory rate of the drug on tumor cell growth. the

The test results show that the 1,3-dihydro-1-oxo-2H-isoindole compounds (I) disclosed in the present invention or their pharmaceutically acceptable salts have an IC ₅₀ of inhibition of these tumor cells greater than 80 μg/ mL, indicating that the cytotoxicity of such compounds is small.

The 1,3-dihydro-1-oxo-2H-isoindole compound (I) or a pharmaceutically acceptable salt thereof disclosed in the present invention has significant integrin α _v β ₃ inhibitory activity and can be used as an integrin Use of α _v β ₃ inhibitors in preventing or/and treating various symptoms or diseases caused by excessive neovascular proliferation, including but not limited to tumor metastasis, tumor growth, solid tumor growth, angiogenesis, retinopathy , macular degeneration, osteoporosis, arthritis, smooth muscle cell migration and atherosclerosis, etc.

Detailed ways

The present invention can be further described by the following examples, however, the scope of the present invention is not limited to the following examples. Those skilled in the art can understand that various changes and modifications can be made in the present invention without departing from the spirit and scope of the present invention. the

Example 1

Preparation of 3-(6-(3-guanidinobenzamido)-1-oxoisoindol-2-yl)-3-phenylpropion hydrochloride (I-1) and trifluoroacetate

Step (a): Add 10.0mmol of 2-formyl-5-nitrobenzoic acid (4), 12.0mmol of β-phenyl-β-alanine methyl ester and 80ml of methanol into the reaction flask, and stir at room temperature for 2.0h , slowly add NaBH ₄ 10.0mmol under cooling in an ice-water bath, keep stirring and react (the reaction process is monitored by TLC), after the reaction is completed, evaporate the solvent under reduced pressure, add 20ml of acetic acid to the residue, keep the reaction at 80°C for 40min, and pour into the reaction flask Add 100ml of deionized water to the mixture, filter the precipitated solid, wash the filter cake with aqueous sodium carbonate solution, and dry to obtain 3-(6-nitro-1-oxoisoindol-2-yl)-3-phenylpropionic acid Methyl ester, yield 90.0%, ESI-MS (m/z, +Q): 341.0[M+H] ⁺ ;

Step (b): Add 10.0mmol of 3-(6-nitro-1-oxoisoindol-2-yl)-3-phenylpropionic acid methyl ester and 200ml of methanol into the reaction flask, stir well, then add 10 %Pd/C0.5g, after replacing the air in the reaction bottle with hydrogen, carry out hydrogen reduction reaction at normal temperature and pressure for 12h (reaction progress is monitored by TLC, petroleum ether/ethyl acetate=1:2v/v), after the reaction , filtered, the filtrate was evaporated to remove the solvent under reduced pressure, and the residue was purified by recrystallization or column chromatography to obtain 3-(6-amino-1-oxoisoindol-2-yl)-3-phenylpropionic acid methyl ester , yield 99.0%, ESI-MS (m/z, +Q): 311.1[M+H] ⁺ ;

Step (c): Add 1.0 mmol of 3-(6-amino-1-oxoisoindol-2-yl)-3-phenylpropionic acid methyl ester, 1.2 m-guanidinobenzoic acid hydrochloride in the reaction flask mmol and pyridine 25ml, stirred at room temperature until the solid was completely dissolved, then added 1.5mmol of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, stirred at room temperature for 24-48h (the reaction process was determined by TLC monitoring, chloroform/methanol=5:1v/v), after the reaction finishes, filter, and the filtrate decompresses to remove solvent, and the residue is purified by recrystallization or column chromatography to obtain 3-(6-(3-guanidinobenzene Formamido)-1-oxoisoindol-2-yl)-3-phenylpropionic acid methyl ester hydrochloride, yield 80.5%, ESI-MS (m/z, +Q): 472.1 [M- Cl] ⁺ ;

Step (d): add 3-(6-(3-guanidinobenzamido)-1-oxoisoindol-2-yl)-3-phenylpropanoic acid methyl ester hydrochloride 0.5 mmol, 10ml of methanol, 5ml of tetrahydrofuran and 5ml of deionized water, stir well at room temperature, add lithium hydroxide 2.0mmol, stir at room temperature for 1.5h (the reaction process is monitored by TLC, chloroform/methanol/HOAc=3/1/0.5v/v ), after the reaction, adjust the pH of the solution to 1-2 with 10% hydrochloric acid aqueous solution, evaporate the solvent under reduced pressure, and the residue is purified by recrystallization or column chromatography to obtain 3-(6-(3-guanidinobenzyl Amino)-1-oxoisoindol-2-yl)-3-phenylpropionium hydrochloride, yield 65.0%, ¹ H NMR (400MHz, DMSO-d ₆ )δ: 12.45 (brs, 1H, COOH) 10.64 (s, 1H, CONH), 10.52 (brs, 1H, HCl), 8.21 (d, 1H, J = 1.6Hz, Ar-H), 7.95 (dd, 1H, _J1 = 1.6Hz, _J2 =8.4Hz, Ar-H), 7.89(d, 1H, J=8.0Hz, Ar-H), 7.86(s, 1H, Ar-H), 7.69(brs, 4H, NH ₂ C(NH)NH) , 7.59(d, 1H, J=8.0Hz, Ar-H), 7.59(t, 1H, J=8.0Hz, Ar-H), 7.53(d, 1H, J=8.0Hz, Ar-H), 7.43 (d, 2H, J=8.0Hz, Ar-H), 7.41-7.27(m, 5H, Ar-H), 5.76(t, 1H, J=8.0Hz, CH), 4.51(d, 1H, J= 17.6Hz, ArCH ₂ ), 4.14 (d, 1H, J=17.6Hz, ArCH ₂ ), 3.18-3.05 (m, 2H, CH ₂ COOH); ESI-MS (m/z): 458.10 [M-Cl] ⁺ ;

In the above step (d), after the hydrolysis is completed, the pH of the solution is adjusted to 1-2 with trifluoroacetic acid, the solvent is evaporated under reduced pressure, and the residue is purified by recrystallization or column chromatography to obtain 3-(6-(3- Guanidinobenzamido)-1-oxoisoindol-2-yl)-3-phenylpropionic acid trifluoroacetate, yield 60.0%. the

Example 2

Preparation of 3-(6-(4-guanidinobenzamido)-1-oxoisoindol-2-yl)-3-phenylpropion hydrochloride (I-2)

The operation process is the same as in Example 1, except that the m-guanidinobenzoic acid hydrochloride in the step (c) is replaced with p-guanidinobenzoic acid hydrochloride, and the condensing agent 1-ethyl-3-(3-dimethyl Aminopropyl) carbodiimide hydrochloride is replaced by DCC to obtain 3-(6-(4-guanidinobenzamido)-1-oxoisoindol-2-yl)-3-phenylpropionic acid Hydrochloride, yield 74.0%, ¹ H NMR (400MHz, DMSO-d ₆ ) δ: 12.42 (brs, 1H, COOH), 11.01 (brs, 1H, HCl), 10.64 (s, 1H, CONH), 8.26 (d, 1H, J = 1.6Hz, Ar-H), 8.11 (d, 2H, J = 8.4Hz, Ar-H), 7.98 (dd, 1H, J ₁ = 1.6Hz, J ₂ = 8.0Hz, Ar -H), 7.97 (brs, 4H, NH ₂ C(NH)NH), 7.52 (d, 1H, J=8.0Hz, Ar-H), 7.33 (d, 2H, J=8.4Hz, Ar-H) , 7.40-7.27(m, 5H, Ar-H), 5.77(t, 1H, J=8.0Hz, CH), 4.52(d, 1H, J=17.6Hz, ArCH ₂ ), 4.14(d, 1H, J =17.6 Hz, ArCH ₂ ), 3.13-3.02 (m, 2H, CH ₂ COOH); ESI-MS (m/z): 458.15 [M-Cl] ⁺ .

Example 3

3-[6-[3-[(4,5-Dihydro-1H-imidazol-2-yl)amino]benzamido]-1-oxoisoindol-2-yl]-3-phenylpropane Preparation of hydrochloride (I-3)

The operation process is the same as in Example 1, except that the m-guanidinobenzoic acid hydrochloride in the step (c) is 3-[(4,5-dihydro-1H-imidazol-2-yl)amino]benzoic acid salt Salt replacement, the condensing agent 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride is replaced by DCC, and 3-[6-[3-[(4,5-dihydro -1H-imidazol-2-yl)amino]benzamido]-1-oxoisoindol-2-yl]-3-phenylpropion hydrochloride, yield 85.0%, ¹ H NMR (400MHz, DMSO-d ₆ ) δ: 12.44 (brs, 1H, COOH), 10.89 (s, 1H, HCl), 10.78 (s, 1H, CONH), 8.58 (s, 2H, NHCNH), 8.25 (d, 1H, J =1.2Hz, Ar-H), 7.95(dd, 1H, J ₁ =1.2Hz, J ₂ =8.4Hz, Ar-H), 7.88-7.86(m, 2H, Ar-H), 7.59(t, 1H , J=8.0Hz, Ar-H), 7.53(d, 1H, J=8.0Hz, Ar-H), 7.45(d, 2H, J=8.0Hz, Ar-H), 7.41-7.28(m, 5H , Ar-H), 5.76 (t, 1H, J=8.0Hz, CH), 4.50 (d, 1H, J=17.6Hz, ArCH ₂ ), 4.12 (d, 1H, J=17.6Hz, ArCH ₂ ), 3.69 (s, 4H, _CH2CH2 ), 3.23-3.09 (m, 2H, _CH2COOH ); ESI-MS (m/z ₎ : 484.20 [M-Cl] ⁺ .

Example 4

3-[6-[4-[(4,5-Dihydro-1H-imidazol-2-yl)amino]benzamido]-1-oxoisoindol-2-yl]-3-phenylpropane Preparation of hydrochloride (I-4)

The operation process is the same as in Example 1, except that the m-guanidinobenzoic acid hydrochloride in the step (c) is replaced with 4-[(4,5-dihydro-1H-imidazol-2-yl)amino]benzoic acid salt Salt replacement, the condensing agent 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride is replaced by DCC, and 3-[6-[4-[(4,5-dihydro -1H-imidazol-2-yl)amino]benzamido]-1-oxoisoindol-2-yl]-3-phenylpropion hydrochloride, yield 78.0%, ¹ H NMR (400MHz, DMSO-d ₆ ) δ: 12.10 (brs, 1H, COOH), 11.00 (s, 1H, HCl), 10.51 (s, 1H, CONH), 8.75 (brs, 2H, NHCNH), 8.22 (d, 1H, J = 1.6Hz, Ar-H), 8.07 (d, 2H, J = 8.8Hz, Ar-H), 7.94 (dd, 1H, J ₁ = 1.2Hz, J ₂ = 8.4Hz, Ar-H), 7.54 ( d, 1H, J=8.4Hz, Ar-H), 7.33(d, 2H, J=8.8Hz, Ar-H), 7.40-7.28(m, 5H, Ar-H), 5.76(t, 1H, J =8.0Hz, CH), 4.51 (d, 1H, J=17.6Hz, ArCH ₂ ), 4.13 (d, 1H, J=17.6Hz, ArCH ₂ ), 3.69 (s, 4H, CH ₂ CH ₂ ), 3.20 -3.07 (m, 2H, _CH2COOH ); ESI-MS (m/z): 484.20 [M-Cl] ⁺ .

Example 5

3-(3,5-Dichlorophenyl)-3-[6-(3-guanidinobenzamido)-1-oxoisoindol-2-yl]propion hydrochloride (I-5) preparation of

Step (a): The operation process is the same as step (a) of Example 1, except that β-phenyl-β-alanine methyl ester is replaced with β-(3,5-dichlorophenyl)-β-alanine methyl ester Ester substitution, 3-(3,5-dichlorophenyl)-3-(6-nitro-1-oxoisoindol-2-yl) methyl propionate, yield 82.0%, ESI-MS ( m/z,+Q): 409.0[M+H] ⁺ ;

Step (b): Dissolve 10.0 mmol of methyl 3-(3,5-dichlorophenyl)-3-(6-nitro-1-oxoisoindol-2-yl)propionate in 150 ml of tetrahydrofuran, Add 25ml of acetic acid and 160.0mmol of zinc powder, heat to reflux for 4h (the reaction process is monitored by TLC, petroleum ether/ethyl acetate=1:2v/v), after the reaction is completed, cool to room temperature, and adjust the pH of the solution with saturated aqueous sodium bicarbonate to alkaline, filter, the filtrate was extracted with 3×70ml CH ₂ Cl ₂ , the organic layers were combined and washed with saturated brine, the organic layer was dried over anhydrous NaSO ₄ , the solvent was evaporated under reduced pressure, and the residue was subjected to recrystallization or column chromatography Purify to obtain 3-(3,5-dichlorophenyl)-3-(6-amino-1-oxoisoindol-2-yl)propionic acid methyl ester with a yield of 96.0%, ESI-MS (m /z,+Q): 365.1[M+H] ⁺ ;

Step (c): The operation process is the same as that of Example 1 step (c), except that 3-(6-amino-1-oxoisoindol-2-yl)-3-phenylpropionic acid methyl ester is used with 3-(3 , 5-dichlorophenyl)-3-(6-amino-1-oxoisoindol-2-yl) methyl propionate instead, 3-(3,5-dichlorophenyl)-3-[ 6-(3-guanidinobenzamido)-1-oxoisoindol-2-yl]propionic acid methyl ester hydrochloride, yield 80.0%, ESI-MS (m/z, +Q): 540.0 [M-Cl] ⁺ ;

Step (d): The operation process is the same as step (d) of Example 1, except that 3-(6-(3-guanidinobenzamido)-1-oxoisoindol-2-yl)-3-phenyl Methyl propionate hydrochloride with 3-(3,5-dichlorophenyl)-3-[6-(3-guanidinobenzamido)-1-oxoisoindol-2-yl]propionic acid Substitution of methyl ester hydrochloride to obtain 3-(3,5-dichlorophenyl)-3-[6-(3-guanidinobenzamido)-1-oxoisoindol-2-yl]propionic acid Hydrochloride, yield 93.0%, ¹ H NMR (400MHz, DMSO-d ₆ ) δ: 12.53 (brs, 1H, COOH), 10.62 (s, 1H, HCl), 10.12 (s, 1H, CONH), 8.23 (d, 1H, J = 1.2Hz, Ar-H), 7.96 (dd, 1H, J ₁ = 2.0Hz, J ₂ = 8.4Hz, Ar-H), 7.91 (d, 1H, J = 8.0Hz, Ar -H), 7.87(s, 1H, Ar-H), 7.63(brs, 4H, NH ₂ C(NH)NH), 7.59(d, 1H, J=8.0Hz, Ar-H), 7.57(d, 1H, J=2.0Hz, Ar-H), 7.56(d, 1H, J=8.4Hz, Ar-H), 7.48(d, 2H, J=1.6Hz, Ar-H), 7.45(d, 1H, J=8.0Hz, Ar-H), 5.65(t, 1H, J=7.6Hz, CH), 4.52(d, 1H, J=17.6Hz, _ArCH2 ), 4.30(d, 1H, J=17.6Hz, ArCH ₂ ), 3.27-3.18 (m, 2H, CH ₂ COOH); ESI-MS (m/z): 526.15 [M-Cl] ⁺ .

Example 6

3-(3,5-Dichlorophenyl)-3-[6-(4-guanidinobenzamido)-1-oxoisoindol-2-yl]propion hydrochloride (I-6) preparation of

The operation process is the same as in Example 5, except that the m-guanidinobenzoic acid hydrochloride in the step (c) is replaced with p-guanidinobenzoic acid hydrochloride, and the condensing agent 1-ethyl-3-(3-dimethyl Aminopropyl) carbodiimide hydrochloride is replaced by DCC to obtain 3-(3,5-dichlorophenyl)-3-[6-(4-guanidinobenzamido)-1-oxoisoind Indol-2-yl]propion hydrochloride, yield 97.0%, ¹ H NMR (400MHz, DMSO-d ₆ ) δ: 12.51 (brs, 1H, COOH), 10.53 (s, 1H, HCl), 10.32 ( brs, 1H, CONH), 8.24 (d, 1H, J = 1.6Hz, Ar-H), 8.08 (d, 2H, J = 8.8Hz, Ar-H), 7.96 (dd, 1H, J ₁ = 2.0Hz , J ₂ =8.4Hz, Ar-H), 7.74 (brs, 4H, NH ₂ C(NH)NH), 7.57 (d, 1H, J=2.0Hz, Ar-H), 7.55 (d, 1H, J =8.4Hz, Ar-H), 7.48(d, 2H, J=1.6Hz, Ar-H), 7.38(d, 2H, J=8.8Hz, Ar-H), 5.65(t, 1H, J=7.6 Hz, CH), 4.52 (d, 1H, J=17.6Hz, ArCH ₂ ), 4.30 (d, 1H, J=17.6Hz, ArCH ₂ ), 3.29-3.27 (m, 2H, CH ₂ COOH); ESI- MS (m/z): 526.10 [M-Cl] ⁺ .

Example 7

3-(3,5-dichlorophenyl)-3-[6-[3-[(4,5-dihydro-1H-imidazol-2-yl)amino]benzamido]-1-oxoiso Preparation of indol-2-yl] propionate hydrochloride (I-7)

The operating process is the same as in Example 5, except that the m-guanidinobenzoic acid hydrochloride in the step (c) is replaced with 3-[(4,5-dihydro-1H-imidazol-2-yl)amino]benzoic acid salt Salt replacement, condensing agent 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride is replaced by DCC, and 3-(3,5-dichlorophenyl)-3-[ 6-[3-[(4,5-Dihydro-1H-imidazol-2-yl)amino]benzamido]-1-oxoisoindol-2-yl]propion hydrochloride, yield 98.0 %, ¹ H NMR (400MHz, DMSO-d ₆ ) δ: 12.53 (brs, 1H, COOH), 10.90 (s, 1H, HCl), 10.81 (s, 1H, CONH), 8.60 (brs, 2H, NHCNH), 8.26(d, 1H, J=2.0Hz, Ar-H), 8.01(dd, 1H, _J1 =2.0Hz, _J2 =8.4Hz, Ar-H), 7.88(d, 1H, J=8.0Hz, Ar-H), 7.87(s, 1H, Ar-H), 7.61(d, 1H, J=8.0Hz, Ar-H), 7.57(d, 1H, J=8.4Hz, Ar-H), 7.56( d, 1H, J=2.0Hz, Ar-H), 7.48(d, 2H, J=2.0Hz, Ar-H), 7.45(d, 1H, J=8.0Hz, Ar-H), 5.65(t, 1H, J=7.6Hz, CH), 4.52(d, 1H, J=17.6Hz, _ArCH2 ), 4.30(d, 1H, J=17.6Hz, _{ArCH2 )} , 3.70(s, 4H, _CH2CH2 ), 3.30-3.18 (m, 2H, CH ₂ COOH); ESI-MS (m/z): 552.20 [M-Cl] ⁺ .

Example 8

3-(3,5-dichlorophenyl)-3-[6-[4-[(4,5-dihydro-1H-imidazol-2-yl)amino]benzamido]-1-oxoiso Preparation of indol-2-yl] propionate hydrochloride (I-8)

The operation process is the same as in Example 5, except that the m-guanidinobenzoic acid hydrochloride in the step (c) is replaced with 4-[(4,5-dihydro-1H-imidazol-2-yl)amino]benzoic acid salt Salt replacement, condensing agent 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride is replaced by DCC, and 3-(3,5-dichlorophenyl)-3-[ 6-[4-[(4,5-Dihydro-1H-imidazol-2-yl)amino]benzamido]-1-oxoisoindol-2-yl]propion hydrochloride, yield 90.0 %, ¹ H NMR (400MHz, DMSO-d ₆ ) δ: 12.49 (brs, 1H, COOH), 11.86 (brs, 1H, HCl), 10.58 (s, 1H, CONH), 8.85 (brs, 2H, NHCNH) , 8.25(d, 1H, J=2.4Hz, Ar-H), 8.08(d, 2H, J=8.8Hz, Ar-H), 7.97(dd, 1H, _J1 =1.2Hz, _J2 =8.0Hz , Ar-H), 7.56(d, 1H, J=1.2Hz, Ar-H), 7.55(d, 1H, J=8.0Hz, Ar-H), 7.47(d, 2H, J=2.4Hz, Ar -H), 7.37 (d, 2H, J=8.8Hz, Ar-H), 5.66 (t, 1H, J=7.6Hz, CH), 4.53 (d, 1H, J=17.6Hz, ArCH ₂ ), 4.31 (d, 1H, J=17.6Hz, ArCH ₂ ), 3.69 (s, 4H, CH ₂ CH ₂ ), 3.24-3.13 (m, 2H, CH ₂ COOH); ESI-MS (m/z): 552.15[ M-Cl] ⁺ .

Example 9

Adopt the similar synthetic method of embodiment 1～8, just replace corresponding reaction substrate, can obtain corresponding 1,3-dihydro-1-oxo-2H-isoindole compound (I), its structural formula as follows:

3-[6-(2-guanidinoacetamido)-1-oxoisoindol-2-yl]propion hydrochloride (I-9) 

Yield 40.5%, ESI-MS (m/z): 320.10[M-Cl] ⁺ ;

3-[6-(3-guanidinopropionamido)-1-oxoisoindol-2-yl]propion hydrochloride (I-10) 

Yield 36.0%, ESI-MS (m/z): 334.20[M-Cl] ⁺ ;

3-[1-Oxo-6-[3-[(1,4,5,6-tetrahydropyrimidin-2-yl)amino]benzamido]isoindol-2-yl]-3-phenyl Propion hydrochloride (I-11)

Yield 79.5%, ESI-MS (m/z): 498.20[M-Cl] ⁺ ;

3-[1-Oxo-6-[4-[(1,4,5,6-tetrahydropyrimidin-2-yl)amino]benzamido]isoindol-2-yl]-3-phenyl Propion hydrochloride (I-12)

Yield 77.0%, ESI-MS (m/z): 498.10[M-Cl] ⁺ ;

3-(3,5-dichlorophenyl)-3-[1-oxo-6-[3-[(1,4,5,6-tetrahydropyrimidin-2-yl)amino]benzamido] Isoindol-2-yl]propion hydrochloride (I-13)

Yield 87.5%, ESI-MS (m/z): 566.10[M-Cl] ⁺ ;

3-(3,5-dichlorophenyl)-3-[1-oxo-6-[4-[(1,4,5,6-tetrahydropyrimidin-2-yl)amino]benzamido] Isoindol-2-yl]propion hydrochloride (I-14)

Yield 90.0%, ESI-MS (m/z): 566.05[M-Cl] ⁺ ;

3-(3,5-dichlorophenyl)-3-[1-oxo-6-[3-[(5-fluoro-1,4,5,6-tetrahydropyrimidin-2-yl)amino]benzene Formamido]isoindol-2-yl]propion hydrochloride (I-15)

Yield 85.6%, ESI-MS (m/z): 584.10[M-Cl] ⁺ ;

3-(3,5-dichlorophenyl)-3-[1-oxo-6-[3-[(5-methyl-1,4,5,6-tetrahydropyrimidin-2-yl)amino] Benzamido]isoindol-2-yl]propion hydrochloride (I-16)

Yield 87.3%, ESI-MS (m/z): 580.20[M-Cl] ⁺ ;

3-(3,5-dichlorophenyl)-3-[1-oxo-6-[3-[(5-trifluoromethyl-1,4,5,6-tetrahydropyrimidin-2-yl) Amino]benzamido]isoindol-2-yl]propion hydrochloride (I-17)

Yield 93.5%, ESI-MS (m/z): 634.10[M-Cl] ⁺ ;

3-(3,5-dichlorophenyl)-3-[1-oxo-6-[3-[(5-hydroxy-1,4,5,6-tetrahydropyrimidin-2-yl)amino]benzene Formamido]isoindol-2-yl]propion hydrochloride (I-18)

Yield 80.5%, ESI-MS (m/z): 582.15[M-Cl] ⁺ ;

3-(4-methoxyphenyl)-3-[6-[3-[(4,5-dihydro-1H-imidazol-2-yl)amino]benzamido]-1-oxoisoindo Indol-2-yl]propion hydrochloride (I-19)

Yield 80.5%, ESI-MS (m/z): 514.20[M-C1] ⁺ ;

3-(4-methoxyphenyl)-3-[1-oxo-6-[3-[(1,4,5,6-tetrahydropyrimidin-2-yl)amino]benzamido]iso Indol-2-yl]propion hydrochloride (I-20)

Yield 77.3%, ESI-MS (m/z): 528.20[M-Cl] ⁺ ;

3-(4-methoxyphenyl)-3-[6-[4-[(4,5-dihydro-1H-imidazol-2-yl)amino]benzamido]-1-oxoisoindo Indol-2-yl]propion hydrochloride (I-21)

Yield 79.5%, ESI-MS (m/z): 514.15[M-Cl] ⁺ ;

3-(4-methoxyphenyl)-3-[1-oxo-6-[3-[(5-fluoro-1,4,5,6-tetrahydropyrimidin-2-yl)amino]benzidine Amino]isoindol-2-yl]propion hydrochloride (I-22)

Yield 85.0%, ESI-MS (m/z): 546.30[M-Cl] ⁺ ;

3-(4-Bromophenyl)-3-[6-[3-[(4,5-dihydro-1H-imidazol-2-yl)amino]benzamido]-1-oxoisoindole- 2-yl] propionate hydrochloride (I-23)

Yield 88.5%, ESI-MS (m/z): 562.20[M-Cl] ⁺ ;

3-(4-Bromophenyl)-3-[1-oxo-6-[3-[(1,4,5,6-tetrahydropyrimidin-2-yl)amino]benzamido]isoindole -2-yl] propionate hydrochloride (I-24) 

Yield 90.0%, ESI-MS (m/z): 576.15[M-Cl] ⁺ ;

3-(4-Bromophenyl)-3-[1-oxo-6-[3-[(5-trifluoromethyl-1,4,5,6-tetrahydropyrimidin-2-yl)amino]benzene Formamido]isoindol-2-yl]propion hydrochloride (I-25)

Yield 82.3%, ESI-MS (m/z): 644.10[M-Cl] ⁺ ;

3-(3-nitrophenyl)-3-[6-[3-[(4,5-dihydro-1H-imidazol-2-yl)amino]benzamido]-1-oxoisoindole -2-yl] propionate hydrochloride (I-26) 

Yield 75.5%, ESI-MS (m/z): 529.20[M-Cl] ⁺ ;

3-(3-nitrophenyl)-3-[1-oxo-6-[3-[(1,4,5,6-tetrahydropyrimidin-2-yl)amino]benzamido]isoindo Indol-2-yl]propion hydrochloride (I-27)

Yield 73.2%, ESI-MS (m/z): 543.25[M-Cl] ⁺ ;

3-(4-methoxy-3-nitrophenyl)-3-[6-[3-[(4,5-dihydro-1H-imidazol-2-yl)amino]benzamido]- 1-Oxoisoindol-2-yl]propion hydrochloride (I-28)

Yield 83.2%, ESI-MS (m/z): 559.20[M-Cl] ⁺ ;

3-(4-methoxy-3-nitrophenyl)-3-[1-oxo-6-[3-[(1,4,5,6-tetrahydropyrimidin-2-yl)amino]benzene Formamido]isoindol-2-yl]propion hydrochloride (I-29)

Yield 75.3%, ESI-MS (m/z): 573.15[M-Cl] ⁺ ;

3-(4-Dimethylamino-3-nitrophenyl)-3-[6-[3-[(4,5-dihydro-1H-imidazol-2-yl)amino]benzamido] -1-Oxoisoindol-2-yl]propion hydrochloride (I-30)

Yield 70.0%, ESI-MS (m/z): 572.25[M-Cl] ⁺ ;

3-(4-Dimethylamino-3-nitrophenyl)-3-[6-[4-[(4,5-dihydro-1H-imidazol-2-yl)amino]benzamido] -1-Oxoisoindol-2-yl]propion hydrochloride (I-31)

Yield 68.5%, ESI-MS (m/z): 572.20[M-Cl] ⁺ ;

3-(4-chloro-3-nitrophenyl)-3-[6-[3-[(4,5-dihydro-1H-imidazol-2-yl)amino]benzamido]-1- Oxyisoindol-2-yl]propion hydrochloride (I-32)

Yield 82.0%, ESI-MS (m/z): 563.15[M-Cl] ⁺ ;

3-(4-chloro-3-nitrophenyl)-3-[6-[4-[(4,5-dihydro-1H-imidazol-2-yl)amino]benzamido]-1- Oxyisoindol-2-yl]propion hydrochloride (I-33)

Yield 75.5%, ESI-MS (m/z): 563.15[M-Cl] ⁺ ;

3-(3,4-dichlorophenyl)-3-[6-[3-[(4,5-dihydro-1H-imidazol-2-yl)amino]benzamido]-1-oxoiso Indol-2-yl]propion hydrochloride (I-34)

Yield 86.0%, ESI-MS (m/z): 552.10[M-Cl] ⁺ ;

3-(3,4-dichlorophenyl)-3-[6-[4-[(4,5-dihydro-1H-imidazol-2-yl)amino]benzamido]-1-oxoiso Indol-2-yl]propion hydrochloride (I-35)

Yield 82.0%, ESI-MS (m/z): 552.20[M-Cl] ⁺ ;

3-(3,4-dimethoxyphenyl)-3-[6-[3-[(4,5-dihydro-1H-imidazol-2-yl)amino]benzamido]-1- Oxyisoindol-2-yl]propion hydrochloride (I-36)

Yield 75.0%, ESI-MS (m/z): 544.30[M-Cl] ⁺ ;

3-(3,4-dimethoxyphenyl)-3-[6-[4-[(4,5-dihydro-1H-imidazol-2-yl)amino]benzamido]-1- Oxyisoindol-2-yl]propion hydrochloride (I-37)

Yield 72.9%, ESI-MS (m/z): 544.20[M-Cl] ⁺ ;

3-(Benzo[d][1,3]diox-5-yl)-3-[6-[3-[(4,5-dihydro-1H-imidazol-2-yl)amino]benzyl Amino]-1-oxoisoindol-2-yl]propion hydrochloride (I-38)

Yield 77.5%, ESI-MS (m/z): 528.20[M-Cl] ⁺ ;

3-(Benzo[d][1,3]diox-5-yl)-3-[6-[4-[(4,5-dihydro-1H-imidazol-2-yl)amino]benzyl Amino]-1-oxoisoindol-2-yl]propion hydrochloride (I-39)

Yield 69.5%, ESI-MS (m/z): 528.10[M-Cl] ⁺ ;

3-(Benzo[d][1,3]diox-4-yl)-3-[6-[3-[(4,5-dihydro-1H-imidazol-2-yl)amino]benzyl Amino]-1-oxoisoindol-2-yl]propion hydrochloride (I-40)

Yield 65.6%, ESI-MS (m/z): 528.05[M-Cl] ⁺ ;

3-(Benzo[d][1,3]diox-4-yl)-3-[6-[4-[(4,5-dihydro-1H-imidazol-2-yl)amino]benzyl Amino]-1-oxoisoindol-2-yl]propion hydrochloride (I-41)

Yield 63.0%, ESI-MS (m/z): 528.20[M-Cl] ⁺ ;

3-(2-furyl)-3-[6-[3-[(4,5-dihydro-1H-imidazol-2-yl)amino]benzamido]-1-oxoisoindole-2 -yl] propionate hydrochloride (I-42)

Yield 55.0%, ESI-MS (m/z): 474.15[M-Cl] ⁺ ;

3-(2-thienyl)-3-[6-[3-[(4,5-dihydro-1H-imidazol-2-yl)amino]benzamido]-1-oxoisoindole-2 -yl] propionate hydrochloride (I-43)

Yield 72.0%, ESI-MS (m/z): 490.20[M-Cl] ⁺ ;

3-(3-thienyl)-3-[6-[3-[(4,5-dihydro-1H-imidazol-2-yl)amino]benzamido]-1-oxoisoindole-2 -yl] propionate hydrochloride (I-44)

Yield 70.3%, ESI-MS (m/z): 490.15[M-Cl] ⁺ ;

3-(pyridin-3-yl)-3-[6-[3-[(4,5-dihydro-1H-imidazol-2-yl)amino]benzamido]-1-oxoisoindole- 2-yl] propionate hydrochloride (I-45)

Yield 62.5%, ESI-MS (m/z): 485.20[M-Cl] ⁺ ;

3-(pyridin-3-yl)-3-[6-[4-[(4,5-dihydro-1H-imidazol-2-yl)amino]benzamido]-1-oxoisoindole- 2-yl] propionate hydrochloride (I-46)

Yield 60.0%, ESI-MS (m/z): 485.20[M-Cl] ⁺ ;

3-(pyridin-4-yl)-3-[6-[3-[(4,5-dihydro-1H-imidazol-2-yl)amino]benzamido]-1-oxoisoindole- 2-yl] propionate hydrochloride (I-47)

Yield 65.5%, ESI-MS (m/z): 485.25[M-Cl] ⁺ ;

3-(pyridin-4-yl)-3-[6-[4-[(4,5-dihydro-1H-imidazol-2-yl)amino]benzamido]-1-oxoisoindole- 2-yl] propionate hydrochloride (I-48)

Yield 57.0%, ESI-MS (m/z): 485.20[M-Cl] ⁺ ;

3-(benzofuran-4-yl)-3-[6-[3-[(4,5-dihydro-1H-imidazol-2-yl)amino]benzamido]-1-oxoisoind Indol-2-yl]propion hydrochloride (I-49)

Yield 85.5%, ESI-MS (m/z): 524.25[M-Cl] ⁺ ;

3-(benzofuran-4-yl)-3-[6-[4-[(4,5-dihydro-1H-imidazol-2-yl)amino]benzamido]-1-oxoisoind Indol-2-yl]propionic acid hydrochloride (I-50)

Yield 87.0%, ESI-MS (m/z): 524.20[M-Cl] ⁺ ;

3-(7-Chloro-benzofuran-4-yl)-3-[6-[3-[(4,5-dihydro-1H-imidazol-2-yl)amino]benzamido]-1 -Oxyisoindol-2-yl]propion hydrochloride (I-51)

Yield 90.0%, ESI-MS (m/z): 558.20[M-Cl] ⁺ ;

3-(7-Chloro-benzofuran-4-yl)-3-[6-[4-[(4,5-dihydro-1H-imidazol-2-yl)amino]benzamido]-1 -Oxyisoindol-2-yl]propion hydrochloride (I-52)

Yield 93.2%, ESI-MS (m/z): 558.10[M-Cl] ⁺ ;

3-(2,3-Dihydrobenzofuran-4-yl)-3-[6-[3-[(4,5-dihydro-1H-imidazol-2-yl)amino]benzamido] -1-Oxoisoindol-2-yl]propion hydrochloride (I-53)

Yield 87.5%, ESI-MS (m/z): 526.20[M-Cl] ⁺ ;

3-(2,3-Dihydrobenzofuran-4-yl)-3-[6-[4-[(4,5-dihydro-1H-imidazol-2-yl)amino]benzamido] -1-Oxoisoindol-2-yl]propion hydrochloride (I-54)

Yield 85.6%, ESI-MS (m/z): 526.20[M-Cl] ⁺ ;

3-(2,3-Dihydrobenzofuran-5-yl)-3-[6-[3-[(4,5-dihydro-1H-imidazol-2-yl)amino]benzamido] -1-Oxoisoindol-2-yl]propion hydrochloride (I-55)

Yield 80.8%, ESI-MS (m/z): 526.25[M-Cl] ⁺ ;

3-(2,3-Dihydrobenzofuran-5-yl)-3-[6-[4-[(4,5-dihydro-1H-imidazol-2-yl)amino]benzamido] -1-Oxoisoindol-2-yl]propion hydrochloride (I-56)

Yield 82.0%, ESI-MS (m/z): 526.15 [M-Cl] ⁺ .

Embodiment 10 biological activity test result

Table 1: Inhibition rate of some targets on the adhesion of melanoma M21 cells on Fibronectin (n=3)

Conclusion: The 1,3-dihydro-1-oxo-2H-isoindole compound (I) or its pharmaceutically acceptable salt disclosed in the present invention can significantly inhibit the adhesion of melanoma cell M21 to fibronectin, and some The inhibitory activity of the target compound was stronger than that of the positive control drug—cyclo(RGDfV). the

Claims (3)

1. A method for preparing 1,3-dihydro-1-oxo- 2H -isoindole compounds represented by general formula ( I ) and pharmaceutically acceptable salts thereof,

In the _formula : R represents H, phenyl, 2-furyl, 2-thienyl, 3-thienyl, 3-pyridyl, 4-pyridyl, 3,4-methylenedioxyphenyl, 2 ,3-methylenedioxyphenyl,

,

,

, or

; R ₃ , R ₄ , and R ₅ each independently represent H, C ₁ ~C ₆ alkyl, C ₁ ~C ₆ alkoxy, hydroxyl, halogen, amino, nitro, cyano, carboxyl, CF ₃ , NR ₇ R ₈ , R ₆ represents H, C ₁ ~C ₆ alkyl, C ₁ ~C ₆ alkoxy, halogen, nitro, cyano or CF ₃ , R ₇ , R ₈ each independently represent C ₁ ~C ₆ alkyl, R ₃ , R ₄ , R ₅ , R ₆ can be in any possible position of the benzene ring, R ₃ , R ₄ , R ₅ can be the same or different; R ₂ means

,

,

, , n represents 1-3, m represents 0-2, R ₉ represents C ₁ ~ C ₆ alkyl, halogen, hydroxyl or CF ₃ ; It is characterized in that comprising the steps of:

( A ) Starting from 2-formyl-5-nitrobenzoic acid ( 4 ) and β-alanine methyl ester derivative ( 5 ), the 3-(6 -methyl nitro-1-oxoisoindol-2-yl)propionate ( 6 ); ( B ) The obtained intermediate ( 6 ) is reduced to an amino group by chemical reduction or catalytic hydrogenation reduction to obtain methyl 3-(6-amino-1-oxoisoindol-2-yl)propionate ( 7 ); ( C ) The intermediate ( 7 ) is condensed with the guanidino-containing carboxylic acid compound ( 8 ) in the presence of a condensing agent to obtain 1,3-dihydro-1-oxo-2 H -isoindolyl methyl ester compound ( 9 ); ( D ) ( 9 ) is hydrolyzed with alkali and neutralized with acid to obtain 1,3-dihydro-1-oxo-2 H -isoindole compound ( I ). 2. The preparation method of 1,3-dihydro-1-oxo- 2H -isoindole compound ( I ) and pharmaceutically acceptable salt thereof as claimed in claim 1, characterized in that step ( A ) Among them, the solvents used in the reaction are: C _1-8 fatty alcohol, N,N -dimethylformamide, diethyl ether, isopropyl ether, methyl tert-butyl ether, tetrahydrofuran, ethylene glycol dimethyl ether, C _1-6 Fatty acids, esters of C _1-6 fatty acids and C _1-6 fatty alcohols, dichloromethane, chloroform, 1,2-dichloroethane, benzene, toluene, xylene, chlorobenzene, o-dichlorobenzene or acetonitrile; The reducing agents used are: KBH ₄ , NaBH ₄ , LiBH ₄ , NaBH ₃ CN; 2-formyl-5-nitrobenzoic acid ( 4 ), β-alanine methyl ester derivative ( 5 ) and moles of reducing agent The feed ratio is 1.0:1.0~5.0:0.5~5.0; the reaction temperature is -20°C~120°C; the reaction time is 30 minutes~72 hours; In step ( B ), when the chemical reduction method is used, the solvent used for the reaction is: C ₁ ~ C ₆ fatty alcohol, tetrahydrofuran, ether, C _1-6 fatty acid, C _1-6 fatty acid and C _1-6 fatty alcohol Esters, benzene, toluene, 1,4-dioxane, ethylene glycol dimethyl ether, dichloromethane, chloroform, 1,2-dichloroethane, hydrochloric acid aqueous solution with a concentration of 1%-36%, the concentration is 1%-30% ammonium chloride aqueous solution, or a mixture of the above solvents; the reducing agent used is: zinc powder, iron powder or tin dichloride; the molar ratio of compound ( 6 ) to reducing agent is 1.0:1.0~10.0 ;The reaction temperature is 0°C~150°C; the reduction reaction time is 0.5~72 hours; When catalytic hydrogenation is used for reduction, the solvent used in the reaction is: C _1-8 fatty alcohol, diethyl ether, isopropyl ether, methyl tert-butyl ether, tetrahydrofuran, ethylene glycol dimethyl ether, C _1-6 fatty acid, C _1-6 Esters of fatty acids and C _1-6 fatty alcohols, benzene, toluene, xylene or acetonitrile; the catalysts used are: 1%~30% Pd-C, 1%~30% Pd(OH) ₂ -C, Raney-Ni ; The mass ratio of the compound ( 6 ) to the catalyst is 1.0:0.01~1.0; the reaction pressure is normal pressure~10.0 MPa; the reaction temperature is room temperature~150°C; the reaction time is 1~48 hours. 3. The preparation method of 1,3-dihydro-1-oxo-2 H -isoindole compound ( I ) and pharmaceutically acceptable salt thereof as claimed in claim 1, characterized in that step ( C ) Among them, the solvents used in the reaction are: pyridine, water, C _1-8 fatty alcohol, N, N-dimethylformamide, C _3-8 fatty ketone, ether, isopropyl ether, methyl tert-butyl ether, tetrahydrofuran, Ethylene glycol dimethyl ether, C _1-6 fatty acid, ester of C _1-6 fatty acid and C _1-6 fatty alcohol, dichloromethane, chloroform, 1,2-dichloroethane, benzene, toluene, xylene , chlorobenzene, o-dichlorobenzene or acetonitrile, the reaction can be carried out in the above-mentioned single solvent, or in the above-mentioned mixed solvent, the volume ratio of the mixed solvent is 1:0.1~10; the condensing agent used is: N -ethoxycarbonyl -2-Ethoxy-1,2-dihydroquinoline, dicyclohexylcarbodiimide, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, cyano Diethyl phosphate, 2-chloro-4,6-dimethoxy-1,3,5-triazine, 4-(4,6-dimethoxy-1,3,5-triazine-chloride 2-yl)-4-methylmorpholine salt; compound ( 7 ): compound ( 8 ): the molar feed ratio of condensing agent is 1.0: 1.0~4.5: 1.0~4.5; the condensation reaction temperature is 0~130°C; The reaction time is 30 minutes to 72 hours; In step ( D ), the base used for hydrolysis is alkali metal or alkaline earth metal hydroxide, alkali metal or alkaline earth metal carbonate, alkali metal or alkaline earth metal bicarbonate; the molar feed ratio of base to compound ( 9 ) is 1.0 ~5.0:1.0; the hydrolysis reaction temperature is room temperature ~150°C; the hydrolysis time is 0.5~24 hours.