CN103159667B - 6-guanidyl-2H-isoindole compound as well as preparation method and application thereof - Google Patents

Abstract

The invention discloses a 6-guanidyl-2H-isoindole compound (I) and pharmaceutically acceptable salts thereof as well as a preparation method and an application of the 6-guanidyl-2H-isoindole compound (I) in preparation of medicines for preventing or/and treating various tumor metastasis, tumor growth, solid tumor growth, angiogenesis, retinopathy, macular degeneration, osteoporosis, arthritis, smooth muscle cell migration and atherosclerosis symptoms or diseases caused by integrin alphaVbeta3 and/or alpha5beta1 mediates.

Description

6-guanidine radicals-2H-isoindoles compound, Preparation Method And The Use

Technical field

The invention belongs to medicinal chemistry art, relate to 6-guanidine radicals-2 h-isoindoles compound ( i) and pharmacy acceptable salt, its preparation method, pharmaceutical composition and preparation prevention or/and treat various because of integrin alpha _vβ ₃and/or α ₅β ₁symptom caused by mediation or the purposes in disease medicament, include but not limited to metastases, tumor growth, implanted solid tumor growth, angiogenesis, retinopathy, macular degeneration, osteoporosis, sacroiliitis, smooth muscle cell migration and atherosclerosis etc.

Background technology

Angiogenesis (angiogenesis) refers to the process producing neovasculature from already present vescular bed, under normal physiological conditions, vasculogenesis is tightly controlled in some of short duration, specific physiological process, as reproduction, growth course and wound healing process etc., and the vasculogenesis of survival is some pathology, as: the characteristic of the malignancy, retinopathy, macular degeneration, sacroiliitis, osteoporosis etc. of tumor tissues.Research shows, the angiogenesis of tumor inducing and the propagation of tumour cell, invade moisten and shift closely related, the malignancy that new vessel is not only tumour cell provides sufficient nutrient material, but also for tumour cell amphi position transfer provide path, therefore, the angiogenesis of Tumor suppression induction, reducing or block the blood supply of tumor tissues and " died of hunger " by tumour cell, is the effective way of preventing and treating metastases.This starvation cure is compared with traditional cell toxicant series antineoplastic medicament, because anti-angiogenic drugs makes tumor cell proliferation cycle stretch-out by reducing or block the blood supply of tumor tissues, promote tumor cell necrosis and work, instead of directly kill tumour cell, therefore, this kind of agents on normal cells, without obvious toxic-side effects, shows good tumor-targeting; And this therapy can overcome the resistance problems in classic chemotherapy drug treatment, and resistance is one of major reason of current chemotherapeutic treatment means failure.

Nearest research shows, the angiogenesis of tumor inducing and metastases and integrin (Integrins) closely related, integrin belongs to cell surface adhesion molecule family, the heterodimer transmembrane glycoprotein be formed by connecting by non covalent bond by α and β two subunits.Found more than 20 kind of integrin hypotype so far altogether, different subtype has different cell characteristics and adhesion characteristics, they by mediated cell and cell, cell and extracellular matrix (extracellular matrix, eCM) adhesion and participate in the signal transduction of cell and important regulating and controlling effect, wherein integrin alpha risen to the adhesion, propagation, transfer, apoptosis etc. of cell _vβ ₃and/or α ₅β ₁in tumor growth, local infiltration, transfer, particularly play an important role in the process such as tumor inducing vasculogenesis.

Integrin alpha _vβ ₃and α ₅β ₁be made up of α and β two kinds of subunits, be divided into extracellular region, cross-film district and intracellular region three part; The structural research of its extracellular region is shown, its extracellular region becomes avette " head " by the N-terminal sections fit of α and β subunit, two almost parallel " tails " are extended from " head ", the combination of part occurs in the zone of action of α and β subunit, and ligand binding domain mainly comprises the β-propeller structural domain of α subunit and the β of β subunit _astructural domain, and the participation needing divalent cation, positively charged ion in ligand binding not only with acidic-group effect mediating ligand and the α of part _vβ ₃or α ₅β ₁combination, can also stable alpha _vβ ₃or α ₅β ₁ligand binding surface.Integrin alpha _vβ ₃and α ₅β ₁cerebrospinal fluid be containing Arg-Gly-Asp in extracellular matrix components (as: fibronectin (fibronectin), laminin (laminin), hyaluronectin (vitronectin) and thrombospondin (thromobospondin) etc.) (arg-Gly-Asp, RGD, 1)the sequence of tripeptides, and the identification of extracellular matrix composition has selectivity, works as α _vβ ₃or α ₅β ₁after RGD in matrix components is combined, make vascular endothelial cell proliferation, differentiation and migration by Mitogen-actived protein kinase approach, promote segment dislocation and become new vessel.Experiment proves, uses exogenous sequence peptide containing RGD or analogue, can the adhesion of inhibition tumor cell and migration, inducing apoptosis of tumour cell and Tumor suppression angiogenesis, finally causes tumor growth to be suppressed, even tumor regression.Due to integrin alpha _vβ ₃normal vascular endothelial cell and most normal organ system are seldom expressed, just high expression level on the osteoclast and invasive tumour cell of the vascular endothelial cell being subject to activating under angiogenesis factor (angiogenic growth factors) stimulates, activation; And neovascular endothelium cell belongs to the non-malignant cell of inheritance stability, the α on its surface _vβ ₃and/or α ₅β ₁acceptor is not undergone mutation, with inhibitor mechanism in can not produce drug resistance problems; In addition, α _vβ ₃and α ₅β ₁acceptor is positioned at cell surface, direct and contacting blood, and medicine is easy to arrive target spot; Therefore, integrin alpha _vβ ₃and α ₅β ₁be that design is efficient, the anti-angiogenic rebirth of highly selective and the comparatively ideal target of medicine for anti transfer of tumor.

Find integrin alpha _vβ ₃and/or α ₅beta inhibitor, for discovery anti-angiogenic rebirth and medicine for anti transfer of tumor significant, the common diseases such as the tumor growth because new vessel hyperplasia causes, implanted solid tumor growth, retinopathy, macular degeneration, osteoporosis, sacroiliitis, smooth muscle cell migration and atherosclerosis can also be used for the treatment of.

。

At present, with integrin alpha _vβ ₃for the anti-angiogenic rebirth of action target spot and medicine for anti transfer of tumor are still in the clinical or preclinical test stage, these inhibitor can be divided three classes, that is: monoclonal antibody, polypeptide and cyclic peptide and stand-in, non-peptide micromolecular.Vitaxin and Vitaxin II is the α of Eli Lilly company exploitation _vβ ₃monoclonal antibody, can with integrin alpha _vβ ₃specific binding, Tumor suppression growth and angiogenesis, just carry out II clinical trial phase at present, is used for treating prostate cancer, the melanoma of transfer, Advanced Colon Cancer and rheumatic arthritis.Research finds, uses the exogenous linear peptides containing RGD sequence or pdef polypeptide, as: RGDS, GRGDS, poly-RGDS etc. can stop the angiogenesis of bFGF, TNF-α and the various tumor fragment induction of people, and on already present blood vessel without impact.But these peptide quasi-molecules are flexible strong, cause integrin alpha _vβ ₃the selectivity of acceptor and avidity are all poor.Cyclic peptide due to its conformation within the specific limits in bond, therefore to different integrins, there is good selectivity.Cyclic peptide cyclo (RGDfV) (cyclo (-Arg-Gly-Asp-D-Phe-Val-), 2)it is the integrin alpha of a kind of high reactivity and highly selective _vβ ₃inhibitor, its vitro inhibition α _vβ ₃with the IC that its ligandin is combined ₅₀be 2.5 nmol/L; Cyclo (RGDfV) peptide backbone methylates by Merck company researchist on this basis, obtains more highly active peptidomimetics cyclo (RGDf-N (Me) V) (3, popular name Cilengitide ), its IC ₅₀be 0.58 nmol/L, can antagonism integrin alpha _vβ ₃the vascular endothelial cell of mediation and the interaction of extracellular matrix, the growth of check melanin knurl and angiogenesis, and the apoptosis of the brain tumor cell grown on vitronectin can be induced, current Cliengitide carries out II/III clinical trial phase, is used for the treatment of lung cancer, carcinoma of the pancreas, mammary cancer, melanoma, tumor of kidney and colorectal carcinoma.Because antibody and peptide medicament exist, vivo degradation, transformation period are short, bioavailability is low needs the deficiency such as intravenously administrable, a large amount of preparation difficulties, therefore, find and find non-peptide integrin like proteins α that is efficient, highly selective Orally-administrable _vβ ₃and/or α ₅β ₁inhibitor is one of key areas of anti-angiogenic rebirth and medicine for anti transfer of tumor research both at home and abroad at present.

Summary of the invention

The object of the invention is to disclose a class and there is 6-guanidine radicals-2 h-isoindoles chemical structure ( i) integrin alpha _vβ ₃and/or α ₅β ₁inhibitor or its pharmacy acceptable salt.

Another object of the present invention is to disclose such 6-guanidine radicals-2 h-isoindoles compound ( i) preparation method.

Another object of the present invention is openly to comprise 6-guanidine radicals-2 h-isoindoles compound ( i) or the pharmaceutical composition of its pharmacy acceptable salt.

Another object of the present invention is open 6-guanidine radicals-2 h-isoindoles compound ( i) or its pharmacy acceptable salt preparation prevention or/and treat various because of integrin alpha _vβ ₃and/or α ₅β ₁symptom caused by mediation or the purposes in disease medicament, include but not limited to metastases, tumor growth, implanted solid tumor growth, angiogenesis, retinopathy, macular degeneration, osteoporosis, sacroiliitis, smooth muscle cell migration and atherosclerosis etc.

6-guanidine radicals-2 disclosed in this invention h-isoindoles compound ( i) chemical structure of general formula be:

In formula: R ₁expression H, phenyl, 2-furyl, 2-thienyl, 3-thienyl, 3-pyridyl, 4-pyridyl, 3,4-methylenedioxyphenyl bases, 2,3-methylenedioxyphenyl bases, , , , or ; R ₃, R ₄, R ₅represent H, C independently of one another ₁~ C ₆alkyl, C ₁~ C ₆alkoxyl group, hydroxyl, halogen, amino, nitro, cyano group, carboxyl, CF ₃, NR ₇r ₈, R ₆represent H, C ₁~ C ₆alkyl, C ₁~ C ₆alkoxyl group, halogen, nitro, cyano group or CF ₃, R ₇, R ₈represent C independently of one another ₁~ C ₆alkyl, R ₃, R ₄, R ₅, R ₆can at any possible position of phenyl ring, R ₃, R ₄, R ₅can be identical, also can be different; R ₂represent , , ; M represents 1 or 2; N represents 0,1 or 2; R ₉represent C ₁~ C ₆alkyl, halogen, hydroxyl or CF ₃.

For 6-guanidine radicals-2 disclosed in this invention h-isoindoles compound ( i) when there is chiral carbon in molecule, compound ( i) be raceme or optically active body.

6-guanidine radicals-2 disclosed in this invention h-isoindoles compound ( i) prepare by following methods:

In formula: R ₁, R ₂with the definition of m and chemical structure of general formula ( i) identical;

With 6-guanidine radicals-1,3-dihydro-1-oxygen-2 h-isoindole carboxylic acid hydrochloride compounds ( 5) and Beta-alanine methyl esters compounds ( 6) raceme or optically active body be starting raw material, condensation under solvent and condensing agent existence condition, obtains 6-guanidine radicals-1,3-dihydro-1-oxygen-2 h-isoindole carboxylic acid methyl esters compounds ( 7) raceme or optically active body, then through basic hydrolysis, acid neutralization, obtain 6-guanidine radicals-2 h-isoindoles compound ( i) raceme or optically active body, gained compound ( i) more conventionally with corresponding sour salify, obtain compound ( i) salt of raceme or optically active body.

The each reactions steps of aforesaid method specifically describes as follows:

a)with 6-guanidine radicals-1,3-dihydro-1-oxygen-2 h-isoindole carboxylic acid hydrochloride compounds ( 5) and Beta-alanine methyl esters compounds ( 6) raceme or optically active body be starting raw material, condensation under solvent and condensing agent existence condition, obtains 6-guanidine radicals-1,3-dihydro-1-oxygen-2 h-isoindole carboxylic acid methyl esters compounds ( 7) raceme or optically active body, wherein, reacting solvent for use is: pyridine, n,N-dimethyl formamide, dimethyl sulfoxide (DMSO), C _3-8aliphatic ketone, ethers (as: ether, isopropyl ether, methyl tertiary butyl ether, tetrahydrofuran (THF), glycol dimethyl ether etc.), C _1-6lipid acid and C _1-6ester that fatty alcohol is formed, halohydrocarbon (as: methylene dichloride, chloroform, 1,2-ethylene dichloride etc.), aromatic hydrocarbon or substituted aroma hydrocarbon (as: benzene,toluene,xylene, chlorobenzene, orthodichlorobenzene 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 be pyridine, tetrahydrofuran (THF), n, n-dimethyl formamide, dimethyl sulfoxide (DMSO), acetone, chlorobenzene or acetonitrile, condensing agent used is: phosphinylidyne diimidazole (CDI), chloroformic acid C _1-8aliphatic alcohol ester compounds (as: Vinyl chloroformate, isobutylchloroformate, chloroformic acid benzyl ester etc.), n-ethoxycarbonyl-2-oxyethyl group-1, 2-dihydroquinoline (EEDQ), carbodiimide compound [as: dicyclohexylcarbodiimide (referred to as DCC), 1-ethyl-3-(3-dimethylamine propyl) carbodiimide hydrochloride (referred to as EDCI)], diethyl phosphorocyanidate (DEPC), 2-chloro-4, 6-dimethoxy-1, 3, 5-triazine (referred to as CDMT), chlorination 4-(4, 6-dimethoxy-1, 3, 5-triazine-2-base)-4-methylmorpholine salt (referred to as DMTMM), preferred condensing agent is: phosphinylidyne diimidazole (CDI), Vinyl chloroformate, dicyclohexylcarbodiimide (DCC), EDCI, DMTMM, compound ( 5): compound ( 6): the molar feed ratio of condensing agent is 1.0:0.8 ~ 10.0:1.0 ~ 10.0, and preferred feed ratio is 1.0:1.0 ~ 3.0:1.0 ~ 3.0, setting-up point is 0 ~ 130 DEG C, and preferable temperature is 0 ~ 50 DEG C, condensation reaction time is 30 minutes ~ 72 hours, and the preferred time is 2 ~ 48 hours.

By step a)6-guanidine radicals-1, the 3-dihydro-1-oxygen-2 obtained h-isoindole carboxylic acid methyl esters compounds ( 7) raceme or optically active body through basic hydrolysis, acid neutralization, obtain 6-guanidine radicals-2 h-isoindoles compound ( i) raceme or optically active body, gained ( i) more conventionally with corresponding sour salify, obtain compound ( i) salt of raceme or optically active body; Wherein, being hydrolyzed alkali used is alkali metal hydroxide, alkaline earth metal hydroxides, basic metal metal carbonate, alkaline earth metal carbonate, alkali metal hydrocarbonate or alkali metal bicarbonates, and preferred bases is: lithium hydroxide, sodium carbonate; Compound ( 7) be 1.0:1.0 ~ 5.0 with the molar feed ratio of alkali, preferred molar feed ratio is 1.0:1.0 ~ 3.0; Hydrolysising reacting temperature is room temperature ~ 150 DEG C, and preferable temperature is room temperature reaction; Hydrolysis time is 0.5 ~ 24 hour, and the preferred time is 1 ~ 7 hour.

6-guanidine radicals-2 disclosed in this invention h-isoindoles compound ( i) raceme or optically active body can obtain acceptable salt on its pharmacology with any suitable acid by pharmaceutically conventional salifying method, described acid is hydrochloric acid, Hydrogen bromide, nitric acid, sulfuric acid, phosphoric acid, formic acid, trifluoroacetic acid, acetic acid, propionic acid, oxalic acid, phenylformic acid, Whitfield's ointment, toxilic acid, fumaric acid, succsinic acid, tartrate, amygdalic acid, citric acid, C _1-6alkylsulphonic acid (as: methanesulfonic, ethane sulfonic acid etc.), camphorsulfonic acid, Phenylsulfonic acid or tosic acid.

Starting raw material of the present invention---6-guanidine radicals-1,3-dihydro-1-oxygen-2 h-isoindole carboxylic acid hydrochloride compounds ( 5) amino-1, the 3-dihydro-1-oxygen-2 of available 6- h-isoindole carboxylic acid compounds ( 4) technology that adopts this area common for substrate, including, but not limited to method [1. T. Suhs, B. Konig, Synthesis of guanidines in solution. disclosed in Publication about Document mini-Rev. Org. Chem. 2006, 3, 315-331; 2. C.A. Maryanoff, R.C. Stanzione, J.N. Plampin, J.E. Mills. A Convenient synthesis of guanidines from thioureas. j. Org. Chem. 1986, 51, 1882-1884; 3. A.R. Katritzky, B. V. Rogovoy. Recent developments in guanylating agents. aRKIVOC 2005, iv, 49-87; 4. A. E. Miller, J.J. Bischoff. A facile conversion of amino acids to guanidino acids. synthesis 1986, 777-779; 5. S.Iwama, T. Kitano, F. Fukuya. Discovery of a potent and selective avb3 integrin antagonist with strong inhibitory activity against neointima formation in rat balloon injury model. bioorg. Med. Chem. Lett. 2004, 14, 2567 – 2570] with corresponding guanidine radicals reagent react, then regulate reaction solution to prepare to strongly-acid with hydrochloric acid; Guanidine radicals reagent used is: , , , , , , , ; N represents 0,1 or 2; R ₉represent C ₁~ C ₆alkyl, halogen, hydroxyl or CF ₃.

The present invention's Beta-alanine methyl esters used compounds ( 6) raceme or optically active body can obtain by the technology that this area is common, including, but not limited to method disclosed in Publication about Document: 1. V.T. Abaev, A.S. Dmitriev, A. V. Gutnov j. Heterocyclic Chem., 43 ( 2006) 1195; 2. C.Y.K. Tana, D.F. Weaver. tetrahedron58 ( 2002) 7449.

Pharmaceutical composition disclosed in this invention comprises one or more 6-guanidine radicals-2 for the treatment of significant quantity h-isoindoles compound ( i) or its pharmacy acceptable salt, this pharmaceutical composition can contain one or more pharmaceutically acceptable carrier or vehicle further.Described " treatment significant quantity " refer to cause investigator or doctor for tissue, the biology of system or animal or the medicine of medicine reaction or the amount of medicament; Described " composition " refers to the product by more than one materials or component being mixed; Described " pharmaceutically acceptable carrier " refers to pharmaceutically acceptable material, composition or carrier, as: liquid or solid weighting agent, thinner, vehicle, solvent or packing material, they carry or transport certain chemical substance.

6-guanidine radicals-2 proposed by the invention h-isoindoles compound ( i) or its pharmacy acceptable salt carried out following bioactivity screening:

(1) solid phase integrin receptor screening

Integrin alpha _vβ ₃albumen Coating Buffer solution (Tris-HCl 20mM, NaCl 150mM, CaCl ₂1mM and NaN ₃0.02%) being made into concentration is 4 μ g/mL, adds in 96 orifice plates, every hole 100 μ L, 4 DEG C of bags are spent the night, negative control 2%BSA with method bag quilt, abandoning supernatant, every hole adds 2% BSA of 100 μ L, closes after 1 hour with Binding Buffer solution (Tris-HCl 50mM, CaCl for 37 DEG C ₂2mM, NaN ₃0.02% and BSA 1mg/mL (0.1%)) cleaning.Compound Binding Buffer solution dilution to be screened is become different concns, take biotin labeled Bio-Fibronectin solution (8 μ g/mL) to mix with the compound to be screened of different concns, add in 96 orifice plates, every hole 100 μ L, negative control group and model group add equivalent Binding Buffer solution, hatch 3 hours for 37 DEG C, after the cleaning of Binding Buffer solution, every hole adds the HRP-Streptavidin (1:500) of 100 μ L, hatch 1 hour for 37 DEG C, three times are cleaned with Binding Buffer solution, every hole adds 3 of 100 μ L, 3 ', 5, 5 '-tetramethyl benzidine stoste (TMB), incubated at room 10 minutes, then 1.0M sulphuric acid soln (every hole 100 μ L) termination reaction is added, microplate reader measures each hole OD value (wavelength is 450 nm), calculate compound to be screened and integrin alpha _vβ ₃albumen in conjunction with inhibiting rate, inhibiting rate (%)=[1-(by test product OD value-feminine gender group OD)/(model group OD-feminine gender group OD)) × 100.

Utilize aforesaid method, determine target compound to integrin alpha _vβ ₃the avidity of albumen, the 6-guanidine radicals-2 of result display disclosed in the embodiment of the present invention h-isoindoles compound ( i) or its pharmacy acceptable salt and integrin alpha _vβ ₃the combination of albumen suppresses IC ₅₀at 3.5nM ~ 1500nM, and the IC of positive control drug cyclo (RGDfV) ₅₀for 15.0nM.

(2) the inhibiting mensuration of cell adhesion

The 96 well culture plates Vitronectin of 1 μ g/mL or 7 μ g/mL Fibronectin bag is spent the night, control wells 1% BSA bag quilt, abandoning supernatant, and every hole adds 1% BSA of 100 μ L, closed 1 hour of room temperature.The PBS solution of compound 0.01M to be screened is made into different concns, and adding concentration is 5 × 10 ⁵in the M21 cell suspension of/ml, positive control cyclo (RGDfV) and SC-68448, the PBS solution of blank 0.01M replaces, hatch 30 minutes for 37 DEG C, then M21 cell suspension is added in above-mentioned 96 well culture plates closed in advance, every hole 100 μ L, the multiple hole of each concentration 6, hatch 1 hour for 37 DEG C, cell suspension gently in sucking-off hole, 3 times are washed with the PBS of pre-temperature, remove the cell do not adhered to, 10% glutaraldehyde that every hole adds 100 μ L fixes 30 minutes, thoroughly clean to glutaraldehyde with deionized water wash, put 37 DEG C of baking oven finish-dryings, the Viola crystallina adding 0.1% dyes to cell, jolting 30 minutes, with distilled water, unnecessary Viola crystallina is cleaned, put 37 DEG C of baking oven finish-dryings, the Viola crystallina of acetic acid to Cell uptake adding 10% is extracted, in microplate reader, OD value (wavelength is 595 nm) is measured after 1 hour, calculate cell adhesion inhibiting rate, can show that target compound is to integrin alpha _vβ ₃and/or α ₅β ₁inhibit activities.

Utilize aforesaid method, determine the adhesion inhibiting activity of target compound to M21 cell.The 6-guanidine radicals-2 of result display disclosed in the embodiment of the present invention h-isoindoles compound ( i) or its pharmacy acceptable salt all can significantly check melanin oncocyte M21 be to the adhesion of Vitronectin (VN) or Fibronectin (FN), the inhibit activities comparatively positive control medicine of partial target compound---cyclo (RGDfV) is strong.Such as: compound disclosed in the embodiment of the present invention 2-17to integrin alpha _vβ ₃and/or α ₅β ₁the inhibit activities result that receptor-mediated human melanoma cell M21 adheres to is as follows:

,

。

(3) mensuration of target compound cytotoxic activity

Caused by cell toxicant factor to get rid of the Anti cell adhesion activity of target compound to M21 cell, for this reason, we test the inhibit activities of target compound to human liver cancer cell HepG2, human A549 cell lines, B16 mouse melanoma cell line, MCF-7 and mouse colonic cell C26.Tumour cell is digested counting respectively, and be inoculated in 96 orifice plates (5000 cells/well), PBS liquid with pH7.4 after 24 hours washs 2 times, add the medicine containing different concns, cultivate 48 hours, after washing, dye 15 minutes with under 0.4% Sulforhodamine (SRB) solution room temperature, 1% Glacial acetic acid washes 3 times, in air, the cell of dyeing is dry, dissolve with the Tris-HCl of 10mM, and survey the absorbance of 540nm wavelength with Dynatech MR 7000 instrument, calculate the inhibiting rate of drug on tumor Growth of Cells.

Result shows the 6-guanidine radicals-2 disclosed in the embodiment of the present invention h-isoindoles compound ( i) or its pharmacy acceptable salt to the IC of these inhibiting tumour cells ₅₀all be greater than 80 μm of ol/L, illustrate that the cell toxicant of this compounds is less.

6-guanidine radicals-2 disclosed in this invention h-isoindoles compound ( i) or its pharmacy acceptable salt there is remarkable integrin alpha _vβ ₃and/or α ₅β ₁inhibit activities, can be used as integrin alpha _vβ ₃and/or α ₅β ₁inhibitor or/and treat various because of the symptom caused by new vessel hyperplasia or the purposes in disease medicament, includes but not limited to metastases, tumor growth, implanted solid tumor growth, angiogenesis, retinopathy, macular degeneration, osteoporosis, sacroiliitis, smooth muscle cell migration and atherosclerosis etc. for preventing.

Accompanying drawing explanation

fig. 1be in embodiment compound 2-17 to the effect diagram of chick chorioallantoic membrane angiogenesis.

Embodiment

Can be conducted further description the present invention by the following examples, but scope of the present invention is not limited to following embodiment.One of skill in the art can understand, and under the prerequisite not deviating from the spirit and scope of the present invention, can carry out various change and modification to the present invention.

embodiment 1

6-guanidine radicals-2 hmethod is led in the preparation of-isoindoles compound (I) and salt thereof

Step ( a): in reaction flask, add 6-guanidine radicals-1,3-dihydro-1-oxygen-2 h-isoindole carboxylic acid hydrochloride compounds ( 5) 1.0 mmol, Beta-alanine methyl esters compounds ( 6) raceme or optically active body 1.2 mmol and pyridine 25ml, after stirring at room temperature is entirely molten to solid, add 1-ethyl-3-(3-dimethylamine propyl) carbodiimide hydrochloride 1.5 mmol, stirring at room temperature 24 ~ 72 hours (reaction process TLC monitors, chloroform/methanol=5:1 v/v), after reaction terminates, filter, filtrate decompression is steamed and is desolventized, and resistates, through recrystallization or column chromatography purification (elutriant: chloroform-methanol=20:1 v/v), obtains dihydro-1-oxygen-2 h-isoindole carboxylic acid methyl esters compounds ( 7) raceme or optically active body, yield 58.5%-97.3%, the equal warp of its chemical structure ¹h-NMR, ¹³c-NMR and ESI-MS confirms.

Step ( b): add in reaction flask according to the dihydro-1-oxygen-2 obtained by above-mentioned steps h-isoindole carboxylic acid methyl esters compounds ( 7) raceme or optically active body 0.5 mmol, methyl alcohol 10ml, tetrahydrofuran (THF) 5ml and deionized water 5ml, add lithium hydroxide 2.0 mmol after stirring at room temperature is even, within 1-3 hour, (reaction process TLC monitors in stirring at room temperature reaction, chloroform/methanol/HOAc=3/1/0.5 v/v), after reaction terminates, with 10% aqueous hydrochloric acid regulator solution pH to 6-7, remove solvent under reduced pressure, resistates, through recrystallization or column chromatography purification (elutriant: chloroform-methanol=5:1 v/v), obtains corresponding 6-guanidine radicals-2 h-isoindoles compound ( i) raceme or optically active body, yield 72.0%-90.5%, the equal warp of its chemical structure ¹h-NMR, ¹³c-NMR and ESI-MS confirms.

Step ( c): add in reaction flask according to above-mentioned steps ( a) obtained by dihydro-1-oxygen-2 h-isoindole carboxylic acid methyl esters compounds ( 7) raceme or optically active body 0.5 mmol, methyl alcohol 10ml, tetrahydrofuran (THF) 5ml and deionized water 5ml, add lithium hydroxide 2.0 mmol after stirring at room temperature is even, within 1-3 hour, (reaction process TLC monitors in stirring at room temperature reaction, chloroform/methanol/HOAc=3/1/0.5 v/v), after reaction terminates, with corresponding acid-conditioning solution pH to 1-2, remove solvent under reduced pressure, resistates, through recrystallization or column chromatography purification (elutriant: chloroform-methanol=5:1 v/v), obtains corresponding 6-guanidine radicals-2 h-isoindoles compound ( i) salt of raceme or optically active body, yield 60.0%-86.5%, the equal warp of its chemical structure ¹h-NMR, ¹³c-NMR and ESI-MS confirms.

6-guanidine radicals-2 h-isoindoles compound ( i) salt of raceme or optically active body also can be prepared by the following method:

Step ( d): add in reaction flask according to above-mentioned steps ( b) obtained by 6-guanidine radicals-2 h-isoindoles compound ( i) raceme or optically active body 0.5 mmol, acetone 10ml, deionized water 5ml, 0.6 mmol is added sour accordingly after stirring at room temperature is even, temperature rising reflux stirring reaction 20 minutes, room temperature is cooled to after reaction terminates, remove solvent under reduced pressure, resistates acetone recrystallization, filters the solid of separating out, obtains corresponding 6-guanidine radicals-2 h-isoindoles compound ( i) salt of raceme or optically active body, yield 80.0%-95.0%, the equal warp of its chemical structure ¹h-NMR, ¹³c-NMR and ESI-MS confirms.

The target compound structure adopting above-mentioned logical method to prepare is as follows:

(connecting table)

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embodiment 2 compound 2-17 is on the impact of angiogenesis

2.1. Pharmaceutical formulations:

With PBS(pH=7.4) preparation compound 2-17by the 1mM storage liquid of test product, get supernatant after centrifugal, through 0.22 μm of millipore filtration Entkeimung, 4 DEG C save backup; Be diluted to desired concn with the low ratio of aseptic PBS before use to use.

chick chorioallantoic membrane (chick chorioallantoic membrane, CAM) model of angiogenesis is set up and administration:

Getting fertilization kind of an egg, cleaning and put into 37 DEG C, the HH-BH.360BS incubator (Shanghai leap Medical Devices Co., Ltd.) of 60% relative humidity hatches, choose well-developed 10 days instar chicken embryos, be divided into administration group and control group at random, often organize 10.After kind of an egg surface is disinfected in alcohol, open on egg embryo top the osculum that a size is 1.5 × 1.5cm by dental burr in SW-CJ Bechtop (SuZhou Antai Air Tech Co., Ltd.), cameral mantle is needled from air chamber and yolk sac separated place with ophthalmic forceps, remove the cameral mantle on upper strata gently, expose the CAM being positioned at cameral mantle lower floor; Be that the load sample filter membrane (PBS or different concns by test product) of 8 mm is placed in CAM and the less position of yolk cyst membrane place blood vessel by diameter, then use sterile transparent rubber seal mouth, 37 DEG C, continue to hatch 48 hours in the incubator of 60% relative humidity.

result is observed:

After hatching, with the eggshell of ophthalmic forceps strip off thereabout gently, expose the chicken embryo around filter membrane, remove the transparent adhesive tape of chick embryo air sac end sealing, throw off the eggshell of surrounding carefully with ophthalmic forceps from air chamber end, fully expose CAM, connect AxioCam MRc5 digital camera with Stemi 2000C stereoscopic microscope (Zeiss, Germany company) and take pictures (its result as shown in Figure 1), from accompanying drawing 1 compound 2-17the angiogenesis of chick chorioallantoic membrane is had to the restraining effect of concentration dependent, compare with Vehicle controls group, obviously reduce by new vessel during test product 1 μM of concentration, when 100 μMs, new vessel clearly reduces.

embodiment 3 compound 2-17 is on the impact of metastases

3.1. mouse melanin tumor cell is cultivated:

B16F10 murine melanoma high-transfer cell, biotherapy National Key Laboratory of Sichuan University provides, with 10% FBS/DMEM substratum, in 37 DEG C/5% CO ₂cellar culture in the incubator of saturated humidity, the tumour cell in vegetative period of taking the logarithm, pancreatin conventional digestion, washing 2 times, adjustment cell concn is 10 ⁷individual/ml.

murine melanoma and Lung metastases model are set up and administration:

The male C57BL/6 mouse of SPF level, in 5 ~ 6 week age, 15 ~ 17 g, Sichuan University's Experimental Animal Center provides, and routine is raised in SPF level Animal House.In mouse left hind sole position inoculation B16F10 tumor cell suspension 50 μ l/ only.Tumor inoculation the 8th day, animal is divided into 3 groups (7 ~ 12/group) at random by body weight, is respectively: Vehicle controls group, 15 mg/kg compound 2-17 groups, and 30 mg/kg compound 2-17 groups.Give by test sample solution or PBS solvent according to the administration volume abdominal injection of 0.1 ml/10 g, every day 1 time, successive administration 4 weeks.

result is observed:

Weigh weekly and regulate administration volume, by last administration after 24 hours, animal overnight fasting, put to death after weighing, cut from mouse left and right hind leg calcaneum and weigh, the two difference is Zuo Zhi foot tumor in situ weight; Get two lung, through Bouin ' s liquid-solid fixed 24 hours, 70% ethanol decolorization, under dissecting microscope, the melanoma tubercle number of every mouse lung surface transfer is observed, counted to blind, calculate and often organize the spontaneous Lung metastases incidence (%) of tumor-bearing mice, result represents with mean ± standard deviation, and SPSS16.0 software statistics analyzes the significance of group difference.Its result is as shown in table 2:

From table 2 result, compare with Vehicle controls group, compound 2-17 has the slight restraining effect of dose-dependently to the melanomatous growth of mouse B16F10 by test product, but has no significant difference (P > 0. 05); In addition, high and low dose is all obviously less than Vehicle controls group by the mouse lung transfer incidence of test product administration group, thus prompting: to metastases, there is good restraining effect by test product, and and dose proportional.

Claims (9)

1. a class have as general formula ( i) shown in 6-guanidine radicals-2 h-isoindoles compound and pharmacy acceptable salt thereof:

In formula: R ₁expression phenyl, 2-furyl, 2-thienyl, 3-thienyl, 3-pyridyl, 4-pyridyl, 3,4-methylenedioxyphenyl bases, 2,3-methylenedioxyphenyl bases, , , , or ; R ₃, R ₄, R ₅represent H, C independently of one another ₁~ C ₆alkyl, C ₁~ C ₆alkoxyl group, hydroxyl, halogen, amino, nitro, cyano group, carboxyl, CF ₃, NR ₇r ₈, R ₆represent H, C ₁~ C ₆alkyl, C ₁~ C ₆alkoxyl group, halogen, nitro, cyano group or CF ₃, R ₇, R ₈represent C independently of one another ₁~ C ₆alkyl, R ₃, R ₄, R ₅, R ₆can at any possible position of phenyl ring, R ₃, R ₄, R ₅can be identical, also can be different; R ₂represent , , ; M represents 1 or 2; N represents 0,1 or 2; R ₉represent C ₁~ C ₆alkyl, halogen, hydroxyl or CF ₃.

2. 6-guanidine radicals-2 as claimed in claim 1 h-isoindoles compound ( i) and pharmacy acceptable salt, when it is characterized in that there is chiral carbon in molecule, compound is raceme or optically active body.

3. the 6-guanidine radicals-2 as described in any one of claim 1-2 h-isoindoles compound ( i) and pharmacy acceptable salt, it is characterized in that described pharmacy acceptable salt is 6-guanidine radicals-2 h-isoindoles compound ( i) and hydrochloric acid, Hydrogen bromide, nitric acid, sulfuric acid, phosphoric acid, formic acid, trifluoroacetic acid, acetic acid, propionic acid, oxalic acid, phenylformic acid, Whitfield's ointment, toxilic acid, fumaric acid, succsinic acid, tartrate, amygdalic acid, citric acid, C _1-6the salt of alkylsulphonic acid, camphorsulfonic acid, Phenylsulfonic acid or tosic acid.

4. 6-guanidine radicals-2 as claimed in claim 1 h-isoindoles compound ( i) and the preparation method of pharmacy acceptable salt, it is characterized in that the preparation method of described compound comprises the steps: ( a) with 6-guanidine radicals-1,3-dihydro-1-oxygen-2 h-isoindole carboxylic acid hydrochloride compounds ( 5) and Beta-alanine methyl esters compounds ( 6) raceme or optically active body be starting raw material, condensation under solvent and condensing agent existence condition, obtains 6-guanidine radicals-1,3-dihydro-1-oxygen-2 h-isoindole carboxylic acid methyl esters compounds ( 7) raceme or optically active body; ( b) gained 6-guanidine radicals-1,3-dihydro-1-oxygen-2 h-isoindole carboxylic acid methyl esters compounds ( 7) raceme or optically active body through basic hydrolysis, acid neutralization, obtain 6-guanidine radicals-2 h-isoindoles compound ( i) raceme or optically active body; Gained ( i) more conventionally with corresponding sour salify, obtain compound ( i) salt of raceme or optically active body.

5. 6-guanidine radicals-2 as claimed in claim 4 h-isoindoles compound ( i) and the preparation method of pharmacy acceptable salt, it is characterized in that step ( a) in, reaction solvent for use is: pyridine, n,N-dimethyl formamide, dimethyl sulfoxide (DMSO), C _3-8aliphatic ketone, ether, isopropyl ether, methyl tertiary butyl ether, tetrahydrofuran (THF), glycol dimethyl ether, C _1-6lipid acid and C _1-6ester that fatty alcohol is formed, methylene dichloride, chloroform, 1,2-ethylene dichloride, benzene,toluene,xylene, chlorobenzene, orthodichlorobenzene or acetonitrile, reaction can be carried out in above-mentioned single solvent, also can carry out in above-mentioned mixed solvent, and mixed solvent volume ratio is 1:0.1 ~ 10; Condensing agent used is: phosphinylidyne diimidazole, chloroformic acid C _1-8aliphatic alcohol ester compounds, n-ethoxycarbonyl-2-oxyethyl group-1,2-dihydroquinoline, dicyclohexylcarbodiimide, 1-ethyl-3-(3-dimethylamine propyl) carbodiimide hydrochloride, diethyl phosphorocyanidate, 2-chloro-4,6-dimethoxy-1,3,5-triazine, chlorination 4-(4,6-dimethoxy-1,3,5-triazines-2-base)-4-methylmorpholine salt; Compound ( 5): compound ( 6): the molar feed ratio of condensing agent is 1.0:0.8 ~ 10.0:1.0 ~ 10.0; Setting-up point is 0 ~ 130 DEG C; Condensation reaction time is 30 minutes ~ 72 hours.

6. 6-guanidine radicals-2 as claimed in claim 4 h-isoindoles compound ( i) and the preparation method of pharmacy acceptable salt, it is characterized in that step ( b) in, being hydrolyzed alkali used is alkali metal hydroxide, alkaline earth metal hydroxides, basic metal metal carbonate, alkaline earth metal carbonate, alkali metal hydrocarbonate or alkali metal bicarbonates; Compound ( 7) be 1.0:1.0 ~ 5.0 with the molar feed ratio of alkali; Hydrolysising reacting temperature is room temperature ~ 150 DEG C; Hydrolysis time is 0.5 ~ 24 hour.

7. a pharmaceutical composition, comprises one or more 6-guanidine radicals-2 as described in any one of claim 1-3 for the treatment of significant quantity h-isoindoles compound ( i) and pharmacy acceptable salt.

8. pharmaceutical composition as claimed in claim 7, is characterized in that this pharmaceutical composition further containing one or more pharmaceutically acceptable carrier or vehicle.

9. the 6-guanidine radicals-2 as described in any one of claim 1-2 h-isoindoles compound ( i) and pharmacy acceptable salt preparation prevention or/and treat various because of integrin alpha _vβ ₃and/or α ₅β ₁purposes in metastases caused by mediation, tumor growth, implanted solid tumor growth, angiogenesis, retinopathy, macular degeneration, osteoporosis, sacroiliitis, smooth muscle cell migration and symptoms of atherosclerosis or disease medicament.