CN109134433A - It is a kind of inhibit ROCK compound and its application - Google Patents

It is a kind of inhibit ROCK compound and its application Download PDF

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CN109134433A
CN109134433A CN201810619518.1A CN201810619518A CN109134433A CN 109134433 A CN109134433 A CN 109134433A CN 201810619518 A CN201810619518 A CN 201810619518A CN 109134433 A CN109134433 A CN 109134433A
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stereoisomer
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李进
张登友
冯静超
廖伟
林丽
李偲
陈伟
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Chengdu Lead Drug Development Corp Ltd
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Abstract

The invention discloses formula (I) compound represented and the preparation methods and purposes of the compound.Good ROCK inhibitory activity is shown, provides a kind of new medicinal possibility to the abnormal relevant disease of ROCK activity for clinical treatment.

Description

It is a kind of inhibit ROCK compound and its application
Technical field
The invention belongs to pharmaceutical synthesis fields, and in particular to it is a kind of inhibit ROCK compound and its treatment and ROCK phase Application in related disorders.
Background technique:
Rho belongs to small molecule list aggressiveness GTPase superfamily, is the mammalian genes homologue of Ras superfamily, passes through Most important effector molecule Rho kinases (Rho-associated coiled-coil containing protein downstream Kinase, ROCK), to adjust the recombination of Cellular actin cytoskeleton, thus wide participation cell mitogen, cytoskeleton tune A series of biological processes such as whole, smooth muscle cell contraction, nerve regneration, the adjusting of tumor cell invasion, Apoptosis.Rho/ A variety of substrates can be acted on after ROCK activation, to generate biological process.Most important two kinds of substrates are that myosin is light Chain (MLC) and Myosin light chain phosphatase (MLCP), the phosphorylation level of MLC are a weights for determining smooth muscle contraction degree Want factor.The site Ser-19 of myosin light chain kinase (MLCK) phosphorylation MLC, leads to smooth muscle contraction；The inhibition of MLCP The contraction of the phosphorylation and smooth muscle that can make MLC further enhances.After ROCK is activated, itself can be by MLC phosphorylation And myofilament contraction occurs；It can also so that MLCP is inactivated MLCP phosphorylation simultaneously, lead to MLC phosphorylation journey in cell cytosol Degree increases, and promotes myofilament to shrink indirectly.
The inhibition of Rho kinase activity shows a variety of benefits for the treatment of human diseases, including angiocarpy in animal model Disease such as pulmonary hypertension, hypertension, atherosclerosis, cardiomegaly, intraocular hypertension, cerebral ischemia, cerebral angiospasm etc., and in Pivot nervous system illness such as neuronal degeneration etc. and tumour.Research shows that ROCK expression and activity are big in spontaneous hypertension It is increased in mouse, illustrates that it has with the generation of these animal hypertension and be associated with (Involvement of Rho-kinase in hypertensive vascular disease:a novel therapeutic target in hypertension [J].FASEB J.,2001,15(6):1062-4).ROCK inhibitor Y-27632 can make three kinds of rat hypertension models (spontaneous Property hypertension, renal hypertension, percorten salt form hypertension) in blood pressure significantly reduce, and to control rats Blood pressure acts on smaller (Calcium sensitization of smooth muscle mediated by a Rho- associated protein kinase in hypertension[J].Nature,1997,389(6654):990-4).? Some researches show that ROCK inhibitors to have preferable effect (Acute vasodilator effects of a to pulmonary hypertension Rho-kinase inhibitor,fasudil,in patients with severe pulmonary hypertension [J].Heart,2005:91(3):391-2)。
The ROCK inhibitor researched and developed at present can be divided into five major class: (1) iloquinoline derivative: such compound structure is special Point is that have an isoquinoline structure and piperazine ring, and the two is connected by sulfonyl.Represent object have method Soviet Union ground you (Uehata M, Ishizaki T,Satoh H,et al.Calcium sensitization of smooth muscle mediated by a Rho-associated protein kinase in hypertension[J].Nature,1997,389:990-994)、H- 1152P(Tamura M,Nakao H,Yoshizaki H,et al.Development of specific Rho-kinase inhibitors and their clinical application[J].Biochim Biophys Acta,2005,1754: 245-252)；(2) 4-aminopyridine class: such compound structure in addition to 4-aminopyridine parent nucleus, the center of molecule also Containing a hexamethylene or benzene ring structure, 4 in hexamethylene have side-chain structure.Represent object have Y-30141 (Takami A, Iwakubo M,Okada Y,et al.Design and synthesis of Rho kinase inhibitors[J] .Bioorg Med Chem,2004,12:2115-2137)；(3) indazole class: such compound is by 5- amino or 5- alkoxy -1H Indazole is as skeleton；(4) amide and ureas: the strand that there is such compound a phthalimide and a urea groups to constitute And structure.(5) other classes: other ROCK inhibitors not comprising above structure, represent object have Rockout (Yarrow JC, Totsukawa G,Charras GT,et al.Screening for cell migration inhibitors via automated microscopy reveals a Rho-kinase inhibitor[J].Chem Biol,2005,12:385- 395)。
The ROCK inhibitor drug listed at present has the Eril of Asahi Kasei company (suitable for cerebral angiospasm Treatment) and Kowa company Glanatec (treatment suitable for ocular hypertension and glaucoma).Wherein Glanatec is only in Japan List marketing.Therefore developmental function is carried out to study in the targeting small-molecule drug of ROCK, obtain active more preferable, selectivity is higher, More hypotoxicity and side effect, more economical ROCK inhibitor have highly important social and economic implications.
Summary of the invention:
The present invention provides a kind of Formulas I compound represented or its stereoisomers:
Wherein,
M is 1 or 2；
N is 1 or 2；
R1Selected from hydrogen, C1~6Alkyl；
A ring is selected from 5~6 yuan of aromatic rings, 5~6 yuan of heteroaromatics；Wherein the aromatic ring, heteroaromatic can be further by 1~4 It is a to be respectively and independently selected from hydroxyl, halogen, amino, nitro, cyano, trifluoromethyl, carboxyl, C1~6Alkyl, C1~6Alkoxy, C1~6Alkane Amino, C1~6Dialkylamino, C1~6Replaced the substituent group of acyl group；
R2Selected from hydrogen, hydroxyl, halogen, amino, nitro, cyano, trifluoromethyl, carboxyl, C1~6Alkyl, C1~6Alkoxy, C1~6Alkylamino, C1~6Dialkylamino, C1~6Acyl group；
Further, m and n adds up to 2 or 3.
Further, A ring is phenyl ring.
Preferably, compound of formula I is as shown in following structural:
The present invention also provides above compound or its stereoisomers or its crystal form, pharmaceutically acceptable salt, hydrate Or purposes of the solvate as preparation treatment and the drug of the abnormal relevant disease of ROCK activity.
Specifically, described is to adjust with cell mitogen, cytoskeleton, is smooth to the abnormal relevant disease of ROCK activity One or more of relevant disease such as Muscle cell contract, nerve regneration, tumor cell invasion, Apoptosis.
Further, the disease is cardiovascular disease, ocular hypertension, glaucoma and cancer.
Further, the disease is pulmonary hypertension, ocular hypertension, glaucoma.
Another aspect of the present invention also provides a kind of drug, it is with above compound and its stereoisomer or its crystalline substance Type, pharmaceutically acceptable salt, hydrate or solvate are active constituent, in addition pharmaceutically acceptable auxiliary material is prepared Preparation.
Compound and derivative provided in the present invention can according to IUPAC (International Union of Pure and Applied Chemistry) or The name of CAS (chemical abstracts service, Columbus, OH) naming system.
About the definition of the invention using term: unless otherwise indicated, group or term herein provide initial Definition is suitable for group or term of entire description；For the term being not specifically defined herein, it should according to open Content and context, their meaning can be given by providing those skilled in the art.
" substitution " refers to that the hydrogen atom in molecule is replaced by other different atoms or molecule.
The minimum value and maximum value of carbon content are indicated by prefix in hydrocarbon group, for example, prefix CA~b) alkyl table Bright any alkyl containing " a " to " b " a carbon atom.Thus, for example, C1~4Alkyl refers to the alkyl comprising 1~4 carbon atom.
" Asia~yl " of the invention refers to that the corresponding hydrocarbon of the group loses the group after two hydrogen atoms, and such as " alkylidene " is pair The alkane answered loses the group obtained after two hydrogen atoms.
C in the present inventionA~bAlkoxy, CA~bCarbalkoxy, CA~bAlkylamino, CA~bAcyl group refers respectively to a to " b " containing " a " The alkyl of carbon atom and corresponding oxygen atom, ester group, amino, acyl group are connected obtained group.
Term " pharmaceutically acceptable " refers to certain carrier, load, diluent, auxiliary material, and/or to be formed by salt usual In chemistry or physically with constitute the other compatible at split-phase of certain pharmaceutical dosage form, and physiologically mutually compatible with receptor.
Term " salt " and " pharmaceutical salt " refer to above compound or its stereoisomer, with inorganic and/or organic acid The acid and/or basic salt formed with alkali also includes amphoteric ion salt (inner salt), further includes quaternary ammonium salt, such as alkylammonium salt.This A little salt can be to be directly obtained in being finally separating and purify of compound.It is also possible to by by above compound or it is vertical Body isomers is obtained by mixing with a certain number of acid or alkali appropriate (such as equivalent).These salt may be in the solution It forms precipitating and is collected with filter method, or recycle obtain after the solvent evaporates, or be freeze-dried after reacting in an aqueous medium It is made.Heretofore described salt can be hydrochloride, sulfate, citrate, benzene sulfonate, hydrobromate, the hydrogen of compound Fluorate, phosphate, acetate, propionate, succinate, oxalates, malate, succinate, fumarate, maleic acid Salt, tartrate or trifluoroacetate.
In some embodiments, one or more compounds of the invention can be used in conjunction with one another.Also may be selected will The compound of the present invention is used in combination with any other active agent, is used to prepare regulating cell function or treats the medicine of disease Object or pharmaceutical composition.If using one group of compound, can by these compounds simultaneously, respectively or in an orderly manner to tested Object is administered
Obviously, above content according to the present invention is not being departed from according to the ordinary technical knowledge and customary means of this field Under the premise of the above-mentioned basic fundamental thought of the present invention, the modification, replacement or change of other diversified forms can also be made.
The specific embodiment of form by the following examples remakes further specifically above content of the invention It is bright.But the range that this should not be interpreted as to the above-mentioned theme of the present invention is only limitted to example below.It is all to be based on above content of the present invention The technology realized all belongs to the scope of the present invention.
Specific embodiment
Compound structure is by nuclear magnetic resonance (NMR) and mass spectrum (MS) come what is determined.NMR is displaced (δ) with 10-6's (ppm) Unit provides.The measurement of NMR is to use (Bruker AvanceIII 400 and Bruker Avance 300) nuclear magnetic resonance spectrometer, is measured molten Agent is deuterated dimethyl sulfoxide (DMSO-d6), deuterated chloroform (CDCl3), deuterated methanol (MeOD), be inside designated as tetramethylsilane (TMS)。
The measurement of LC-MS uses Shimadzu LC-MS instrument (Shimadzu LC-MS 2020 (ESI)).
The measurement of HPLC uses Shimadzu high pressure liquid chromatograph (Shimadzu LC-20A).
MPLC (middle to suppress standby chromatography) uses Gilson GX-281 reversed-phase preparative chromatography instrument.
Column chromatography is generally carrier using 200~300 mesh silica gel of Yantai Huanghai Sea silica gel.
Tlc silica gel plate isolates and purifies product with Yantai Huanghai Sea HSGF254 or Qingdao GF254 silica gel plate, thin-layer chromatography The specification of use is 0.4mm~0.5mm.
Raw material, equipment used in the specific embodiment of the invention are known product, are obtained by purchase commercial product. Known starting material can be used or be synthesized according to methods known in the art, or can purchase in the resistance to Jilin Chemical of peace, Chengdu The companies such as section's dragon chemical industry, splendid remote chemical science and technology, lark prestige science and technology.
Room temperature is optimum reaction temperature, is 20 DEG C~30 DEG C.Without specified otherwise in embodiment, M is mole every liter.It is real It applies without specified otherwise in example, solution refers to aqueous solution.
Write a Chinese character in simplified form explanation: DCM is methylene chloride；EA, EtOAc are ethyl acetate；PE is petroleum ether；THF is tetrahydrofuran； DMF is N,N-dimethylformamide；DIEA, DIPEA are diisopropylethylamine；DMAP is 4-dimethylaminopyridine；EDCI is 1- Ethyl-(3- dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate；HOBT is I-hydroxybenzotriazole；HBTU is three nitrogen of benzo Azoles-N, N, N', N'- tetramethylurea hexafluorophosphate.
The preparation of embodiment 1,6- isoquinolin-((S) -2- phenyl -1,4- azacycloalkyl)-ketone
Step 1, (S)-N- benzylidene -2- methylpropane -2- sulfonamide
(S) tert-butyl Asia sulphur is added into tetrahydrofuran (150mL) solution of benzaldehyde (30g, 0.28mmol) at room temperature Amide (51g, 0.42mmol) and tetraisopropyl titanate (100g, 0.35mmol), after stirring 4 hours in 60 DEG C plus water filters, and uses Ethyl acetate extraction, then water phase is extracted with ethyl acetate twice, merging organic phase is simultaneously dry with anhydrous sodium sulfate, removes under reduced pressure Solvent.Column chromatographic purifying obtains (S)-N- benzylidene -2- methylpropane -2- sulfonamide (54g, 0.23mmol, yield 82%).
MS (ESI) m/z=210 (M+1)+。
The preparation of step 2, (S) -2- methyl-N- (2- nitro -1- phenethyl)-propane -2- sulfenamide
(S)-N- benzylidene -2- methylpropane -2- sulfonamide (48g, 0.23mmol) is dissolved in tetrahydrofuran (500mL), Under nitrogen protection in 0 DEG C of addition potassium tert-butoxide (38.5g, 0.34mmol), nitromethane is added after stirring 1 hour in reaction solution (140g, 2.29mol) is stirred at room temperature 24 hours.Reaction solution ethyl acetate and water extraction, then water phase is extracted with ethyl acetate Twice, merge organic phase and, evaporating solvent under reduced pressure dry with anhydrous sodium sulfate.Column chromatographic purifying obtains (S) -2- methyl-N- (2- Nitro -1- phenethyl)-propane -2- sulfenamide (18g, 0.67mmol, yield 29%).
MS (ESI) m/z=271 (M+1)+。
The preparation of step 3, (S) -2- methyl-N- (2- amido -1- phenethyl)-propane -2- sulfenamide
(S) -2- methyl-N- (2- nitro -1- phenethyl)-propane -2- sulfenamide (8.0g, 30mmol) is dissolved in methanol (50mL) is added Raney's nickel (0.80g, 10%), is stirred at room temperature 24 hours.Reaction solution is filtered with diatomite, evaporating solvent under reduced pressure Obtain (S) -2- methyl-N- (2- amido -1- phenethyl)-propane -2- sulfenamide (6.0g, 21.5mmol, yield 86%).
MS (ESI) m/z=241 (M+1)+。
The preparation of step 4, benzyl-N- (((S)-t-butyl sulfonamide) -2- phenethyl) carbamate
(S) -2- methyl-N- (2- amido -1- phenethyl)-propane -2- sulfenamide (7.00g, 29.1mmol) is dissolved in Tetrahydrofuran (70.0mL), addition triethylamine (11.8g, 116mmol) and benzene methoxy carbonyl acyl succinimide (8.70g, 34.9mmol), it is extracted after stirring 1 hour with ethyl acetate and water, then water phase is extracted with ethyl acetate twice, merge organic phase And, evaporating solvent under reduced pressure dry with anhydrous sodium sulfate.Column chromatographic purifying obtains benzyl-N- (((S)-t-butyl sulfonamide) -2- benzene Ethyl) carbamate (5.9g, 12.6mmol, yield 43%).
MS (ESI) m/z=375 (M+1)+。
The preparation of step 5, benzyl-N- (2- amino -2- phenyl-ethyl group) carbamate
Benzyl-N- (((S)-t-butyl sulfonamide) -2- phenethyl) carbamate (5.0g, 13.4mmol) is dissolved in Methanol (10mL) is added Isosorbide-5-Nitrae-dioxane hydrochloric acid solution (40ml, 5mol/L), and evaporating solvent under reduced pressure obtains after stirring 1 hour Benzyl-N- (2- amino -2- phenyl-ethyl group) carbamate (3.02g, 10.3mmol, yield 77%).
MS (ESI) m/z=271 (M+1)+。
The preparation of step 6, benzyl-N- ((S) -2- (isoquinolin -6- formamide) -2- phenyl-ethyl group) carbamate
Benzyl-N- (2- amino -2- phenyl-ethyl group) carbamate (2.00g, 7.40mmol) is dissolved in DMF Benzotriazole-N, N, N', N'- tetramethylurea hexafluorophosphoric acid ester (2.07g, 8.14mmol), isoquinolin-is added in (10.0mL) 6- formic acid (1.28g, 7.40mmol) and n,N-diisopropylethylamine (3.38g, 29.6mmol) use acetic acid second after stirring 1 hour Ester and water extraction, then water phase is extracted with ethyl acetate twice, merging organic phase is simultaneously dry with anhydrous sodium sulfate, removes under reduced pressure molten Agent.Column chromatographic purifying obtain benzyl-N- ((S) -2- (isoquinolin -6- formamide) -2- phenyl-ethyl group) carbamate (1.98g, 4.21mmol, yield 57%).
MS (ESI) m/z=426 (M+1)+。
The preparation of step 7, phenyl-(S) -4- (isoquinolin -6- carbonyl) -3- benzyl -1,4- diazacyclo
By benzyl-N- ((S) -2- (isoquinolin -6- formamide) -2- phenyl-ethyl group) carbamate (1.00g, It 2.35mmol) is dissolved in DMF (5.00mL), it under a nitrogen will after 0 DEG C of addition sodium hydrogen (226mg, 9.40mmol), stirring 1 hour 1,3- dibromopropane (712mg, 3.53mmol) is added drop-wise in reaction solution, and reaction is quenched after 2 hours with saturated ammonium chloride solution, second Acetoacetic ester extraction, then water phase is extracted with ethyl acetate twice, merging organic phase is simultaneously dry with anhydrous sodium sulfate, removes under reduced pressure molten Agent.Column chromatographic purifying obtains phenyl-(S) -4- (isoquinolin -6- carbonyl) -3- benzyl -1,4- diaza cycloalkanes (500mg, 970 μ Mol, yield 41%).
MS (ESI) m/z=466 (M+1)+。
The preparation of step 8,6- isoquinolin-((S) -2- phenyl -1,4- azacycloalkyl)-ketone
Phenyl (S) -4- (isoquinolin -6- carbonyl) -3- benzyl -1,4- diaza cycloalkanes (170mg, 360 μm of ol) is dissolved in Acetic acid (5.00mL) is added hydrobromic acid acetum (2.5ml, 33%), evaporating solvent under reduced pressure after stirring 1 hour, then uses reverse phase MPLC purifies to obtain 6- isoquinolin-((S) -2- phenyl -1,4- azacycloalkyl)-ketone (45mg, 110 μm of ol, yield 33%).
MS (ESI) m/z=368 (M+1)+。
1H NMR(400MHz,DMSO-d6+D2O): δ=9.86 (s, 1H), 8.33 (s, 1H), 8.70-8.71 (d, J= 6.4Hz, 1H), 8.58-8.61 (d, J=8.8Hz, 2H), 8.53-8.54 (m, 1H), 8.39-8.41 (d, J=8.0Hz, 1H), 7.48-7.50(m,2H),7.39-7.43(m,2H),7.34-7.36(m,1H),5.89-5.96(m,1H),5.43-5.53(m, 3H),3.75-3.76(m,1H),3.67-3.69(m,2H),3.50-3.53(m,1H)。
The embodiment 2, (preparation of -6 base of (S)-isoquinolin (2- phenylpiperazine -1- base) ketone
The preparation of step 1, (S) -4- (isoquinolin -6- formoxyl) -3- phenylpiperazine -1- carboxylic acid tert-butyl ester
At room temperature toward isoquinolin -6- formic acid (100mg, 577 μm of ol) and (S) -3- phenylpiperazine -1- carboxylic acid tert-butyl ester Be added in DMF (4.00mL) solution of (152mg, 577 μm of ol) HBTU (176mg, 693 μm of ol) and DIPEA (373mg, 2.89mmol, 504 μ L), it is stirred at room temperature 2 hours.It is quenched with water (50.0mL), ethyl acetate extracts (50mL × 2).Merge organic Successively saturated salt solution (50mL × 1), water (50mL × 1) are washed after phase, and organic phase anhydrous sodium sulfate dries, filters rear filtrate decompression Solvent is evaporated off.Column chromatographic purifying obtain (S) -4- (isoquinolin -6- formoxyl) -3- phenylpiperazine -1- carboxylic acid tert-butyl ester (220mg, 485 μm of ol, yield 84%).
MS (ESI) m/z=418 (M+1)+。
The preparation of step 2, -6 base of (S)-isoquinolin (2- phenylpiperazine -1- base) ketone
At room temperature toward (S) -4- (isoquinolin -6- formoxyl) -3- phenylpiperazine -1- carboxylic acid tert-butyl ester (220mg, 527 μ Mol concentrated hydrochloric acid (2.00mL) is added in methanol (10.0mL) solution), evaporating solvent under reduced pressure after 1 hour is stirred at room temperature.Through preparing HPLC purifies -6 base of (S)-isoquinolin (2- phenylpiperazine -1- base) ketone (77mg, 193 μm of ol, yield 37%).
MS (ESI) m/z=318 (M+1)+。
1H NMR(400MHz,DMSO-d6+D2O): δ=9.81 (s, 1H), 8.64 (d, J=6.44Hz, 1H), 8.58 (t, J =4.44Hz, J=3.8Hz, 1H), 8.49 (d, J=5.88Hz, 1H), 8.45 (s, 1H), 8.06 (d, J=8.28Hz, 1H), 7.49 (t, J=8.04Hz, J=7.32Hz, 2H), 7.38 (t, J=7.36Hz, J=7.12Hz, 3H), 3.99-4.06 (m, 3H), 3.61 (dd, J=4.76Hz, J=4.76Hz, 1H), 3.38-3.46 (w, 1H), 3.22-3.29 (m, 1H), 3.09-3.15 (w,1H)。
The preparation of embodiment 3,6- isoquinolin-((S) -2- (3- bromophenyl) -1,4- azacycloalkyl)-ketone
Step 1, (S)-benzyl ester -3- (3- bromophenyl) -4- (isoquinolin -6- carbonyl) -1,4- azepan -1- formic acid Preparation
According to the method for step 1 to 7 in embodiment 1, the benzaldehyde in step 1 is replaced with into 3- bromobenzaldehyde, is obtained (S)-benzyl ester -3- (3- bromophenyl) -4- (isoquinolin -6- carbonyl)-Isosorbide-5-Nitrae-azepan -1- formic acid (1.28g, gross production rate 2.1%).
The preparation of step 2,6- isoquinolin-((S) -2- (3- bromophenyl) -1,4- azacycloalkyl)-ketone
By (S)-benzyl ester -3- (3- bromophenyl) -4- (isoquinolin -6- carbonyl) -1,4- azepan -1- formic acid (200mg, 0.367mmol) is dissolved in acetic acid (5mL), is added hydrobromic acid acetum (2.5ml, 33%), and reaction solution stirs one After hour.Reaction solution obtains 6- isoquinolin-((S) -2- (3- bromophenyl) -1,4- azepine with Rotary Evaporators organic solvent of going out Cycloalkanes)-ketone (27mg, 59 μm of ol, yield 16%).
MS (ESI) m/z=410,412 (M+1)+
1H NMR (400MHz, MeOD): δ=9.91 (s, 1H), 9.00 (s, 1H), 8.64-8.70 (m, 3H), 8.52- 8.54(m,1H),7.78-7.79(m,1H),7.56-7.58(m,2H),7.37-7.41(m,1H),5.98-5.69(m,1H), 5.54-5.65(m,3H),3.76-3.84(m,3H),3.51-3.56(m,1H),3.33-3.34(m,1H)。
The preparation of embodiment 4, (S)-isoquinolin -6- base (4- methyl -2- phenyl -1,4- azacycloalkyl)-ketone
6- isoquinolin-((S) -2- phenyl-Isosorbide-5-Nitrae-azacycloalkyl)-ketone (30mg, 90umol) is dissolved in methanol (1.5mL), is added acetic acid (3.4mg, 45umol) and formalin (58uL, 13M, 0.76mmol), and stirring is added after 1 hour Sodium cyanoborohydride (228mg, 0.36mmol) is extracted after the reaction was continued 2 hours with ethyl acetate and water, then by water phase acetic acid Ethyl ester is extracted twice, and merges organic phase and, evaporating solvent under reduced pressure dry with anhydrous sodium sulfate.Column chromatographic purifying obtains (S)-isoquinoline Quinoline -6- base (4- methyl -2- phenyl -1,4- azacycloalkyl)-ketone (1.41mg, 3.96umol, yield 4.4%).
MS (ESI) m/z=346 (M+1)+。
1H NMR(400MHz,DMSO-d6): δ=9.80 (s, 1H), 8.93-8.95 (m, 1H), 8.72-8.73 (d, J= 6.0Hz, 1H), 8.50-8.52 (m, 1H), 8.43-8.45 (d, J=8.4Hz, 1H), 8.38-8.40 (d, J=6.4Hz, 1H), 7.57-7.61(m,2H),7.34-7.44(m,3H),6.06-6.13(m,1H),5.52-5.70(m,3H),3.96-4.02(m, 1H),3.82-3.85(m,3H),3.41-3.53(m,1H),2.83-2.89(m,3H)。
In order to illustrate beneficial effects of the present invention, the present invention provides following tests example:
The detection of test example 1, ROCK2 inhibitory activity
ROCK2 can phosphorylation S6K (KRRRLASLR) peptide substrate, ATP is converted to ADP.After kinase reaction, add Enter ADP-GloTMReagent terminates kinase reaction, and runs out of remaining ATP.Kinase assay reagent is added, it makes ADP turns While being melted into ATP, ATP is again by Ultra-GloTMLuciferase is converted to light luminous signal, and luminous signal and kinase activity are just It is related.
The detection of ROCK2 inhibitory activity is carried out according to the following steps:
1.Assay Buffer:40mM Tris pH 7.5,20mM MgCl2,0.1%BSA (w/v), 50 μM of DTT；
2. 12 μ L2.5x0.1 μ g/ml ROCK2 working solutions is added to enter 96 hole PCR plates；
3. adding 6 μ L6x compound working solutions to mix into 96 hole PCR plates, 25 DEG C of preincubate 10min；
4. 12 μ L 2.5x, 37.5 μ g/mlS6K substrate and 12.5 μM of ATP hybrid working liquid, 30 DEG C of incubation 60min are added；
5. taking 25 μ L reaction mixtures to a new 96 hole PCR plate, and 25 μ L ADP-Glo are addedTMReagent mixes, and 25 DEG C It is incubated for 40min and terminates reaction；
6. 40 μ L is taken to terminate reaction mixture to a new 96 hole PCR plate, and 40 μ L kinase assay reagents are added and mix, 25 DEG C be incubated for 40min；
7. reading luminescence (cold light) signal value, inhibiting rate is calculated.
The detection of ROCK2 inhibitory activity is carried out to the compound of embodiment preparation according to the method described above, test result is shown in Table 1, Wherein measure the IC of each compound50According to illustrating to classify, in table 1:
"+" indicates IC50Measured value is greater than 250nM；
" ++ " indicates IC50Measured value is less than 250nM and is greater than 50nM；
" +++ " indicates IC50Measured value is less than 50nM.
Table 1, compound are to the inhibitory activity of ROCK2
Embodiment ROCK2 Embodiment ROCK2
1 +++ 2 ++
3 +++ 4 +
Experiments have shown that the compound of the embodiment of the present invention have good ROCK inhibitory activity, can effective for The treatment of ROCK activity abnormal diseases.
In conclusion noval chemical compound shown in Formulas I disclosed by the invention, shows good ROCK inhibitory activity, is Clinical treatment provides a kind of new medicinal possibility to the abnormal relevant disease of ROCK activity.

Claims (9)

1. a kind of Formulas I compound represented or its stereoisomer:
Wherein,
M is 1 or 2；
N is 1 or 2；
R1Selected from hydrogen, C1~6Alkyl；
A ring is selected from 5~6 yuan of aromatic rings, 5~6 yuan of heteroaromatics；Wherein the aromatic ring, heteroaromatic can be further by 1~4 points It is not independently selected from hydroxyl, halogen, amino, nitro, cyano, trifluoromethyl, carboxyl, C1~6Alkyl, C1~6Alkoxy, C1~6Alkane ammonia Base, C1~6Dialkylamino, C1~6Replaced the substituent group of acyl group；
R2Selected from hydrogen, hydroxyl, halogen, amino, nitro, cyano, trifluoromethyl, carboxyl, C1~6Alkyl, C1~6Alkoxy, C1~6Alkane Amino, C1~6Dialkylamino, C1~6Acyl group.
2. compound according to claim 1 or its stereoisomer, it is characterized in that: m and n add up to 2 or 3.
3. compound according to claim 1 or its stereoisomer, it is characterized in that: A ring is phenyl ring.
4. compound according to claim 1 or its stereoisomer, it is characterized in that: the compound such as following structural It is shown:
5. compound described in claims 1 or 2 any one or its stereoisomer or its crystal form, pharmaceutically acceptable salt, The purposes of hydrate or solvate as preparation treatment and the drug of the abnormal relevant disease of ROCK activity.
6. purposes according to claim 4, it is characterised in that: the disease relevant to ROCK activity exception is that have with cell The relevant diseases such as silk division, cytoskeleton adjustment, smooth muscle cell contraction, nerve regneration, tumor cell invasion, Apoptosis One or more of.
7. purposes according to claim 5, it is characterised in that: the disease is cardiovascular disease, ocular hypertension, glaucoma And cancer.
8. purposes according to claim 6, it is characterised in that: the disease is pulmonary hypertension, ocular hypertension, glaucoma.
9. a kind of drug, it is characterised in that: it is with compound described in claims 1 or 2 any one or its stereoisomer Or its crystal form, pharmaceutically acceptable salt, hydrate or solvate are active constituent, in addition pharmaceutically acceptable auxiliary material system Preparation made of standby.
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