CN109134433B - Compound for inhibiting ROCK and application thereof - Google Patents

Compound for inhibiting ROCK and application thereof Download PDF

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CN109134433B
CN109134433B CN201810619518.1A CN201810619518A CN109134433B CN 109134433 B CN109134433 B CN 109134433B CN 201810619518 A CN201810619518 A CN 201810619518A CN 109134433 B CN109134433 B CN 109134433B
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李进
张登友
冯静超
廖伟
林丽
李偲
陈伟
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Abstract

The invention discloses a compound shown as a formula (I), and a preparation method and application of the compound. Shows good ROCK inhibitory activity, and provides a new medicinal possibility for clinically treating diseases related to ROCK activity abnormity.

Description

Compound for inhibiting ROCK and application thereof
Technical Field
The invention belongs to the field of drug synthesis, and particularly relates to a compound for inhibiting ROCK and application thereof in treating ROCK-related diseases.
Background art:
rho belongs to a small molecule single-polymer GTPase superfamily, is a mammalian gene homolog of a Ras superfamily, and regulates the recombination of a cell actin framework through a main downstream effector Rho kinase (ROCK), so that Rho can be widely involved in a series of biological processes such as mitosis, cytoskeletal regulation, smooth muscle cell contraction, nerve regeneration, tumor cell infiltration, apoptosis regulation and the like. Rho/ROCK can be activated to act on a variety of substrates, thereby generating a biological process. The two most prominent substrates are Myosin Light Chain (MLC), the level of phosphorylation of which is an important factor in determining the degree of smooth muscle contraction, and Myosin Light Chain Phosphatase (MLCP). Myosin Light Chain Kinase (MLCK) phosphorylates the Ser-19 site of MLC, leading to smooth muscle contraction; inhibition of MLCP can further enhance phosphorylation of MLC and contraction of smooth muscle. After the ROCK is activated, MLC can be phosphorylated to generate myofilament contraction; meanwhile, MLCP can be phosphorylated to inactivate the MLCP, so that the phosphorylation degree of MLC in cytoplasm of cells is increased, and myofilament contraction is indirectly promoted.
Inhibition of Rho kinase activity in animal models has shown various benefits in the treatment of human diseases including cardiovascular diseases such as pulmonary hypertension, atherosclerosis, cardiac hypertrophy, ocular hypertension, cerebral ischemia, cerebral vasospasm, and the like, and central nervous system disorders such as neuronal degeneration and the like, as well as tumors. ROCK expression and activity have been shown to be elevated in spontaneously hypertensive rats, suggesting an association with the development of hypertension in these animals (invasion of Rho-kinase in hypertensive vascular disease: a novel therapeutic target in hypertension [ J ]. FASEB J.,2001,15(6): 1062-4). The ROCK inhibitor Y-27632 can significantly lower blood pressure in three rat hypertension models (spontaneous hypertension, renal hypertension, deoxycorticosterone acetate type hypertension), while having less effect on blood pressure in control rats (Calcium sensitivity of small cardiac media hypertension by a Rho-associated protein kinase in hypertension [ J ] Nature,1997,389(6654): 990-4). It has also been shown that ROCK inhibitors have a better effect on pulmonary hypertension (Acute vasodialator effects of a Rho-kinase inhibitor, facial, in tissues with a segment pulmonary hypertension [ J ]. Heart,2005:91(3): 391-2).
ROCK inhibitors that have been studied and developed to date can be divided into five major classes: (1) isoquinolines: the compound has the structural characteristics that the compound has an isoquinoline structure and a piperazine ring which are connected through a sulfonyl group. Representatives are fasudil (Uehata M, Ishizaki T, Satoh H, et al. calcium transduction of small sized media by a Rho-associated protein kinase in hypertension [ J ]. Nature,1997,389: 990-; (2) 4-aminopyridines: the structure of the compound contains a cyclohexane or benzene ring structure at the central position of a molecule besides a 4-aminopyridine mother nucleus, and a side chain structure is arranged at the 4-position of cyclohexane. Representatives are 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) indazoles: such compounds have 5-amino or 5-alkoxy-1H indazoles as backbone; (4) amides and ureas: the compound has a structure formed by a phthalimide and a carbamide. (5) Other classes: other ROCK inhibitors not comprising the above structure are represented by Rockout (Yarrow JC, Totsukawa G, Charras GT, et al, screening for cell migration inhibitors via automated microscopical improvements a Rho-kinase inhibitor [ J ]. Chem Biol,2005,12: 385-.
ROCK inhibitor drugs are currently marketed by the company Asahi Kasei (for the treatment of cerebral vasospasm) and Kowa Glanatec (for the treatment of ocular hypertension and glaucoma). Of which Glanatec is only commercially available in japan. Therefore, the research of developing targeted micromolecular medicines acting on ROCK is carried out, and the ROCK inhibitor with better activity, higher selectivity, lower toxicity and side effect and more economy is obtained, thereby having very important social and economic significance.
The invention content is as follows:
the invention provides a compound shown as a formula I or a stereoisomer thereof:
Figure BDA0001696876810000021
wherein,
m is 1 or 2;
n is 1 or 2;
R1selected from hydrogen, C1～6An alkyl group;
the ring A is selected from a 5-6-membered aromatic ring and a 5-6-membered aromatic heterocycle; wherein the aromatic ring and the aromatic heterocycle can be further independently selected from 1 to 4 of hydroxyl, halogen, amino, nitro, cyano, trifluoromethyl, carboxyl and C1～6Alkyl radical, C1～6Alkoxy radical, C1～6Alkylamino radical, C1～6Dialkylamino group, C1～6Acyl is substituted by a substituent;
R2selected from hydrogen, hydroxy, halogen, amino, nitro, cyano, trifluoromethyl, carboxy, C1～6Alkyl radical, C1～6Alkoxy radical, C1～6Alkylamino radical, C1～6Dialkylamino group, C1～6An acyl group;
further, m and n are added to 2 or 3.
Further, ring A is a benzene ring.
Preferably, the compounds of formula I are represented by the following structural formula:
Figure BDA0001696876810000031
the invention also provides application of the compound or the stereoisomer or the crystal form, the pharmaceutically acceptable salt, the hydrate or the solvate thereof in preparing a medicament for treating diseases related to ROCK activity abnormity.
Specifically, the diseases related to abnormal ROCK activity are one or more of diseases related to cell mitosis, cytoskeleton regulation, smooth muscle cell contraction, nerve regeneration, tumor cell infiltration, apoptosis and the like.
Further, the diseases are cardiovascular diseases, ocular hypertension, glaucoma and cancer.
Further, the disease is pulmonary hypertension, ocular hypertension, glaucoma.
On the other hand, the invention also provides a medicament which is a preparation prepared by taking the compound and the stereoisomer or the crystal form, the pharmaceutically acceptable salt, the hydrate or the solvate thereof as active ingredients and adding pharmaceutically acceptable auxiliary materials.
The compounds and derivatives provided in the present invention may be named according to the IUPAC (international union of pure and applied chemistry) or CAS (chemical abstracts service, Columbus, OH) naming system.
Definitions of terms used in connection with the present invention: the initial definitions provided herein for a group or term apply to that group or term throughout the specification unless otherwise indicated; for terms not specifically defined herein, the meanings that would be given to them by a person skilled in the art are to be given in light of the disclosure and the context.
"substituted" means that a hydrogen atom in a molecule is replaced by a different atom or molecule.
The minimum and maximum values of the carbon atom content in the hydrocarbon group are indicated by a prefix, e.g. prefix Ca～b) Alkyl means any alkyl group containing from "a" to "b" carbon atoms. Thus, for example, C1～4The alkyl group means an alkyl group having 1 to 4 carbon atoms.
The term "arylene" as used herein refers to a group wherein the corresponding hydrocarbon of the group has been deprived of two hydrogen atoms, e.g., "alkylene" refers to a group wherein the corresponding alkane has been deprived of two hydrogen atoms.
In the invention Ca～bAlkoxy radical, Ca～bAlkyl ester group, Ca～bAlkylamino radical, Ca～bAcyl is a group formed by connecting alkyl containing carbon atoms from "a" to "b" with corresponding oxygen atom, ester group, amino group and acyl.
The term "pharmaceutically acceptable" means that the carrier, cargo, diluent, adjuvant, and/or salt formed is generally chemically or physically compatible with the other ingredients comprising a pharmaceutical dosage form and physiologically compatible with the recipient.
The terms "salt" and "pharmaceutically acceptable salt" refer to acid and/or base salts of the above compounds or stereoisomers thereof, with inorganic and/or organic acids and bases, as well as zwitterionic (inner) salts, and also quaternary ammonium salts, such as alkylammonium salts. These salts can be obtained directly in the final isolation and purification of the compounds. The compound or a stereoisomer thereof may be obtained by appropriately (e.g., equivalently) mixing the above compound or a stereoisomer thereof with a predetermined amount of an acid or a base. These salts may form precipitates in the solution which are collected by filtration, or they may be recovered after evaporation of the solvent, or they may be prepared by reaction in an aqueous medium followed by lyophilization. The salt in the invention can be hydrochloride, sulfate, citrate, benzene sulfonate, hydrobromide, hydrofluoride, phosphate, acetate, propionate, succinate, oxalate, malate, succinate, fumarate, maleate, tartrate or trifluoroacetate of the compound.
In certain embodiments, one or more compounds of the present invention may be used in combination with each other. Alternatively, the compounds of the present invention may be used in combination with any other active agent for the preparation of a medicament or pharmaceutical composition for modulating cellular function or treating a disease. If a group of compounds is used, the compounds may be administered to a subject simultaneously, separately or sequentially
Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.
The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.
Detailed Description
The structure of the compounds was determined by Nuclear Magnetic Resonance (NMR) and Mass Spectrometry (MS). NMR shifts (. delta.) are given in units of 10-6 (ppm). NMR was measured using a (Bruker AvanceIII 400 and Bruker Avance 300) nuclear magnetic instrument using deuterated dimethyl sulfoxide (DMSO-d6), deuterated chloroform (CDCl)3) Deuterated methanol (MeOD), internal standard Tetramethylsilane (TMS).
LC-MS was measured using Shimadzu LC-MS 2020 (ESI).
HPLC was performed using Shimadzu high pressure liquid chromatograph (Shimadzu LC-20A).
MPLC (Medium pressure preparative chromatography) Gilson GX-281 reverse phase preparative chromatography was used.
The column chromatography generally uses 200-300 mesh silica gel of the Tibet Huanghai silica gel as a carrier.
The thin layer chromatography silica gel plate is a tobacco yellow sea HSGF254 or Qingdao GF254 silica gel plate, and the specification of the thin layer chromatography separation and purification product is 0.4 mm-0.5 mm.
The raw materials and equipment used in the embodiment of the present invention are known products and obtained by purchasing commercially available products. Known starting materials can be synthesized by or according to methods known in the art or can be purchased from companies such as Enfagi chemistry, Chengdong chemical, Shaoshi chemical technology, Bailingwei technology, and the like.
The room temperature is the most suitable reaction temperature and is 20-30 ℃. In the examples, M is mole per liter, unless otherwise specified. In the examples, the solution means an aqueous solution unless otherwise specified.
Brief description: DCM is dichloromethane; EA. EtOAc is ethyl acetate; PE is petroleum ether; THF is tetrahydrofuran; DMF is N, N-dimethylformamide; DIEA and DIPEA are diisopropylethylamine; DMAP is 4-dimethylaminopyridine; EDCI is 1-ethyl- (3-dimethylaminopropyl) carbonyldiimine hydrochloride; HOBT is 1-hydroxybenzotriazole; HBTU is benzotriazole-N, N, N ', N' -tetramethyluronium hexafluorophosphate.
Example 1 preparation of 6-isoquinoline- ((S) -2-phenyl-1, 4-azacycloalkane) -methanone
Step 1, (S) -N-benzylidene-2-methylpropane-2-sulfonamide
Figure BDA0001696876810000051
To a solution of benzaldehyde (30g,0.28mmol) in tetrahydrofuran (150mL) at room temperature were added (S) tert-butylsulfinamide (51g,0.42mmol) and tetraisopropyl titanate (100g,0.35mmol), stirred at 60 ℃ for 4 hours, filtered with water, extracted with ethyl acetate, the aqueous phase extracted twice with ethyl acetate, the organic phases combined and dried over anhydrous sodium sulfate, and the solvent evaporated under reduced pressure. Purification by column chromatography gave (S) -N-benzylidene-2-methylpropane-2-sulfonamide (54g,0.23mmol, 82% yield).
MS(ESI)m/z＝210(M+1)+。
Step 2 preparation of (S) -2-methyl-N- (2-nitro-1-phenylethyl) -propane-2-sulfinamide
Figure BDA0001696876810000052
(S) -N-benzylidene-2-methylpropane-2-sulfonamide (48g,0.23mmol) was dissolved in tetrahydrofuran (500mL), potassium tert-butoxide (38.5g,0.34mmol) was added at 0 ℃ under nitrogen protection, and after the reaction mixture was stirred for 1 hour, nitromethane (140g,2.29mol) was added and the mixture was stirred at room temperature for 24 hours. The reaction solution was extracted with ethyl acetate and water, the aqueous phase was extracted twice with ethyl acetate, the organic phases were combined and dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure. Purification by column chromatography gave (S) -2-methyl-N- (2-nitro-1-phenylethyl) -propane-2-sulfinamide (18g,0.67mmol, 29% yield).
MS(ESI)m/z＝271(M+1)+。
Step 3 preparation of (S) -2-methyl-N- (2-amino-1-phenylethyl) -propane-2-sulfinamide
Figure BDA0001696876810000053
(S) -2-methyl-N- (2-nitro-1-phenylethyl) -propane-2-sulfinamide (8.0g,30mmol) was dissolved in methanol (50mL), Raney' S nickel (0.80g, 10%) was added, and the mixture was stirred at room temperature for 24 hours. The reaction mixture was filtered through celite, and the solvent was evaporated under reduced pressure to give (S) -2-methyl-N- (2-amino-1-phenylethyl) -propane-2-sulfinamide (6.0g,21.5mmol, yield 86%).
MS(ESI)m/z＝241(M+1)+。
Step 4 preparation of benzyl-N- (((S) -tert-butylsulfinamide) -2-phenylethyl) carbamate
Figure BDA0001696876810000061
(S) -2-methyl-N- (2-amino-1-phenylethyl) -propane-2-sulfinamide (7.00g,29.1mmol) was dissolved in tetrahydrofuran (70.0mL), triethylamine (11.8g,116mmol) and benzyloxycarbonyl succinimide (8.70g,34.9mmol) were added, and after stirring for 1 hour, extraction was performed with ethyl acetate and water, the aqueous phase was extracted twice with ethyl acetate, the organic phases were combined and dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure. Purification by column chromatography gave benzyl-N- (((S) -tert-butylsulfinamide) -2-phenylethyl) carbamate (5.9g,12.6mmol, 43% yield).
MS(ESI)m/z＝375(M+1)+。
Step 5 preparation of benzyl-N- (2-amino-2-phenyl-ethyl) carbamate
Figure BDA0001696876810000062
benzyl-N- (((S) -tert-butylsulfinamide) -2-phenylethyl) carbamate (5.0g,13.4mmol) was dissolved in methanol (10mL), a 1, 4-dioxane hydrochloric acid solution (40mL,5mol/L) was added, and after stirring for 1 hour, the solvent was distilled off under reduced pressure to give benzyl-N- (2-amino-2-phenyl-ethyl) carbamate (3.02g,10.3mmol, 77% yield).
MS(ESI)m/z＝271(M+1)+。
Step 6 preparation of benzyl-N- ((S) -2- (isoquinoline-6-carboxamide) -2-phenyl-ethyl) carbamate
Figure BDA0001696876810000063
benzyl-N- (2-amino-2-phenyl-ethyl) carbamate (2.00g,7.40mmol) was dissolved in DMF (10.0mL), benzotriazole-N, N, N ', N' -tetramethyluronium hexafluorophosphate (2.07g,8.14mmol), isoquinoline-6-carboxylic acid (1.28g,7.40mmol) and N, N-diisopropylethylamine (3.38g,29.6mmol) were added, after stirring for 1 hour extraction with ethyl acetate and water, the aqueous phase was extracted twice with ethyl acetate, the organic phases were combined and dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure. Purification by column chromatography gave benzyl-N- ((S) -2- (isoquinoline-6-carboxamide) -2-phenyl-ethyl) carbamate (1.98g,4.21mmol, 57% yield).
MS(ESI)m/z＝426(M+1)+。
Step 7, preparation of phenyl- (S) -4- (isoquinoline-6-carbonyl) -3-benzyl-1, 4-diazacyclo
Figure BDA0001696876810000071
benzyl-N- ((S) -2- (isoquinoline-6-carboxamide) -2-phenyl-ethyl) carbamate (1.00g,2.35mmol) was dissolved in DMF (5.00mL), sodium hydride (226mg,9.40mmol) was added at 0 ℃ under nitrogen, 1, 3-dibromopropane (712mg,3.53mmol) was added dropwise to the reaction after stirring for 1 hour, after 2 hours of reaction quenched with saturated ammonium chloride solution, extracted with ethyl acetate, the aqueous phase was extracted twice with ethyl acetate, the organic phases were combined and dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure. Purification by column chromatography gave phenyl- (S) -4- (isoquinoline-6-carbonyl) -3-benzyl-1, 4-diazacycloalkane (500mg, 970. mu. mol, 41% yield).
MS(ESI)m/z＝466(M+1)+。
Step 8, preparation of 6-isoquinoline- ((S) -2-phenyl-1, 4-azacycloalkane) -methanone
Figure BDA0001696876810000072
Phenyl (S) -4- (isoquinoline-6-carbonyl) -3-benzyl-1, 4-diazacycloalkane (170mg, 360. mu. mol) was dissolved in acetic acid (5.00mL), a hydrobromic acid acetic acid solution (2.5mL, 33%) was added, the solvent was evaporated under reduced pressure after stirring for 1 hour, and the mixture was purified by reverse-phase MPLC to give 6-isoquinoline- ((S) -2-phenyl-1, 4-azacycloalkane) -methanone (45mg, 110. mu. mol, 33% yield).
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)。
Example 2 preparation of ((S) -isoquinolin-6-yl (2-phenylpiperazin-1-yl) methanone
Step 1 preparation of tert-butyl (S) -4- (isoquinoline-6-formyl) -3-phenylpiperazine-1-carboxylate
Figure BDA0001696876810000073
To a solution of isoquinoline-6-carboxylic acid (100mg, 577. mu. mol) and tert-butyl (S) -3-phenylpiperazine-1-carboxylate (152mg, 577. mu. mol) in DMF (4.00mL) was added HBTU (176mg, 693. mu. mol) and DIPEA (373mg,2.89mmol, 504. mu.L) at room temperature, and the mixture was stirred at room temperature for 2 hours. Quenched with water (50.0mL) and extracted with ethyl acetate (50 mL. times.2). The organic phases were combined, washed with saturated brine (50 mL. times.1) and water (50 mL. times.1) in this order, dried over anhydrous sodium sulfate, filtered, and the filtrate was evaporated under reduced pressure to remove the solvent. Purification by column chromatography gave (S) -4- (isoquinoline-6-formyl) -3-phenylpiperazine-1-carboxylic acid tert-butyl ester (220mg, 485. mu. mol, 84% yield).
MS(ESI)m/z＝418(M+1)+。
Step 2 preparation of (S) -isoquinolin-6-yl (2-phenylpiperazin-1-yl) methanone
Figure BDA0001696876810000081
To a solution of (S) -4- (isoquinoline-6-formyl) -3-phenylpiperazine-1-carboxylic acid tert-butyl ester (220mg, 527. mu. mol) in methanol (10.0mL) was added concentrated hydrochloric acid (2.00mL) at room temperature, and after stirring at room temperature for 1 hour, the solvent was evaporated under reduced pressure. Purification by preparative HPLC afforded (S) -isoquinolin-6-yl (2-phenylpiperazin-1-yl) methanone (77mg, 193. mu. mol, 37% yield).
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)。
Example 3 preparation of 6-isoquinoline- ((S) -2- (3-bromophenyl) -1, 4-azacycloalkane) -methanone
Step 1 preparation of (S) -benzyl ester-3- (3-bromophenyl) -4- (isoquinoline-6-carbonyl) -1, 4-azepane-1-carboxylic acid
Figure BDA0001696876810000082
Following the procedure of steps 1 to 7 in example 1, benzaldehyde in step 1 was replaced with 3-bromobenzaldehyde to give (S) -benzyl ester-3- (3-bromophenyl) -4- (isoquinoline-6-carbonyl) -1, 4-azepane-1-carboxylic acid (1.28g, overall yield 2.1%).
Step 2, preparation of 6-isoquinoline- ((S) -2- (3-bromophenyl) -1, 4-azacycloalkane) -methanone
Figure BDA0001696876810000083
(S) -benzyl ester-3- (3-bromophenyl) -4- (isoquinoline-6-carbonyl) -1, 4-azepane-1-carboxylic acid (200mg,0.367mmol) was dissolved in acetic acid (5mL), and hydrobromic acid acetic acid solution (2.5mL, 33%) was added and the reaction was stirred for one hour. The organic solvent was removed from the reaction solution by a rotary evaporator to obtain 6-isoquinoline- ((S) -2- (3-bromophenyl) -1, 4-azacycloalkane) -methanone (27mg, 59. mu. mol, 16% yield).
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)。
EXAMPLE 4 preparation of (S) -isoquinolin-6-yl (4-methyl-2-phenyl-1, 4-azacycloalkane) -methanone
Figure BDA0001696876810000091
6-isoquinoline- ((S) -2-phenyl-1, 4-azacycloalkane) -methanone (30mg, 90. mu. mol) was dissolved in methanol (1.5mL), acetic acid (3.4mg, 45. mu. mol) and aqueous formaldehyde (58uL, 13M, 0.76mmol) were added, after stirring for 1 hour, sodium cyanoborohydride (228mg,0.36mmol) was added, the reaction was continued for 2 hours and extracted with ethyl acetate and water, the aqueous phase was extracted twice with ethyl acetate, the organic phases were combined and dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure. Purification by column chromatography gave (S) -isoquinolin-6-yl (4-methyl-2-phenyl-1, 4-azacycloalkane) -methanone (1.41mg,3.96umol, 4.4% yield).
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)。
To illustrate the advantageous effects of the present invention, the present invention provides the following test examples:
test example 1 detection of ROCK2 inhibitory Activity
ROCK2 is capable of phosphorylating the S6K (KRRRLASLR) polypeptide substrate, converting ATP to ADP. After the kinase reaction, ADP-Glo was addedTMReagents to terminate the kinase reaction and consume excess ATP. Adding a kinase detection reagent which converts ADP to ATP and simultaneously converts ATP to Ultra-GloTMThe luciferase is converted into a luminescent signal, which is positively correlated with kinase activity.
The ROCK2 inhibitory activity was measured as follows:
1.Assay Buffer:40mM Tris pH 7.5，20mM MgCl2，0.1％BSA(w/v)，50μM DTT；
2. adding 12 mu L2.5x0.1 mu g/ml ROCK2 working solution into a 96-well PCR plate;
3. adding 6 μ L of 6x compound working solution, mixing with 96-well PCR plate, and pre-incubating at 25 deg.C for 10 min;
4. adding 12 μ L of mixed working solution of 2.5 × 37.5 μ g/ml S6K substrate and 12.5 μ MATP, and incubating at 30 deg.C for 60 min;
5. 25 μ L of the reaction mixture was transferred to a new 96-well PCR plate and 25 μ L of ADP-Glo was addedTMMixing the reagents uniformly, and incubating for 40min at 25 ℃ to terminate the reaction;
6. taking 40 mu L of termination reaction mixture to a new 96-well PCR plate, adding 40 mu L of kinase detection reagent, mixing uniformly, and incubating for 40min at 25 ℃;
7. the luminescence signal value was read and the inhibition ratio was calculated.
ROCK2 inhibitory Activity of the compounds prepared in the examples was measured according to the above-mentioned method, and the results are shown in Table 1, wherein IC of each compound was measured50Sorted by description, in table 1:
"+" denotes IC50The assay value was greater than 250 nM;
"+ +" denotes IC50A measurement of less than 250nM and greater than 50 nM;
"+ + + +" denotes IC50The assay was less than 50 nM.
TABLE 1 inhibitory Activity of Compounds on ROCK2
Examples ROCK2 Examples ROCK2
1 +++ 2 ++
3 +++ 4 +
Experiments show that the compound provided by the embodiment of the invention has good ROCK inhibitory activity and can be effectively used for treating diseases with abnormal ROCK activity.
In conclusion, the novel compound shown in the formula I shows good ROCK inhibitory activity, and provides a novel medicinal possibility for clinically treating diseases related to ROCK activity abnormity.

Claims (7)

1. A compound of formula I or a stereoisomer thereof:
Figure DEST_PATH_IMAGE001
wherein,
m is 2;
n is 1;
R1is hydrogen;
the ring A is a benzene ring or is independently selected from 1 to 4 of hydroxyl, halogen, amino, nitro, cyano, trifluoromethyl, carboxyl and C1~6Alkyl radical, C1~6Alkoxy radical, C1~6Alkylamino radical, C1~6Dialkylamino group, C1~6A benzene ring substituted with a substituent of an acyl group;
R2is hydrogen.
2. A compound according to claim 1, or a stereoisomer thereof, characterized by: the compound is represented by the following structural formula:
Figure 212368DEST_PATH_IMAGE002
、
Figure DEST_PATH_IMAGE003
。
3. use of a compound according to any one of claims 1 to 2, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the treatment of a disease associated with abnormal ROCK activity.
4. Use according to claim 3, characterized in that: the diseases related to abnormal ROCK activity are one or more of diseases related to cell mitosis, cytoskeleton regulation, smooth muscle cell contraction, nerve regeneration, tumor cell infiltration and cell apoptosis.
5. Use according to claim 4, characterized in that: the diseases are cardiovascular diseases, ocular hypertension, glaucoma and cancer.
6. Use according to claim 4, characterized in that: the diseases are pulmonary hypertension, ocular hypertension and glaucoma.
7. A medicament, characterized by: the compound or the stereoisomer or the pharmaceutically acceptable salt thereof as claimed in any one of claims 1 to 2 is used as an active ingredient, and is added with pharmaceutically acceptable auxiliary materials to prepare the preparation.
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EP1878732A1 (en) * 2005-04-25 2008-01-16 D. Western Therapeutics Institute HIGHLY SELECTIVE Rho-KINASE INHIBITOR
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EP1878732A1 (en) * 2005-04-25 2008-01-16 D. Western Therapeutics Institute HIGHLY SELECTIVE Rho-KINASE INHIBITOR
CN102448941A (en) * 2009-06-19 2012-05-09 株式会社D.西医疗法研究所 Substituted isoquinoline derivative

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