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

Abstract

The present invention discloses a compound represented by formula (I) and a preparation method and application of the compound. It shows good ROCK inhibitory activity, which provides a new medicinal possibility for clinical treatment of diseases related to abnormal ROCK activity.

Description

A compound for inhibiting ROCK and its application

technical field

The invention belongs to the field of drug synthesis, in particular to a compound that inhibits ROCK and its application in the treatment of diseases related to ROCK.

Background technique:

Rho belongs to the small-molecule monomer GTPase superfamily and is a mammalian gene homolog of the Ras superfamily. It regulates cellular actuation through the most important downstream effector molecule, Rho kinase (Rho-associated coiled-coil containing proteinkinase, ROCK). The reorganization of the protein skeleton is widely involved in a series of biological processes such as cell mitosis, cytoskeleton adjustment, smooth muscle cell contraction, nerve regeneration, tumor cell infiltration, and regulation of apoptosis. After activation, Rho/ROCK can act on a variety of substrates, resulting in biological processes. The two most important substrates are myosin light chain (MLC) and myosin light chain phosphatase (MLCP). The phosphorylation level of MLC is an important factor in determining the degree of smooth muscle contraction. Myosin light chain kinase (MLCK) phosphorylates the Ser-19 site of MLC, resulting in smooth muscle contraction; inhibition of MLCP can further enhance MLC phosphorylation and smooth muscle contraction. After ROCK is activated, it can phosphorylate MLC to produce myofilament contraction; at the same time, it can also phosphorylate MLCP to inactivate MLCP, resulting in increased MLC phosphorylation in the cell cytoplasm, which indirectly promotes myofilament contraction.

Inhibition of Rho kinase activity in animal models has demonstrated multiple benefits in the treatment of human diseases, including cardiovascular diseases such as pulmonary hypertension, hypertension, atherosclerosis, cardiac hypertrophy, ocular hypertension, cerebral ischemia, cerebral vasospasm, etc. , and central nervous system disorders such as neuronal degeneration, and tumors. Studies have shown that ROCK expression and activity are elevated in spontaneously hypertensive rats, indicating that it is associated with the occurrence of hypertension in these animals (Involvement of Rho-kinasein hypertensive vascular disease: a novel therapeutic target in hypertension[J]. FASEB J., 2001, 15(6):1062-4). The ROCK inhibitor Y-27632 significantly reduced blood pressure in three rat models of hypertension (spontaneous hypertension, renal hypertension, and deoxycorticosterone acetate-type hypertension), while the effect on blood pressure in control rats was more pronounced than in control rats. Small (Calcium sensitization of smooth muscle mediated by a Rho-associated protein kinase in hypertension[J].Nature,1997,389(6654):990-4). Some studies have also shown that ROCK inhibitors have a good effect on pulmonary hypertension (Acute vasodilator effects of aRho-kinase inhibitor, fasudil, in patients with severe pulmonary hypertension [J]. Heart, 2005:91(3):391-2).

The ROCK inhibitors that have been researched and developed can be divided into five categories: (1) Isoquinolines: These compounds are characterized in that they have an isoquinoline structure and a piperazine ring, which are connected by a sulfonyl group. Representatives include fasudil (Uehata M, Ishizaki T, Satoh H, et al.Calcium sensitization of smooth muscle mediated by aRho-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-kinaseinhibitors and their clinical application[J]. Biochim Biophys Acta, 2005, 1754:245-252); (2) 4-aminopyridines : In addition to the 4-aminopyridine core, the structure of this compound also contains a cyclohexane or benzene ring structure in the center of the molecule, and a side chain structure 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 use 5-amino or 5-alkoxy-1H indazole as the backbone; (4) amides and ureas: such compounds have a skein structure composed of a phthalimide and a urea group. (5) Other categories: Other ROCK inhibitors that do not contain the above structures, represented by 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).

Currently listed ROCK inhibitor drugs include Asahi Kasei's Eril (for the treatment of cerebral vasospasm) and Kowa's Glanatec (for the treatment of ocular hypertension and glaucoma). Among them, Glanatec is only available in Japan. Therefore, it is of great social and economic significance to develop targeted small molecule drugs that act on ROCK to obtain ROCK inhibitors with better activity, higher selectivity, lower toxicity and side effects, and more economical.

Invention content:

The present invention provides a compound of formula I or a stereoisomer thereof:

in,

m is 1 or 2;

n is 1 or 2;

R ¹ is selected from hydrogen, C _1-6 alkyl;

Ring A is selected from 5-6 membered aromatic ring and 5-6 membered aromatic heterocycle; wherein the aromatic ring and aromatic heterocycle can be further independently selected from hydroxyl, halogen, amino, nitro, cyano substituted by substituents of base, trifluoromethyl, carboxyl, C _1-6 alkyl, C _1-6 alkoxy, C _1-6 alkylamino, C _1-6 dialkylamino and C _1-6 acyl;

R ² is selected from hydrogen, hydroxyl, halogen, amino, nitro, cyano, trifluoromethyl, carboxyl, C _1-6 alkyl, C _1-6 alkoxy, C _1-6 alkylamino, C _{1- 6} dialkylamino, C _1-6 acyl;

Further, m and n add up to 2 or 3.

Further, the A ring is a benzene ring.

Preferably, the compound of formula I is shown in the following structural formula:

The present invention also provides the use of the above-mentioned compound or its stereoisomer or its crystal form, pharmaceutically acceptable salt, hydrate or solvate as a medicine for the treatment of diseases related to abnormal ROCK activity.

Specifically, the disease related to abnormal ROCK activity is one or more diseases related to cell mitosis, cytoskeleton adjustment, smooth muscle cell contraction, nerve regeneration, tumor cell infiltration, apoptosis and the like.

Further, the diseases are cardiovascular disease, ocular hypertension, glaucoma and cancer.

Further, the disease is pulmonary hypertension, ocular hypertension, and glaucoma.

In another aspect of the present invention, there is also provided a medicament, which uses the above-mentioned compounds and their stereoisomers or their crystal forms, pharmaceutically acceptable salts, hydrates or solvates as active ingredients, plus pharmaceutically acceptable salts, hydrates or solvates. Formulations prepared from acceptable excipients.

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) nomenclature system.

Definitions of terms used in the present invention: Unless otherwise specified, the initial definitions of groups or terms provided herein apply to the groups or terms throughout the specification; for terms that are not specifically defined herein, they should be based on the disclosure and context. , give their meanings that those skilled in the art can give them.

"Substitution" means that a hydrogen atom in a molecule is replaced by a different atom or molecule.

Minimum and maximum carbon atom content in a hydrocarbon group is indicated by a prefix, eg, the prefix C _a~b ) alkyl denotes any alkyl group containing "a" to "b" carbon atoms. Thus, for example, C _1-4 alkyl refers to an alkyl group containing 1 to 4 carbon atoms.

In the present invention, "~idene" refers to a group obtained after the corresponding hydrocarbon of the group loses two hydrogen atoms, for example, "alkylene" is a group obtained after the corresponding alkane loses two hydrogen atoms.

In the present invention, C _a-b alkoxy group, C _a-b alkyl ester group, C _a-b alkylamino group, C _a-b acyl group respectively refer to an alkyl group containing "a" to "b" carbon atoms and the corresponding The group obtained by connecting the oxygen atom, ester group, amino group and acyl group.

The term "pharmaceutically acceptable" means that a carrier, vehicle, diluent, adjuvant, and/or salt formed is generally chemically or physically compatible with the other ingredients that make up a pharmaceutical dosage form, and is physiologically Compatible with receptors.

The terms "salts" and "pharmaceutically acceptable salts" refer to the above-mentioned compounds or their stereoisomers, acid and/or base salts with inorganic and/or organic acids and bases, and also zwitterionic salts (internal). salts), also including quaternary ammonium salts such as alkylammonium salts. These salts can be obtained directly in the final isolation and purification of the compounds. It can also be obtained by mixing the above-mentioned compound, or a stereoisomer thereof, with a certain amount of acid or base as appropriate (for example, an equivalent amount). These salts may be precipitated in solution and collected by filtration, recovered after evaporation of the solvent, or obtained by lyophilization after reaction in an aqueous medium. The salts described in the present invention can be hydrochloride, sulfate, citrate, benzenesulfonate, hydrobromide, hydrofluoride, phosphate, acetate, propionate, succinate of the compound salt, oxalate, malate, succinate, fumarate, maleate, tartrate or trifluoroacetate.

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 medicaments or pharmaceutical compositions for modulating cellular function or treating diseases. If a group of compounds is used, the compounds may be administered to the subject simultaneously, separately or sequentially

Obviously, according to the above-mentioned content of the present invention, according to the common technical knowledge and conventional means in the field, without departing from the above-mentioned basic technical idea of the present invention, other various forms of modification, replacement or change can also be made.

The above content of the present invention will be further described in detail below through the specific implementation in the form of examples. However, this should not be construed as limiting the scope of the above-mentioned subject matter of the present invention to the following examples. All technologies implemented based on the above content of the present invention belong to the scope of the present invention.

Detailed ways

Compound structures were 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 (Bruker AvanceIII 400 and Bruker Avance 300) nuclear magnetic instruments, and the solvents were deuterated dimethyl sulfoxide (DMSO-d6), deuterated chloroform (CDCl ₃ ), deuterated methanol (MeOD), and internal standard. For tetramethylsilane (TMS).

For the measurement of LC-MS, a Shimadzu LC-MS 2020 (ESI) was used.

For the HPLC measurement, a Shimadzu high pressure liquid chromatograph (Shimadzu LC-20A) was used.

MPLC (medium pressure preparative chromatography) used a Gilson GX-281 reverse phase preparative chromatograph.

Column chromatography generally uses Yantai Huanghai silica gel 200-300 mesh silica gel as the carrier.

The thin layer chromatography silica gel plate is made of Yantai Huanghai HSGF254 or Qingdao GF254 silica gel plate, and the specifications of the thin layer chromatography separation and purification products are 0.4mm ~ 0.5mm.

The raw materials and equipment used in the specific embodiments of the present invention are all known products, which are obtained by purchasing commercially available products. Known starting materials can be synthesized by using or according to methods known in the art, or can be purchased from companies such as Annagy Chemical, Chengdu Kelong Chemical, Shaoyuan Chemical Technology, and Bailingwei Technology.

Room temperature is the most suitable reaction temperature, which is 20°C to 30°C. There is no special description in the examples, and M is moles per liter. There is no special description in the examples, and the solution refers to an aqueous solution.

Abbreviations: DCM is dichloromethane; EA and EtOAc are ethyl acetate; PE is petroleum ether; THF is tetrahydrofuran; DMF is N,N-dimethylformamide; DIEA and DIPEA are diisopropylethylamine; DMAP 4-dimethylaminopyridine; EDCI is 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride; HOBT is 1-hydroxybenzotriazole; HBTU is benzotriazepine Azole-N,N,N',N'-tetramethylurea hexafluorophosphate.

Example 1. Preparation of 6-isoquinoline-((S)-2-phenyl-1,4-azacycloalkane)-methanone

Step 1, (S)-N-benzylidene-2-methylpropane-2-sulfonamide

To a solution of benzaldehyde (30 g, 0.28 mmol) in tetrahydrofuran (150 mL) was added (S) tert-butylsulfinamide (51 g, 0.42 mmol) and tetraisopropyl titanate (100 g, 0.35 mmol) at room temperature, and the solution was added at 60 After stirring at °C for 4 hours, water was added and filtered, extracted with ethyl acetate, and 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)-N-benzylidene-2-methylpropane-2-sulfonamide (54 g, 0.23 mmol, 82% yield).

MS(ESI) m/z=210(M+1) ⁺ .

Step 2. Preparation of (S)-2-methyl-N-(2-nitro-1-phenethyl)-propane-2-sulfinamide

(S)-N-benzylidene-2-methylpropane-2-sulfonamide (48 g, 0.23 mmol) was dissolved in tetrahydrofuran (500 mL), potassium tert-butoxide (38.5 g, 0.34 mmol), the reaction solution was stirred for 1 hour, then nitromethane (140 g, 2.29 mol) was added, and the mixture was stirred at room temperature for 24 hours. The reaction solution was extracted with ethyl acetate and water, and 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)-2-methyl-N-(2-nitro-1-phenethyl)-propane-2-sulfinamide (18 g, 0.67 mmol, 29% yield).

MS(ESI) m/z=271(M+1) ⁺ .

Step 3. Preparation of (S)-2-methyl-N-(2-amino-1-phenethyl)-propane-2-sulfinamide

(S)-2-methyl-N-(2-nitro-1-phenethyl)-propane-2-sulfinamide (8.0 g, 30 mmol) was dissolved in methanol (50 mL), Raney nickel ( 0.80 g, 10%), stirred at room temperature for 24 hours. The reaction solution was filtered with celite, and the solvent was evaporated under reduced pressure to obtain (S)-2-methyl-N-(2-amino-1-phenethyl)-propane-2-sulfinamide (6.0 g, 21.5 mmol, 86% yield).

MS(ESI) m/z=241(M+1) ⁺ .

Step 4. Preparation of benzyl-N-(((S)-tert-butylsulfinamide)-2-phenethyl)carbamate

(S)-2-Methyl-N-(2-amino-1-phenethyl)-propane-2-sulfinamide (7.00 g, 29.1 mmol) was dissolved in tetrahydrofuran (70.0 mL), triethyl Amine (11.8 g, 116 mmol) and benzyloxycarbonyl succinimide (8.70 g, 34.9 mmol), stirred for 1 hour, extracted with ethyl acetate and water, and the aqueous phase was extracted twice with ethyl acetate and combined The organic phase was dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure. Purification by column chromatography gave benzyl-N-(((S)-tert-butylsulfinamide)-2-phenethyl)carbamate (5.9 g, 12.6 mmol, 43% yield).

MS(ESI) m/z=375(M+1) ⁺ .

Step 5. Preparation of benzyl-N-(2-amino-2-phenyl-ethyl)carbamate

Benzyl-N-(((S)-tert-butylsulfinamide)-2-phenethyl)carbamate (5.0 g, 13.4 mmol) was dissolved in methanol (10 mL), 1,4-bisulfite was added The hydrochloric acid solution of oxane (40ml, 5mol/L) was stirred for 1 hour and the solvent was evaporated under reduced pressure to obtain benzyl-N-(2-amino-2-phenyl-ethyl)carbamate (3.02g, 10.3 mmol, 77% yield).

MS(ESI) m/z=271(M+1) ⁺ .

Step 6. Preparation of benzyl-N-((S)-2-(isoquinoline-6-carboxamide)-2-phenyl-ethyl)carbamate

Benzyl-N-(2-amino-2-phenyl-ethyl)carbamate (2.00 g, 7.40 mmol) was dissolved in DMF (10.0 mL) and benzotriazole-N,N,N was added ',N'-tetramethylurea hexafluorophosphate (2.07g, 8.14mmol), isoquinoline-6-carboxylic acid (1.28g, 7.40mmol) and N,N-diisopropylethylamine (3.38g, 29.6 mmol), stirred for 1 hour, extracted with ethyl acetate and water, and then 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. Purified by column chromatography to give benzyl-N-((S)-2-(isoquinoline-6-carboxamide)-2-phenyl-ethyl)carbamate (1.98 g, 4.21 mmol, yield 57 %).

MS(ESI) m/z=426(M+1) ⁺ .

Step 7. Preparation of phenyl-(S)-4-(isoquinoline-6-carbonyl)-3-benzyl-1,4-diazacycle

Benzyl-N-((S)-2-(isoquinoline-6-carboxamide)-2-phenyl-ethyl)carbamate (1.00 g, 2.35 mmol) was dissolved in DMF (5.00 mL) , sodium hydrogen (226 mg, 9.40 mmol) was added at 0 °C under nitrogen, and after stirring for 1 hour, 1,3-dibromopropane (712 mg, 3.53 mmol) was added dropwise to the reaction solution, and after 2 hours of reaction, saturated chlorinated The ammonium solution was quenched, 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 (500 mg, 970 μmol, 41% yield).

MS(ESI) m/z=466(M+1) ⁺ .

Step 8. Preparation of 6-isoquinoline-((S)-2-phenyl-1,4-azacycloalkane)-methanone

Phenyl(S)-4-(isoquinoline-6-carbonyl)-3-benzyl-1,4-diazacycloalkane (170 mg, 360 μmol) was dissolved in acetic acid (5.00 mL) and hydrobromic acid was added Acetic acid solution (2.5ml, 33%), stirred for 1 hour, evaporated the solvent under reduced pressure, and purified by reverse-phase MPLC to obtain 6-isoquinoline-((S)-2-phenyl-1,4-azaheterocycle alkane)-methanone (45 mg, 110 μmol, 33% yield).

MS(ESI) m/z=368(M+1) ⁺ .

¹ H NMR (400 MHz, DMSO-d ₆ +D ₂ O): δ=9.86 (s, 1H), 8.33 (s, 1H), 8.70-8.71 (d, J=6.4 Hz, 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).

Embodiment 2, the preparation of ((S)-isoquinoline-6 base (2-phenylpiperazine-1-yl) ketone

Step 1. Preparation of (S)-4-(isoquinoline-6-formyl)-3-phenylpiperazine-1-carboxylic acid tert-butyl ester

HBTU ( 176 mg, 693 μmol) and DIPEA (373 mg, 2.89 mmol, 504 μL), stirred at room temperature for 2 hours. Quench with water (50.0 mL) and extract with ethyl acetate (50 mL x 2). The organic phases were combined, washed with saturated brine (50 mL×1) and water (50 mL×1) in sequence, the organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was evaporated to remove the solvent under reduced pressure. Purified by column chromatography to obtain (S)-tert-butyl 4-(isoquinoline-6-formyl)-3-phenylpiperazine-1-carboxylate (220 mg, 485 μmol, 84% yield).

MS(ESI) m/z=418(M+1) ⁺ .

Step 2, the preparation of (S)-isoquinolin-6 base (2-phenylpiperazine-1-yl) ketone

To a solution of (S)-tert-butyl 4-(isoquinoline-6-formyl)-3-phenylpiperazine-1-carboxylate (220 mg, 527 μmol) in methanol (10.0 mL) was added concentrated hydrochloric acid at room temperature (2.00 mL), stirred at room temperature for 1 hour, and then evaporated the solvent under reduced pressure. Purification by preparative HPLC gave (S)-isoquinolin-6yl(2-phenylpiperazin-1-yl)methanone (77 mg, 193 μmol, 37% yield).

MS(ESI) m/z=318(M+1) ⁺ .

¹ H NMR (400 MHz, DMSO-d ₆ +D ₂ O): δ=9.81 (s, 1H), 8.64 (d, J=6.44 Hz, 1H), 8.58 (t, J=4.44 Hz, J=3.8 Hz ,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

Following the procedures of steps 1 to 7 in Example 1, substituting benzaldehyde in step 1 with 3-bromobenzaldehyde to obtain (S)-benzyl ester-3-(3-bromophenyl)-4-(isoquinoline oxoline-6-carbonyl)-1,4-azepane-1-carboxylic acid (1.28 g, 2.1% overall yield).

Step 2. Preparation of 6-isoquinoline-((S)-2-(3-bromophenyl)-1,4-azacycloalkane)-methanone

(S)-Benzyl ester-3-(3-bromophenyl)-4-(isoquinoline-6-carbonyl)-1,4-azepane-1-carboxylic acid (200 mg, 0.367 mmol) was dissolved in In acetic acid (5 mL), acetic acid hydrobromic acid solution (2.5 mL, 33%) was added, and the reaction solution 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 (27 mg, 59 μmol, yield 16%).

MS(ESI)m/z=410,412(M+1) ⁺

¹ H 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

6-Isoquinoline-((S)-2-phenyl-1,4-azacycloalkane)-methanone (30 mg, 90 umol) was dissolved in methanol (1.5 mL), and acetic acid (3.4 mg, 45 umol) was added and aqueous formaldehyde solution (58uL, 13M, 0.76mmol), stir for 1 hour, add sodium cyanoborohydride (228mg, 0.36mmol), continue to react for 2 hours, extract with ethyl acetate and water, and then use ethyl acetate for the aqueous phase After extraction twice, the organic phases were combined and dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure. Purified by column chromatography to obtain (S)-isoquinolin-6-yl(4-methyl-2-phenyl-1,4-azacycloalkane)-methanone (1.41mg, 3.96umol, yield 4.4%) ).

MS(ESI) m/z=346(M+1) ⁺ .

¹ H NMR (400 MHz, DMSO-d ₆ ): δ=9.80 (s, 1H), 8.93-8.95 (m, 1H), 8.72-8.73 (d, J=6.0 Hz, 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 the beneficial effects of the present invention, the present invention provides the following test examples:

Test Example 1. Detection of ROCK2 Inhibitory Activity

ROCK2 phosphorylates S6K (KRRRLASLR) polypeptide substrates, converting ATP to ADP. After the kinase reaction, ADP-Glo ^™ reagent is added to stop the kinase reaction and consume the remaining ATP. When adding kinase detection reagent, it converts ADP into ATP at the same time, ATP is then converted into light luminescence signal by Ultra-Glo ^TM luciferase, and the luminescence signal is positively correlated with kinase activity.

The assay of ROCK2 inhibitory activity was performed as follows:

1. Assay Buffer: 40mM Tris pH 7.5, 20mM MgCl2, 0.1%BSA (w/v), 50μM DTT;

2. Add 12μL 2.5x0.1μg/ml ROCK2 working solution into the 96-well PCR plate;

3. Add 6μL of 6x compound working solution into the 96-well PCR plate and mix, and pre-incubate at 25°C for 10min;

4. Add 12μL of 2.5x 37.5μg/ml S6K substrate and 12.5μM ATP mixed working solution, incubate at 30°C for 60min;

5. Take 25μL of the reaction mixture to a new 96-well PCR plate, add 25μL of ADP-Glo ^TM reagent and mix, incubate at 25°C for 40min to terminate the reaction;

6. Take 40 μL of the termination reaction mixture to a new 96-well PCR plate, add 40 μL of kinase detection reagent, mix well, and incubate at 25°C for 40 minutes;

7. Read the luminescence signal value and calculate the inhibition rate.

The compounds prepared in the examples were tested for ROCK2 inhibitory activity according to the above method. The test results are shown in Table 1. The _IC50 of each compound was determined and classified according to the instructions. In Table 1:

"+" indicates that the _IC50 determination value is greater than 250nM;

"++" indicates that the _IC50 value is less than 250nM and greater than 50nM;

"+++" indicates an _IC50 determination of less than 50 nM.

Table 1. Inhibitory activity of compounds on ROCK2

Example ROCK2 Example ROCK2 1 +++ 2 ++ 3 +++ 4 +

Tests show that the compounds of the examples of the present invention have good ROCK inhibitory activity, and can be effectively used for the treatment of diseases with abnormal ROCK activity.

To sum up, the new compound represented by formula I disclosed in the present invention exhibits good ROCK inhibitory activity, and provides a new medicinal possibility for clinical treatment of diseases related to abnormal ROCK activity.

Claims (7)

1. A compound of formula I or a stereoisomer thereof:

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, C_1~6Alkylamino radical, C_1~6Dialkylamino group, C_1~6A 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:

、

。

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.