A nitrocatechol derivative, a pharmaceutical composition containing the same and its application

Abstract

The present invention provides a nitrocatechol derivative, a pharmaceutical composition containing the same and application thereof. The nitrocatechol derivative includes a compound as shown in formula (I) or formula (II), a pharmaceutically acceptable salt thereof or a solvate thereof, which has COMT / MAO-B dual-target inhibitory activity and also has important application value for treating PD.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical chemistry, and specifically relates to a nitrocatechol derivative, a pharmaceutical composition containing the same, and its application Background Art

[0002] Parkinson's disease (PD) is a serious progressive neurodegenerative movement disorder, and its cause is still unclear. The degeneration of dopamine neurons in the substantia nigra and striatum leads to DA deficiency, which is the cause of PD movement disorders. Its main symptoms are usually akinesia, rigidity and tremor. The main strategy for treating Parkinson's disease is to increase the level of dopamine (DA) in the brain. Clinical drug treatment is mainly based on the precursor drug levodopa (L-Dopamine). In addition, it also includes DA receptor agonists, catechol-O-methyltransferase (COMT) inhibitors, monoamine oxidase B (MAO-B) inhibitors, anticholinergic drugs, etc.

[0003] COMT is an enzyme responsible for metabolizing endogenous neurotransmitters. It transfers a methyl group from its coenzyme S-adenosyl-L-methionine (SAM) to a hydroxyl group of the endogenous neurotransmitter through magnesium ion catalysis, thereby terminating its biological activity. Currently, most known COMT inhibitors are nitrocatechol-based compounds, such as the marketed drugs Tolcapone and Entacapone, which can inhibit the activity of COMT enzymes in peripheral and central tissues, block the peripheral degradation of levodopa, and increase its plasma half-life, thereby allowing more oral levodopa to cross the blood-brain barrier (BBB) ​​and enter the brain. Therefore, they are used as an adjunct to levodopa in the treatment of Parkinson's disease. Monoamine oxidase is a mitochondrial enzyme that plays an important role in the in vivo inactivation of biogenic amines in peripheral and central nervous tissues. In the central nervous system (CNS), dopamine is mainly metabolized by MAO-B. Currently, MAO-B inhibitors such as rasagiline, selegiline, and safinamide have been developed for clinical use and are often used alone or in combination with levodopa to treat early PD. In addition, hydrogen peroxide (H2O2) is a byproduct of MAO-B metabolism of dopamine, which may cause oxidative damage to dopaminergic neurons.

[0004] In theory, COMT / MAO-B dual-target inhibitors can effectively inhibit the metabolism of levodopa and dopamine in peripheral and central tissues through multiple pathways, thereby prolonging the half-life of levodopa and inhibiting DA metabolism, thereby increasing and maintaining the DA content in the body through two pathways: increasing exogenous DA and reducing endogenous DA metabolism. In addition, as MAO-B inhibitors, they can also reduce the hydrogen peroxide (H2O2) generated in dopamine metabolism, thereby preventing dopaminergic neurons from being damaged by oxidation. Therefore, the development of COMT / MAO-B dual-target inhibitors is reasonable for the treatment of PD and has important research and application value. At present, there are no COMT / MAO-B dual-target inhibitors. Summary of the invention

[0005] The object of the present invention is to provide a nitrocatechol derivative having COMT / MAO-B enzyme inhibitory activity and a pharmaceutical composition comprising the same. Another object of the present invention is to provide a preparation method and application of the nitrocatechol derivative.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is:

[0007] A nitrocatechol derivative, comprising a compound as shown in formula (I) or (II), a pharmaceutically acceptable salt thereof or a solvate thereof, wherein R1 is selected from substituted or unsubstituted amide, substituted or unsubstituted C1-6 alkyl, substituted or unsubstituted C1-6 alkoxy, substituted or unsubstituted amine, substituted or unsubstituted phenyl, ester, cyano, nitro or halogen, the substitution is mono- or poly-substituted, and the substituent is C1-6 alkyl, halogen, cyano, phenyl or nitro;

[0008]

[0009] R2 is selected from H, CN, nitro or halogen.

[0010] As a further improvement of the technical solution, R1 is selected from -OCH3, Where Y is Z is

[0011] R2 is selected from H or CN.

[0012] As a further improvement of the technical solution, it is selected from any one of the following compounds:

[0013]

[0014] A method for preparing the nitrocatechol derivative comprises the following steps:

[0015]

[0016] (1) Compound 1 and active methylene undergo Knoevenagel condensation reaction to obtain compound 2; Compound 1 undergoes Pinnick oxidation reaction with NaClO2 to obtain compound 3;

[0017] (2) Compound 2 is subjected to condensation reaction with amine and alcohol respectively to obtain a compound as shown in formula (I); Compound 3 is subjected to condensation reaction with amine and alcohol respectively to obtain a compound as shown in formula (II).

[0018] The present invention also discloses a pharmaceutical composition, which comprises the nitrocatechol derivative and one or more pharmaceutically acceptable excipients. Pharmaceutically acceptable excipients refer to excipients and additives used in the production of drugs and the preparation of prescriptions, including solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, adhesives, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, aromatics, anti-adhesive agents, integrators, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants and deflocculating agents, filter aids, release retardants, etc., preferably oral pharmaceutical excipients such as disintegrants, cosolvents, solvents, lubricants, etc.

[0019] As a further improvement of the technical solution, the dosage form of the pharmaceutical composition is capsule, powder, pill, tablet, granule, suspension or injection.

[0020] Since the nitrocatechol derivatives of the present invention have both COMT and MAO inhibitory activities, they have application prospects for Parkinson's disease. The present invention also discloses the use of the nitrocatechol derivatives in the preparation of drugs for preventing and / or treating Parkinson's disease.

[0021] Since the nitrocatechol derivatives of the present invention also have COMT inhibitory activity, the present invention also discloses a use of the nitrocatechol derivatives in the preparation of COMT inhibitors.

[0022] Since the nitrocatechol derivatives of the present invention also have MAO inhibitory activity, the present invention also discloses a use of the nitrocatechol derivatives in the preparation of MAO inhibitors.

[0023] The present invention has outstanding substantive features and significant progress compared to the prior art. Specifically, the dual-target compound of the present invention has COMT and MAO inhibitory activity. Furthermore, the compound of the present invention has good BBB permeability and has important application value in the preparation of drugs for preventing or treating Parkinson's syndrome. Furthermore, the preparation method of the compound of the present invention is simple and convenient for industrial transformation and application. DETAILED DESCRIPTION

[0024] The technical solution of the present invention is further described in detail below through specific implementation methods.

[0025] The experimental methods in the examples of this disclosure that do not specify specific conditions are usually carried out under conventional conditions or under conditions recommended by raw material or product manufacturers. Reagents that do not specify specific sources are conventional reagents purchased from the market.

[0026] The structures of the compounds were determined by nuclear magnetic resonance (NMR) using a Bruker AVANCE-300 / 500 NMR spectrometer. The solvent used was CDCl3 or DMSO-d6, and the internal standard was TMS.

[0027] Embodiment 1:

[0028] (4-{[(3-Fluorophenyl)methyl]oxy}phenyl)methanamine (4a)

[0029]

[0030] Compound 4-[[tert-Butyloxyamide]methyl]-phenol (2.23 g, 10 mmol) was dissolved in acetonitrile, K2CO3 (1.94 g, 15 mmol) was added and stirred for 3 min, 3-fluorobenzyl bromide (1.46 ml, 10 mmol) was added, and the mixture was heated under reflux for 6 h. The reaction solution was filtered, the filtrate was concentrated, dissolved in DCM (20 ml), an equal volume of TFA was added dropwise, and the reaction was carried out at room temperature for 3 h. The reaction solution was concentrated to obtain compound 4a with a yield of 90%. 1 H NMR (300MHz, DMSO-d6, δppm): 8.10 (s, 2H, -NH2), 7.50 (dd, J=8.5, 5.7Hz, 2H, Ar-H), 7.38 (d, J=8.6Hz, 2H, Ar-H),7.23(t,J=8.9Hz,2H,Ar-H),7.06(d,J=8.6Hz,2H,Ar-H),5.12(s,2H,-OCH2-),3.96(s,2H,-CH2-).

[0031] Embodiment 2:

[0032] (4-{[(4-Fluorophenyl)methyl]oxy}phenyl)methanamine (4b)

[0033]

[0034] Substituting 4-fluorobenzyl bromide for 3-fluorobenzyl bromide and keeping other conditions unchanged, the procedures of Example 1 were repeated to obtain compound 4b. 1HNMR (300MHz, DMSO-d6, δppm): 8.12 (s, 2H, -NH2), 7.49–7.40 (m, 1H, Ar-H), 7.38 (d, J = 8.7Hz, 2H, Ar-H), 7.30 (s, 1H, Ar-H), 7 .27(dd,J=5.3,3.3Hz,1H,Ar-H),7.20–7.12(m,1H,Ar-H),7.09–7.03(m,2H,Ar-H),5.17(s,2H,-OCH2-),3.96(s,2H,-CH2-).

[0035] Embodiment 3:

[0036] 4-(Aminomethyl)-N-(prop-2-ynyl)benzamide (5a)

[0037]

[0038] 4-[(tert-Butyloxycarbonylamino)methyl]benzoic acid (2.5 g, 10 mmol) was dissolved in DMF (6 mL), DIEA (1653 μL, 10 mmol) was added, PyBOP (5.2 g, 10 mmol) was added in an ice-water bath, and the mixture was stirred for 0.5 h. Propargylamine (768 μL, 12 mmol) was added, and the mixture was reacted at room temperature for 6 h. 30 mL of water was added to precipitate a white solid, which was filtered off with suction. The filter cake was dried and dissolved in DCM (20 ml). An equal volume of TFA was added dropwise thereto, and the mixture was reacted at room temperature for 1 h. The reaction solution was concentrated and recrystallized from DCM to obtain compound 5a with a yield of 80%. 1 HNMR(300MHz,DMSO-d6,δppm):9.02(t,J=5.5Hz,1H,-CONH-),8.31(s,2H,-NH2),7.90(d,J=8.3 Hz,2H,Ar-H),7.55(d,J=8.3Hz,2H,Ar-H),4.11(s,2H,-CH2-),4.07(dd,J=5.5,2.5Hz,2H,-CONH CH2 -),3.16(t,J=2.5Hz,1H,-CCH).

[0039] Embodiment 4:

[0040] (2S)-2-({[4-(Aminomethyl)phenyl]carbonyl}amino)propanamide (5b)

[0041]

[0042] The procedure of Example 3 was repeated with L-alaninamide hydrochloride replacing propargylamine and other conditions remaining unchanged to obtain compound 5b. 1H NMR (300MHz, DMSO-d6, δppm): 8.49 (d, J=7.5Hz, 1H, -CONH-), 8.32 (s, 2H, -NH2), 7.94 (d, J=8.2Hz, 2H, Ar-H),7.54(d,J=8.2Hz,2H,Ar-H),7.43(s,1H,-CONH2),7.03(s,1H,-CONH2),4.42(m,J=7.2Hz,1H,- CH CH3),4.11(s,2H,CH2Ar),1.34(d,J=7.2Hz,3H,-CH CH3 ).

[0043] Embodiment 5:

[0044] (2E)-2-Cyano-3-(3,4-dihydroxy-5-nitrophenyl)-N-(prop-2-ynyl)prop-2-enamide (C1)

[0045]

[0046] 2a (250 mg, 1.0 mmol) was dissolved in DMF (2 mL), DIEA (174 μL, 1 mmol) was added, PyBOP (520 mg, 1.0 mmol) dissolved in DCM (2 mL) was added in an ice-water bath, stirred for 0.5 h, and then propargylamine (77 μL, 1.2 mmol) was added, and the reaction was carried out at room temperature for 6 h. After low pressure concentration, ethyl acetate (30 mL) was added, and the mixture was washed with water (3×10 mL), HCl 1M (3×10 mL) and brine (10 mL) in sequence, and the organic layer was concentrated and recrystallized from DCM to obtain product C1 with a yield of 25%, mp 200-202°C, HPLC (93% methanol in water with 0.1% HCOOH): 99.1%, t R =3.130min. 1 H NMR(300MHz,DMSO-d6,δppm):10.93(br s,1H,OH),8.88(t,J=5.5Hz,1H,-CONH-),8.09(s,1H,CHAr),7.97(d,J=2.1Hz,1H,Ar-H),7.80(d,J=2.2Hz,1H,Ar-H),3.99(dd,J=2.5Hz,2H,-NH CH2 -),3.18(t,J=2.5Hz,1H,-CCH). 13C NMR(75MHz,DMSO-d6,δppm):161.52,149.83,148.66,146.46,137.68,122.19,12 0.17,118.23,116.68,104.02,81.05,73.76,29.57.HRMS(m / z):[MH]-calcd.for C 13 H9N3O5: 286.0469; found, 286.0495.

[0047] Embodiment 6:

[0048] N-[(2E)-2-Cyano-3-(3,4-dihydroxy-5-nitrophenyl)-1-oxyylideneprop-2-enyl]-L-alanine methyl ester (C2)

[0049]

[0050] 2a (250 mg, 1.0 mmol) was dissolved in DMF (2 mL), DIEA (174 μL, 1 mmol) was added, PyBOP (520 mg, 1.0 mmol) dissolved in DCM (2 mL) was added in an ice-water bath, stirred for 0.5 h, then L-alanine methyl ester hydrochloride (167 mg, 1.2 mmol) was added, and the reaction was carried out at room temperature for 6 h. Concentrate under low pressure, add ethyl acetate (30 mL), wash with water (3×10 mL), HCl 1M (3×10 mL) and brine (10 mL) in sequence, concentrate the organic layer, and recrystallize from DCM to obtain product C2 with a yield of 30%, mp 162-164°C, HPLC (93% methanol in water with 0.1% HCOOH): 97.8%, t R =3.206min. 1 H NMR(300MHz,DMSO-d6,δppm):10.98(br s,2H,2×OH),8.78(d,J=6.9Hz,1H,-CONH-),8.09(s,1H,CHAr),7.96(d,J=2.1Hz,1H,Ar-H) ,7.81(d,J=2.1Hz,1H,Ar-H),4.41(m,1H,-CHCH3),3.66(s,3H,-OCH3),1.39(d,J=7.3Hz,3H m,1H,-CHCH 3 ). 13C NMR(75MHz,DMSO-d6,δppm):172.96,161.87,149.71,148.69,137.67,136.80,122.1 7,120.28,118.07,116.64,104.12,52.53,49.11,17.05.HRMS(m / z):[MH]-calcd.for C 14 H 13 N3O7: 334.0681; found, 334.0713.

[0051] Embodiment 7:

[0052] ((2S)-2-{[(2E)-2-Cyano-3-(3,4-dihydroxy-5-nitrophenyl)-1-oxyylideneprop-2-enyl]amino}propanamide (C3)

[0053]

[0054] 2a (250 mg, 1.0 mmol) was dissolved in DMF (2 mL), DIEA (174 μL, 1 mmol) was added, PyBOP (520 mg, 1.0 mmol) dissolved in DCM (2 mL) was added in an ice-water bath, and stirred for 0.5 h; L-alaninamide hydrochloride (249 mg, 2 mmol) was dissolved in DMF (2 mL), DIEA (348 μL, 2 mmol) was added, and stirred at room temperature for 0.5 h, and then added to the DMF solution of 12b, and reacted at room temperature for 6 h. Concentrate under low pressure, add ethyl acetate (30 mL), wash with water (3×10 mL), HCl 1M (3×10 mL) and brine (10 mL) in sequence, concentrate the organic layer, and recrystallize from methanol to obtain product C3 with a yield of 15%, mp 236-238 ° C, HPLC (93% methanol in water with 0.1% HCOOH): 99.4%, t R =2.987min. 1 H NMR(300MHz,DMSO-d6,δppm):10.96(br s,2H,2×OH),8.30(d,J=7.2Hz,1H,-CONH-),8.11(s,1H,CHAr),7.95(d,J=2.1Hz,1H,A r-H),7.81(d,J=2.2Hz,1H,Ar-H),7.50(s,1H,-NH2),7.15(s,1H,-NH2),4.31(m,1H,- CH CH3), 1.32(d, J=7.1Hz, 3H, -CH CH3 ). 13C NMR(75MHz,DMSO-d6,δppm):174.11,161.26,149.17,148.59,146.02,137.68,12 2.51,119.99,118.23,116.86,104.80,49.56,18.56.HRMS(m / z):[MH]-calcd.for C 13 H 12 N4O6: 319.0684; found, 319.0713.

[0055] Embodiment 8:

[0056] (2E)-3-(3,4-Dihydroxy-5-nitrophenyl)-N-(prop-2-ynyl)prop-2-enamide (C4)

[0057]

[0058] 2b (225 mg, 1.0 mmol) was dissolved in DMF (2 mL), DIEA (174 μL, 1 mmol) was added, PyBOP (520 mg, 1.0 mmol) dissolved in DCM (2 mL) was added in an ice-water bath, stirred for 0.5 h, and then propargylamine (77 μL, 1.2 mmol) was added, and the reaction was carried out at room temperature for 6 h. After low pressure concentration, ethyl acetate (30 mL) was added, and the mixture was washed with water (3×10 mL), HCl 1M (3×10 mL) and brine (10 mL) in sequence, and the organic layer was concentrated and recrystallized from DCM to obtain product C4 with a yield of 24%, mp 202-204°C, HPLC (93% methanol in water with 0.1% HCOOH): 85.7%, t R =2.950min. 1 H NMR (300MHz, DMSO-d6, δppm): 10.53 (br s, 2H, 2×OH), 8.53 (t, J = 5.5Hz, 1H, -CONH-), 7.56 (d, J = 1.9Hz, 1H, Ar-H), 7.36 (d, J = 15.8Hz, 1H, - CH CHCO-),7.26(d,J=1.9Hz,1H,Ar-H),6.48(d,J=15.8Hz,-CH CH CO-),3.99(dd,J=5.4,2.4Hz,2H,-NH CH2 -),3.17(t,J=2.4Hz,1H,-CCH). 13C NMR (75MHz, DMSO-d6, δppm): 164.98, 148.47, 143.73, 138.31, 137.79, 125.72, 121.10, 117.07, 115.58, 81.49, 73.70, 28.47. HRMS (m / z): [MH]-calcd.for C 12 H 10 N2O5: 261.0517; found, 261.0541.

[0059] Embodiment 9:

[0060] N-[(2E)-3-(3,4-dihydroxy-5-nitrophenyl)-1-oxyprop-2-enyl]-L-alanine methyl ester (C5)

[0061]

[0062] 2b (225 mg, 1.0 mmol) was dissolved in DMF (2 mL), DIEA (174 μL, 1 mmol) was added, PyBOP (520 mg, 1.0 mmol) dissolved in DCM (2 mL) was added in an ice-water bath, stirred for 0.5 h, then L-alanine methyl ester hydrochloride (167 mg, 1.2 mmol) was added, and the reaction was carried out at room temperature for 6 h. After low pressure concentration, ethyl acetate (30 mL) was added, and the mixture was washed with water (3×10 mL), HCl 1M (3×10 mL) and brine (10 mL) in sequence, the organic layer was concentrated, and the product C5 was recrystallized from DCM with a yield of 31%, mp 184-186°C, HPLC (93% methanol in water with 0.1% HCOOH): 99.6%, t R =3.008min. 1 H NMR (300MHz, DMSO-d6, δppm): 10.57 (br s, 2H, 2×OH), 8.53 (d, J = 7.0Hz, 1H, -CONH-), 7.57 (d, J = 1.9Hz, 1H, Ar-H), 7.34 (d, J = 15.8Hz, 1H, - CH CHCO-),7.26(d,J=2.0Hz,1H,Ar-H),6.54(d,J=15.8Hz,1H,-CH CH CO-),4.39(m,1H,- CH CH3),3.64(s,3H,-O CH3 ),1.33(d,J=7.3Hz,3H,-CH CH3 ). 13C NMR(75MHz,DMSO-d6,δppm):173.63,165.09,148.50,143.79,138.35,137.76,12 5.71,121.06,117.06,115.57,52.39,48.21,17.54.HRMS(m / z):[MH]-calcd.for C 13 H 14 N2O7: 309.0728; found, 309.0758.

[0063] Embodiment 10:

[0064] (2S)-2-{[(2E)-3-(3,4-dihydroxy-5-nitrophenyl)-1-oxyprop-2-enyl]amino}propanamide (C6)

[0065]

[0066] 2b (225 mg, 1.0 mmol) was dissolved in DMF (2 mL), DIEA (174 μL, 1 mmol) was added, PyBOP (520 mg, 1.0 mmol) dissolved in DCM (2 mL) was added in an ice-water bath, and stirred for 0.5 h; L-alaninamide hydrochloride (249 mg, 2 mmol) was dissolved in DMF (2 mL), DIEA (348 μL, 2 mmol) was added, and stirred at room temperature for 0.5 h, and then added to the DMF solution of 12a, and reacted at room temperature for 6 h. Concentrate under low pressure, add ethyl acetate (30 mL), wash with water (3×10 mL), HCl 1M (3×10 mL) and brine (10 mL) in sequence, concentrate the organic layer, and recrystallize from methanol to obtain product C6 with a yield of 16%, mp 220-222°C, HPLC (93% methanol in water with 0.1% HCOOH): 99.7%, t R =2.800min. 1 H NMR (300MHz, DMSO-d6, δppm): 10.53 (br s, 2H, 2×OH), 8.22 (d, J = 7.7Hz, 1H, -CONH-), 7.55 (d, J = 1.9Hz, 1H, Ar-H), 7.44 (s, 1H, -NH2), 7.30 (d, J = 15.8Hz, 1H, - CH CHCO-),7.26(d,J=1.9Hz,1H,Ar-H),7.03(s,1H,-NH2),6.63(d,J=15.7Hz,1H,-CH CH CO-),4.35(m,1H,- CHCH3), 1.25(d, J=7.1Hz, 3H, -CH CH3 ). 13 C NMR (75MHz, DMSO-d6, δppm): 174.70, 164.77, 148.39, 143.35, 137.68 (d, J = 18.5Hz), 126.18, 122.11, 117.32, 115.33, 48.50, 19.05. HRMS (m / z): [M+H] + calcd.for C 12 H 13 N3O6: 296.0877; found, 296.0881.

[0067] Embodiment 11:

[0068] 3,4-Dihydroxy-5-nitro-N-(prop-2-ynyl)benzamide (C7)

[0069]

[0070] 3 (199 mg, 1.0 mmol) was dissolved in DMF (2 mL), DIEA (174 μL, 1 mmol) was added, PyBOP (520 mg, 1.0 mmol) dissolved in DCM (2 mL) was added in an ice-water bath, stirred for 0.5 h, and then propargylamine (77 μL, 1.2 mmol) was added, and the reaction was carried out at room temperature for 6 h. The mixture was concentrated under low pressure, ethyl acetate (30 mL) was added, and the mixture was washed with water (3×10 mL), HCl 1M (3×10 mL) and brine (10 mL) in sequence, the organic layer was concentrated, and the product C7 was recrystallized from DCM with a yield of 24%, mp 158-160°C, HPLC (93% methanol in water with 0.1% HCOOH): 99.8%, t R =2.892min. 1 H NMR(300MHz,DMSO-d6,δppm):10.68(br s,2H,2×OH),9.00(t,J=5.4Hz,1H,-CONH-),7.95(d,J=2.1Hz,1H,Ar-H),7.59(d,J=2.1Hz,1H,Ar-H),4.02(dd,J=5.4,2.5Hz,2H,-NH CH2 -),3.14(t,J=2.5Hz,1H,-CCH). 13C NMR (75MHz, DMSO-d6, δppm): 164.52, 147.95, 144.84, 137.10, 124.32, 118.68, 114.60, 81.63, 73.38, 29.04. HRMS (m / z): [MH] - calcd.for C 10 H8N2O5: 235.0360; found, 235.0382.

[0071] Embodiment 12:

[0072] N-[(3,4-Dihydroxy-5-nitrophenyl)carbonyl]-L-alanine methyl ester (C8)

[0073]

[0074] 3 (199 mg, 1.0 mmol) was dissolved in DMF (2 mL), DIEA (174 μL, 1 mmol) was added, PyBOP (520 mg, 1.0 mmol) dissolved in DCM (2 mL) was added in an ice-water bath, stirred for 0.5 h, then L-alanine methyl ester hydrochloride (167 mg, 1.2 mmol) was added, and the reaction was carried out at room temperature for 6 h. Concentrate under low pressure, add ethyl acetate (30 mL), wash with water (3×10 mL), HCl 1M (3×10 mL) and brine (10 mL) in sequence, concentrate the organic layer, and recrystallize from DCM to obtain product C8 with a yield of 28%, mp 120-122°C, HPLC (93% methanol in water with 0.1% HCOOH): 99.8%, t R =2.942min. 1 H NMR (300MHz, DMSO-d6, δppm): 10.67 (br s, 2H, 2×OH), 8.87 (d, J=6.8Hz, 1H, -CONH-), 8.01 (d, J=1.7Hz, 1H, Ar-H), 7.59 (d, J=1.7Hz, 1H, Ar-H), 4.45 (m, 1H, - CH CH3),3.64(s,3H,-OCH3),1.39(d,J=7.3Hz,3H,-CH CH3 ). 13 C NMR (75MHz, DMSO-d6, δppm): 173.60, 164.79, 147.97, 145.01, 137.10, 123.99, 118.69, 114.79, 52.38, 48.82, 17.13. HRMS (m / z): [MH] - calcd.for C11 H 12 N2O7: 283.0572; found, 283.0598.

[0075] Embodiment 13:

[0076] (2S)-2-{[(3,4-dihydroxy-5-nitrophenyl)carbonyl]amino}propanamide (C9)

[0077]

[0078] 3 (199 mg, 1.0 mmol) was dissolved in DMF (2 mL), DIEA (174 μL, 1 mmol) was added, PyBOP (520 mg, 1.0 mmol) dissolved in DCM (2 mL) was added in an ice-water bath, and stirred for 0.5 h; L-alaninamide hydrochloride (249 mg, 2 mmol) was dissolved in DMF (2 mL), DIEA (348 μL, 2 mmol) was added, and stirred at room temperature for 0.5 h, and then added to the DMF solution of 19, and reacted at room temperature for 6 h. Concentrate under low pressure, add ethyl acetate (30 mL), wash with water (3×10 mL), HCl 1M (3×10 mL) and brine (10 mL) in sequence, concentrate the organic layer, and recrystallize from methanol to obtain product C9 with a yield of 16%, mp218-220°C, HPLC (93% methanol in water with 0.1% HCOOH): 99.6%, t R =2.750min. 1 H NMR(300MHz,DMSO-d6,δppm):10.59(br s,2H,2×OH),8.53(d,J=7.4Hz,1H,-CONH-),8.02(d,J=2.1Hz,1H,Ar-H),7.5 9(d,J=2.1Hz,1H,Ar-H),7.39(s,1H,-NH2),6.99(s,1H,-NH2),4.36(m,1H,- CH CH3), 1.31(d, J=7.2Hz, 3H, -CH CH3 ). 13 C NMR (75MHz, DMSO-d6, δppm): 174.90, 164.56, 147.79, 144.74, 137.09, 124.58, 118.78, 114.88, 49.36, 18.34. HRMS (m / z): [MH] - calcd.forC 10 H 11 N3O6: 268.0575; found, 268.0600.

[0079] Embodiment 14:

[0080] (2E)-2-Cyano-3-(3,4-dihydroxy-5-nitrophenyl)prop-2-enoic acid methyl ester (C10)

[0081]

[0082] 2a (250 mg, 1.0 mmol) was dissolved in DMF (2 mL), DIEA (174 μL, 1 mmol) was added, PyBOP (520 mg, 1.0 mmol) dissolved in methanol (2 mL) was added in an ice-water bath, and the mixture was reacted at room temperature for 6 h. The mixture was concentrated under low pressure, and water was added to precipitate a yellow solid, which was filtered and the filter cake was recrystallized from DCM to obtain product C10 with a yield of 70%, mp 220-222°C, HPLC (93% methanol inwater with 0.1% HCOOH): 94.2%, t R =3.217min. 1 H NMR (300MHz, DMSO-d6, δppm): 10.92 (br s, 2H, 2×OH), 8.31 (s, 1H, CHAr), 8.12 (d, J = 2.1Hz, 1H, Ar-H), 7.90 (d, J = 2.2Hz, 1H, Ar-H), 3.85 (s, 3H, -CH3). 13 C NMR (75MHz, DMSO-d6, δppm): 163.01, 153.89, 148.61, 147.09, 137.79, 121.90, 121.64, 118.34, 116.18, 100.36, 53.72. HRMS (m / z): [MH] - calcd.forC 11 H8N2O6: 263.0310; found, 263.0334.

[0083] Embodiment 15:

[0084] (2E)-2-Cyano-3-(3,4-dihydroxy-5-nitrophenyl)-N-[(4-{[(3-fluorophenyl)methyl]oxy}phenyl)methyl]prop-2-enamide (C11)

[0085]

[0086] 2a (250 mg, 1.0 mmol) was dissolved in DMF (2 mL), DIEA (174 μL, 1 mmol) was added, PyBOP (520 mg, 1.0 mmol) dissolved in DCM (2 mL) was added in an ice-water bath, stirred for 0.5 h, then 4a (278 mg, 1.2 mmol) was added, and the mixture was reacted at room temperature for 6 h. The mixture was concentrated under low pressure, ethyl acetate (30 mL) was added, and the mixture was washed with water (3×10 mL), HCl 1M (3×10 mL) and brine (10 mL) in sequence, the organic layer was concentrated, and the product C11 was recrystallized from DCM with a yield of 26%, mp 206-208°C, HPLC (93% methanol in water with 0.1% HCOOH): 98.8%, t R =3.980min. 1 H NMR (300MHz, DMSO-d6, δppm): 8.63 (t, J=5.6Hz, 1H, -CONH-), 7.93 (s, 1H, CHAr), 7.88(d,J=1.6Hz,1H,Ar-H),7.88(d,J=1.6Hz,1H,Ar-H),7.56(d,J=1.6Hz,1H,Ar -H),7.48–7.37(m,1H,Ar-H),7.33–7.21(m,4H,Ar-H),7.15(t,J=8.2Hz,1H,Ar- H),6.98(d,J=8.4Hz,2H,Ar-H),5.12(s,2H,-OCH2-),4.32(d,J=5.4Hz,2H,-CONH CH2 -). 13 C NMR (75MHz, DMSO-d6, δppm): 162.64 (d, J = 243.7Hz), 162.61, 157.50, 151.51 ,150.36,140.62(d,J=7.9Hz),135.50,135.47,132.16,130.93(d,J=8.4Hz), 129.28 ,125.18,123.88(d,J=2.3Hz),118.26,115.43, 115.07 ,114.99(d,J=24.2Hz),114.82,114.57(d,J=21.9Hz),105.06,68.72,42.95.HRMS(m / z):[MH] - calcd.for C 24 H 18 FN3O6: 426.1107; found, 462.1149.

[0087] Embodiment 16:

[0088] (2E)-2-Cyano-3-(3,4-dihydroxy-5-nitrophenyl)-N-[(4-{[(4-fluorophenyl)methyl]oxy}phenyl)methyl]prop-2-enamide (C12)

[0089]

[0090] 2a (250 mg, 1.0 mmol) was dissolved in DMF (2 mL), DIEA (174 μL, 1 mmol) was added, PyBOP (520 mg, 1.0 mmol) dissolved in DCM (2 mL) was added in an ice-water bath, stirred for 0.5 h, then 4b (278 mg, 1.2 mmol) was added, and the mixture was reacted at room temperature for 6 h. The mixture was concentrated under low pressure, ethyl acetate (30 mL) was added, and the mixture was washed with water (3×10 mL), HCl 1M (3×10 mL) and brine (10 mL) in sequence, the organic layer was concentrated, and the product C12 was recrystallized from DCM with a yield of 23%, mp 208-210°C, HPLC (93% methanol in water with 0.1% HCOOH): 95.4%, t R =3.990min. 1 H NMR(300MHz,DMSO-d6,δppm):8.86(t,J=5.5Hz,1H,-CONH-),8.09(s,1H,CHAr),8.01(d,J=1.5Hz,1H,Ar-H),7.78(d,J=1.5Hz,1H,Ar-H), 7.57(dd,J=8.0,5.9Hz,2H,Ar-H),7.37–7.25(m,4H,Ar-H),7.05(d,J=8.4Hz,2H,Ar-H),5.18(s,2H,-OCH2-),4.41(d,J=5.4Hz,2H,-CONH CH2 -). 13 CNMR (75MHz, DMSO-d6, δppm): 162.17 (d, J = 243.8Hz), 161.97, 157.68, 150.90, 149.83, 136.72, 133.81 (d, J = 3.0Hz), 131.79, 130.30 (d,J=8.3Hz), 129.31 ,122.14,119.54,117.46,115.83, 115.69 (d,J=21.1Hz),115.20, 115.08 ,101.92,68.87,43.05.HRMS(m / z):[M+H] +calcd.for C 24 H 18 FN3O6: 464.1252; found, 464.1252.

[0091] Embodiment 17:

[0092] (2E)-3-(3,4-dihydroxy-5-nitrophenyl)-N-[(4-{[(3-fluorophenyl)methyl]oxy}phenyl)methyl]prop-2-enamide (C13)

[0093]

[0094] 2b (225 mg, 1.0 mmol) was dissolved in DMF (2 mL), DIEA (174 μL, 1 mmol) was added, PyBOP (520 mg, 1.0 mmol) dissolved in DCM (2 mL) was added in an ice-water bath, stirred for 0.5 h, then 4a (278 mg, 1.2 mmol) was added, and the mixture was reacted at room temperature for 6 h. The mixture was concentrated under low pressure, ethyl acetate (30 mL) was added, and the mixture was washed with water (3×10 mL), HCl 1M (3×10 mL) and brine (10 mL) in sequence, the organic layer was concentrated, and the product C13 was recrystallized from DCM with a yield of 26%, mp 202-204°C, HPLC (93% methanol in water with 0.1% HCOOH): 98.4%, t R =3.728min. 1 H NMR(300MHz,DMSO-d6,δppm):10.54(br s,2H,2×OH),8.53(t,J=5.8Hz,1H,-CONH-),7.55(d,J=1.9Hz,1H,Ar-H),7.47-7.40(m,1H,Ar-H),7.35(d,J=15.7Hz,1H,- CH CHCO-),7.27(d,J=8.7Hz,2H,Ar-H),7.26(d,J=2.0Hz,1H,Ar-H),7.22(d,J=8.7Hz,2H,Ar-H ),7.15(td,J=8.2,2.1Hz,1H,Ar-H),6.98(d,J=8.7Hz,2H,Ar-H),6.53(d,J=15.7Hz,1H,-CH CH CO-),5.13(s,2H,-OCH2-),4.32(d,J=5.7Hz,2H,-CONH CH2 -). 13C NMR (75MHz, DMSO-d6, δppm): 165.17, 162.64 (d, J = 243.7Hz), 157.51, 148.45 ,143.51,140.58(d,J=7.4Hz),137.78,137.76,132.16,130.88(d,J=8.3Hz), 129.25 ,126.03,123.82(d,J=2.7Hz),121.89,117.28,115.23(d,J=21.9Hz), 115.14 ,114.80,114.54(d,J=21.9Hz),68.71,42.26.HRMS(m / z):[MH] - calcd.for C 23 H 19 FN2O6: 437.1154; found, 437.1196.

[0095] Embodiment 18:

[0096] 4-({[(2E)-3-(3,4-dihydroxy-5-nitrophenyl)-1-oxyprop-2-enyl]amino}methyl)-N-(prop-2-ynyl)benzamide (C14)

[0097]

[0098] 2b (225 mg, 1.0 mmol) was dissolved in DMF (2 mL), DIEA (174 μL, 1 mmol) was added, PyBOP (520 mg, 1.0 mmol) dissolved in DCM (2 mL) was added in an ice-water bath, stirred for 0.5 h, then 4b (278 mg, 1.2 mmol) was added, and the mixture was reacted at room temperature for 6 h. The mixture was concentrated under low pressure, ethyl acetate (30 mL) was added, and the mixture was washed with water (3×10 mL), HCl 1M (3×10 mL) and brine (10 mL) in sequence, the organic layer was concentrated, and the product C14 was recrystallized from DCM with a yield of 26%, mp 204-206°C, HPLC (93% methanol in water with 0.1% HCOOH): 91.6%, t R =3.670min. 1 H NMR (300MHz, DMSO-d6, δppm): 10.59(br s,2H,2×OH),8.52(s,1H,-CONH-),7.56(s,1H,Ar-H),7.50(d,J=5.9Hz,2H,Ar-H),7.35(d,J=15.8Hz,1H,- CHCHCO-),7.25(s,1H,Ar-H),7.25–7.16(m,4H,Ar-H),6.97(d,J=7.5Hz,2H,Ar-H),6.54(d,J=15.3Hz,1H,-CH CH CO-),5.08(s,2H,-OCH2-),4.32(d,J=4.0Hz,2H,-CONH CH2 -). 13 C NMR (75MHz, DMSO-d6, δppm): 165.11 (s), 162.63 (d, J = 237.00Hz), 157.65, 148.40, 143.37, 137.84, 137.75, 133.83 (d, J = 2.9Hz), 132.05, 130.30 (d,J=8.3Hz), 129.23 ,126.07,121.92,117.32, 115.69 (d,J=21.1Hz),115.30, 115.16 ,68.88,42.23.HRMS(m / z):[MH] - calcd.for C 23 H 19 FN2O6: 437.1154; found, 437.1196.

[0099] Embodiment 19:

[0100] N-[(4-{[(3-fluorophenyl)methyl]oxy}phenyl)methyl]-3,4-dihydroxy-5-nitrobenzamide (C15)

[0101]

[0102] 3 (199 mg, 1.0 mmol) was dissolved in DMF (2 mL), DIEA (174 μL, 1 mmol) was added, PyBOP (520 mg, 1.0 mmol) dissolved in DCM (2 mL) was added in an ice-water bath, stirred for 0.5 h, then 4a (278 mg, 1.2 mmol) was added, and the mixture was reacted at room temperature for 6 h. The mixture was concentrated under low pressure, ethyl acetate (30 mL) was added, and the mixture was washed with water (3×10 mL), HCl 1M (3×10 mL) and brine (10 mL) in sequence, the organic layer was concentrated, and the product C15 was recrystallized from DCM with a yield of 23%, mp 180-182°C, HPLC (93% methanol in water with 0.1% HCOOH): 97.4%, t R =3.574min. 1H NMR(300MHz,DMSO-d6,δppm):10.61(br s,2H,2×OH),9.02(t,J=5.8Hz,1H,-CONH-),7.97(d,J=2.1Hz,1H,Ar-H),7.59(d,J=2.0Hz,1H,Ar-H),7.43(td,J=8.0,6.1Hz,1H,Ar-H), 7.29–7.22(m,4H,Ar-H),7.15(td,J=8.4,1.9Hz,1H,Ar-H),6.97(d,J=8.6Hz,2H,Ar-H),5.12(s,2H,-OCH2-),4.37(d,J=5.7Hz,2H,-CONH CH2 -). 13 C NMR (75MHz, DMSO-d6, δppm): 164.68, 164.64 (d, J = 243.7Hz), 157.47, 148.06, 145.12, 140.59 (d, J = 7.4Hz), 136.98, 132.41, 130.88 (d, J = 8.3Hz), 129.17 ,124.65,123.82(d,J=2.7Hz),118.56, 115.08 ,114.68(d,J=18.1Hz),114.54(d,J=21.9Hz),68.73,42.64.HRMS(m / z):[MH] - calcd.for C 21 H 17 FN2O6: 411.0998; found, 411.1037.

[0103] Embodiment 20:

[0104] N-[(4-{[(4-fluorophenyl)methyl]oxy}phenyl)methyl]-3,4-dihydroxy-5-nitrobenzamide (C16)

[0105]

[0106] 3 (199 mg, 1.0 mmol) was dissolved in DMF (2 mL), DIEA (174 μL, 1 mmol) was added, PyBOP (520 mg, 1.0 mmol) dissolved in DCM (2 mL) was added in an ice-water bath, stirred for 0.5 h, then 4b (278 mg, 1.2 mmol) was added, and the mixture was reacted at room temperature for 6 h. The mixture was concentrated under low pressure, ethyl acetate (30 mL) was added, and the mixture was washed with water (3×10 mL), HCl 1M (3×10 mL) and brine (10 mL) in sequence, the organic layer was concentrated, and the product C16 was recrystallized from DCM with a yield of 24%, mp 176-178°C, HPLC (93% methanol in water with 0.1% HCOOH): 96.0%, t R =3.524min. 1 H NMR(300MHz,DMSO-d6,δppm):10.60(br s,2H,2×OH),9.02(t,J=5.7Hz,1H,-CONH-),7.97(d,J=1.8Hz,1H,Ar-H),7.60(d,J=1.8Hz,1H,Ar-H),7.49(dd,J=8.3,5.7Hz,2 H,Ar-H),7.24-7.18(dd,J=9.6,8.5Hz,4H,Ar-H),6.96(d,J=8.5Hz,2H,Ar-H),5.07(s,2H,-OCH2-),4.37(d,J=5.6Hz,2H,-CONH CH2 -). 13 C NMR (75MHz, DMSO-d6, δppm): 164.62, 162.16 (d, J = 243.0Hz), 157.59, 147.99, 144.94, 137.04, 133.83 (d, J = 2.9Hz), 132.30, 130.30 (d,J=8.3Hz), 129.14 ,124.70,118.56, 115.69 (d,J=21.1Hz), 115.08 ,114.49,68.87,42.62.HRMS(m / z):[MH] - calcd.for C 21 H 17 FN2O6: 411.0998; found, 411.1026.

[0107] Embodiment 21:

[0108] 4-({[(2E)-3-(3,4-dihydroxy-5-nitrophenyl)-1-oxyylideneprop-2-enyl]amino}methyl)-N-(prop-2-ynyl)benzamide (C17)

[0109]

[0110] 2b (225 mg, 1.0 mmol) was dissolved in DMF (2 mL), DIEA (174 μL, 1 mmol) was added, PyBOP (520 mg, 1.0 mmol) dissolved in DCM (2 mL) was added in an ice-water bath, stirred for 0.5 h, and then 5a (226 mg, 1.2 mmol) was added, and the reaction was carried out at room temperature for 6 h. The mixture was concentrated under low pressure, ethyl acetate (30 mL) was added, and the mixture was washed with water (3×10 mL), HCl 1M (3×10 mL) and brine (10 mL) in sequence, the organic layer was concentrated, and the product C17 was recrystallized from DCM with a yield of 24%, mp 242-244°C, HPLC (93% methanol in water with 0.1% HCOOH): 96.1%, t R =2.840min. 1 H NMR (300MHz, DMSO-d6, δppm): 10.56 (br s, 2H, 2×OH), 8.91 (t, J=5.5Hz, 1H, -CHCHCO NH -),8.67(t,J=6.0Hz,1H,Ar-CONH),7.83(d,J=8.3Hz,2H,Ar-H),7.58(d,J=1.9Hz,1H,Ar-H),7.38(d,J=15.7Hz,1H,- CH CHCONH-),7.38(d,J=8.3Hz,2H,Ar-H),7.27(d,J=2.0Hz,1H,Ar-H),6.56(d,J=15.7Hz,1H,-CH CH CONH-),4.46(d,J=5.8Hz,2H,CH2-Ar),4.05(dd,J=5.5,2.4Hz,2H,- CH2 CCH),3.13(t,J=2.5Hz,1H,-CH2C CH ). 13 C NMR (75MHz, DMSO-d6, δppm):166.15,165.38,148.43,143.52,143.44,138.04,137.81,132.85, 127.85 , 127.59,125.96,121.66,117.32,115.41,81.84,73.31,42.46,28.92.HRMS(m / z):[MH] - calcd.for C 20 H 17 N3O6: 394.1045; found, 394.1079.

[0111] Embodiment 22:

[0112] (2S)-2-({[4-({[(2E)-3-(3,4-dihydroxy-5-nitrophenyl)-1-oxyprop-2-enyl]amino}methyl)phenyl]carbonyl}amino)propanamide (C18)

[0113]

[0114] 2b (225 mg, 1.0 mmol) was dissolved in DMF (2 mL), DIEA (174 μL, 1 mmol) was added, PyBOP (520 mg, 1.0 mmol) dissolved in DCM (2 mL) was added in an ice-water bath, stirred for 0.5 h, then 5b (266 mg, 1.2 mmol) was added, and the mixture was reacted at room temperature for 6 h. The mixture was concentrated under low pressure, ethyl acetate (30 mL) was added, and the mixture was washed with water (3×10 mL), HCl 1M (3×10 mL) and brine (10 mL) in sequence, the organic layer was concentrated, and the product C18 was recrystallized from DCM with a yield of 23%, mp 178-180°C, HPLC (93% methanol in water with 0.1% HCOOH): 99.3%, t R =2.075min. 1 H NMR (300MHz, DMSO-d6, δppm): 10.54 (br s, 2H, 2×OH), 8.67 (t, J=6.0Hz, 1H, -CHCHCO NH -),8.38(d,J=7.5Hz,1H,Ar-CONH),7.87(d,J=8.3Hz,2H,Ar-H),7.58(d,J=1.9Hz,1H,Ar-H),7.38(d,J=15.7Hz,1H,- CH CHCONH-),7.37(d,J=8.5Hz,2H,Ar-H),7.38(s,1H,NH2),7.27(d,J=2.0Hz,1H,Ar-H),7.01(s,1H,NH2),6.57(d,J=15.7Hz,1H,-CH CHCONH-),4.46(d,J=5.8Hz,2H,CH2-Ar),4.40(m,1H,- CH CH3), 1.33(d, J=7.2Hz, 3H, -CH CH3 ). 13 C NMR (75MHz, DMSO-d6, δppm):174.93,166.17,165.36,148.42,143.47,143.19,138.01,137.83,133.20, 128.06 , 127.39 ,125.98,121.69,117.33,115.39,49.17,42.48,18.53.HRMS(m / z):[MH] - calcd.forC 20 H 20 N4O7: 427.1259; found, 427.1296.

[0115] Example 23: In vitro (COMT) inhibitory activity test

[0116] Drugs and reagents: compounds prepared in the examples, rat liver cytoplasmic COMT enzyme (homemade, SD rats, SPF level, purchased from Jiangsu Huachuang Xinnuo Pharmaceutical Technology Co., Ltd.), substrate aesculetin (Jiangsu Aikon Biopharmaceutical Research and Development Co., Ltd.), SAM (Shanghai Bid Pharmaceutical Technology Co., Ltd.), dithiothreitol (Shanghai Xianding Biotechnology Co., Ltd.).

[0117] Instruments: Multifunctional microplate reader (Synergy 2, BioTek Instruments, Inc), high-speed refrigerated microcentrifuge (D3024R, Scilogex), homogenizer (F6 / 10, Shanghai Jingxin Industrial Development Co., Ltd.).

[0118] Preparation of rat liver cytoplasmic COMT enzyme: Rats were anesthetized, blood was collected from the abdominal aorta, and the liver was washed with ice-cold saline, dried with filter paper, and frozen at -20℃ for 1h. The frozen liver tissue was cut into small pieces and homogenized in 2 volumes of PBS (50mM, pH 7.5, containing 0.5mM dithiothreitol) for 2 minutes. The homogenate was centrifuged at 4℃, 10000×g for 15min, and the floating fat on the upper layer was removed. The supernatant was centrifuged at 4℃, 21000×g for 15min. The supernatant was rat liver cytoplasmic COMT enzyme, which was packaged into 2ml / tube (each tube for 100 wells analysis) and frozen at -80℃.

[0119] COMT enzyme activity test steps: (1) Add 170 μL of PBS (25 mM, pH 7.8, containing 2 mM MgCl2, 20 mM L-cysteine, 1 mM SAM, 20 μM fraxin) and 10 μL of inhibitor to a black 96-well plate and pre-incubate for 15 min at 37°C; (2) Add 20 μL of rat liver cytoplasmic COMT enzyme to start the reaction, making the final reaction volume 200 μL; (3) Continuously record the fluorescence intensity (λ ex =360nm,λ em =485nm) for 500s; (4) construct the fluorescence intensity-time curve, and take the slope of the linear reaction period curve, that is, the change of fluorescence intensity per unit time ΔInt / min as the reporting unit; (5) the inhibitory ability is expressed by the residual activity (Ra%), the formula is: Ra% = (ΔInt / min of the well with inhibitor) / (ΔInt / min of the well without inhibitor). The results were obtained by GraphPadPrism TM The software calculated the corresponding IC using a non-liner regression analysis model. 50 The values ​​are shown in Table 1.

[0120] Example 24: In vitro (MAO) inhibitory activity test

[0121] Drugs and reagents: compounds prepared in the examples, rat brain MAO enzyme (self-made, SD rats, SPF level, purchased from Jiangsu Huachuang Xinnuo Pharmaceutical Technology Co., Ltd.), MAO-B substrate benzylamine hydrochloride (Shanghai Bid Pharmaceutical Technology Co., Ltd.), MAO-A substrate benzyl 5-hydroxytryptamine (Shanghai Jizhi Biochemical Co., Ltd.), Amplex Red (Shanghai Xianding Biotechnology Co., Ltd.), HRP (Biyuntian Biotechnology),

[0122] Instruments: Multifunctional microplate reader (Synergy 2, BioTek Instruments, Inc), high-speed refrigerated microcentrifuge (D3024R, Scilogex), homogenizer (F6 / 10, Shanghai Jingxin Industrial Development Co., Ltd.)

[0123] Preparation of rat brain mitochondrial MAO enzyme: After the rats were killed, the whole brain was removed to remove the olfactory bulb, washed with ice-cold saline, dried with filter paper, frozen at -20°C for 1 hour, and homogenized in 5 volumes of PBS (100mM, pH 7.4, 0.1% TritonX-114) for 2 minutes. The homogenate was centrifuged at 4°C, 3000×g for 20 minutes, and the supernatant was rat brain mitochondrial MAO enzyme, which was frozen at -80°C.

[0124] MAO enzyme activity test steps: (1) Add 90 μL of rat brain mitochondrial MAO enzyme and 10 μL of inhibitor diluted with methanol to a flat-bottom black 96-well microplate, and pre-incubate for 10 minutes at 37°C in the dark; (2) After pre-incubation, add 100 μL of MAO-B colorimetric reagent (AR / HRP / benzylamine) or MAO-A colorimetric reagent (AR / HRP / 5-HT) to a final volume of 200 μL and incubate for 15 minutes; (3) Measure the fluorescence (λ) generated by resorufin ex =530nm,λ em =590nm); (4) blank value (b), 1XBuffer was used instead of MAO enzyme; V i is the velocity in the presence of the test compound, V o is the control rate when methanol replaces the inhibitor; (5) The inhibitory capacity is expressed by the residual activity, the formula is: Ra = (V i -b) / (V o -b); the results were analyzed by GraphPad Prism TM The software calculated the corresponding IC using a non-liner regression analysis model. 50 The values ​​are shown in Table 1 and Table 2.

[0125] Table 1 Inhibitory activity of compounds on rat liver COMT, rat brain MAO-A and rat brain MAO-B

[0126]

[0127]

[0128] Result analysis: From the overall results of the activity test, compounds C1-C18 all have excellent COMT inhibitory activity. When the compound is administered at a concentration of 10 μM, the residual activity of MAO-A is greater than 90%, and the residual activity of MAO-B is between 50% and 90%, indicating that the compounds have a certain ability to selectively inhibit MAO-B. Among them, C11 and C12 have the strongest inhibitory ability on MAO-B. Further IC 50 The values ​​were determined as shown in Table 2.

[0129] Table 2 IC50 values ​​of preferred compounds for rat liver COMT and rat brain MAO-B

[0130]

[0131] Result analysis: Compounds C11 and C12 have similar inhibitory abilities on MAO-B, and compound C12 has stronger COMT inhibitory activity than C11.

[0132] Example 25: Detection of the amount in the blood and brain at different times after oral administration of Example 18 (Compound C11)

[0133] Eight male ICR mice were fasted for 12 h, and each mouse was given a single dose of the compound 20 mg / kg orally by gavage (using V DMSO :V PEG400 :V 生理盐水 =1:49:50 to make a solution with a concentration of 2 mg / mL), and 1 mL of blood samples were collected from the orbital sinus in heparinized tubes at 30 min, 50 min, 80 min, and 120 min. The plasma supernatant was separated by centrifugation, and the supernatant was diluted with methanol to 1:3, vortexed for 5 min, and centrifuged at 4°C, 15000 rpm for 15 min before injection and analysis. At the same time, brain tissue samples were taken, and then the brain was rolled on filter paper, weighed (recorded brain weight), placed in a -80°C refrigerator for 15 min, and directly diluted with 1-fold methanol: saline (3:7, v / v), and each tissue sample was homogenized and centrifuged at 4°C, 15000 rpm for 15 min. Then, the supernatant was taken and an equal volume of methanol was added to precipitate protein, vortexed for 5 min, and centrifuged at 4°C, 15000 rpm for 15 min. The supernatant was filtered through a membrane and injected for analysis by LC-MS / MS.

[0134] The LC-MS / MS conditions were as follows: the chromatographic column was a C18 column (150×4.6 mm, 5 μM); the mobile phase was: phase A was pure water, and phase B was an organic phase (methanol); isocratic elution was used: the organic phase ratio was 93%; flow rate: 0.5 mL / min, injection volume: 10.0 μL, and column temperature: 35°C.

[0135] The results are shown in the table. The highest C11 content was detected in the blood 30 minutes after administration, and then rapidly dropped to the lowest at 50 minutes, and then increased again at 80 minutes, which may be due to enterohepatic circulation; the C11 content detected in the brain tissue was 20-300 ng·g -1 Within the range of 30 to 180 minutes, the content of C11 in the brain first increased and then decreased with time, and the highest content was 298.47±176.41ng·g at 120 minutes. -1 At this time, the concentration of C11 in the brain is about twice that in the serum, indicating that C11 has good BBB permeability. At the same time point of 120 minutes, the concentration of compound C11 in brain tissue is higher than that of C12, and C11 may have a better effect of entering the brain.

[0136] Table 3 Concentrations of compounds C11 and C12 in mouse serum and brain tissue at different times after oral administration

[0137] Compound Time / min <![CDATA[Serum concentration / ng·ml -1 > <![CDATA[Brain tissue concentration / ng·g -1 > C11 30 1068.70±332.15 26.58±14.59 C11 50 85.68±42.11 48.21±24.54 C11 80 252.80±8.80 232.80±20.00 C11 120 159.32±140.59 298.47±176.41 C11 180 249.80±143.4 102.20±17.40 C12 120 46.78±13.60 97.40±3.68

[0138] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, ordinary technicians in the field should understand that the specific implementation methods of the present invention can still be modified or some technical features can be replaced by equivalents without departing from the spirit of the technical solution of the present invention, which should be included in the scope of the technical solution for protection of the present invention.

Claims

1. A nitrocatechol derivative, comprising a compound represented by formula (I) or (II), or a pharmaceutically acceptable salt thereof: In the formula, R1 is selected from Where Y is Z is R2 is selected from H or CN.

2. The nitrocatechol derivative according to claim 1, characterized in that It is selected from any of the following compounds:

3. The method for preparing the nitrocatechol derivative according to claim 1 or 2, characterized in that: The following steps are involved: (1) Compound 1 and an acid undergo a Knoevenagel condensation reaction to obtain compound 2; Compound 1 undergoes a Pinnick oxidation reaction with NaClO2 to obtain compound 3; (2) Compound 2 or 3 is subjected to a condensation reaction with an amine or an alcohol to obtain a final product 4 or 5.

4. A pharmaceutical composition, characterized in that It comprises the nitrocatechol derivative according to claim 1 or 2 and one or more pharmaceutically acceptable excipients.

5. The pharmaceutical composition according to claim 4, characterized in that The dosage form of the pharmaceutical composition is capsule, powder, pill, tablet, granule, suspension or injection.

6. Use of the nitrocatechol derivative according to claim 1 or 2 in the preparation of a drug for preventing and / or treating Parkinson's disease.

7. Use of the nitrocatechol derivative according to claim 1 or 2 in the preparation of a COMT inhibitor.

8. Use of the nitrocatechol derivative according to claim 1 or 2 in the preparation of MAO inhibitors.

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