A selective monoamine oxidase B inhibitor and its pharmaceutical use

Abstract

The present invention discloses a β-carboline compound or a pharmaceutically acceptable salt thereof having a structure as shown in formula (I), R 1 is halogen; X is O or NH; n is 1 or 2; Ring A is a 3- to 6-membered cycloalkyl, a 4- to 6-membered heterocyclic group, an aryl group, or a heteroaryl group; R 2 The compounds of the present invention or their salts exhibit highly selective MAO-B inhibition, with in vitro MAO-B inhibitory activity and selectivity significantly superior to those of safinamide. They also exhibit reversible MAO-B inhibition and can be used to treat neurodegenerative diseases such as Parkinson's disease and Alzheimer's disease. The present invention also discloses the use of the β-carboline compounds or their pharmaceutically acceptable salts in the preparation of MAO-B inhibitors or in the preparation of drugs for the treatment and / or prevention of diseases modulated by monoamine oxidase B inhibitors. #imgabs0#

Description

Technical Field

[0001] The present invention belongs to the field of medicine and relates to β-carboline compounds or pharmaceutically acceptable salts thereof and their use as selective monoamine oxidase B (MAO-B) inhibitors. Background Art

[0002] Monoamine oxidases (MAOs) are microsomal enzymes widely distributed throughout the human body, primarily expressed on the outer membrane of mitochondria. They catalyze the oxidative deamination of monoamines. This catalytic process is accompanied by the production of byproducts such as hydrogen peroxide, amines, and aldehydes, which are associated with intracellular oxidative stress. Abnormal expression of MAOs in the body can cause dysfunction in neurotransmitter transmission, impair the normal function of the nervous system, and even cause neuronal damage, which is closely linked to the development and progression of neurodegenerative diseases (Expert Opin Ther Pat. 2018, 28, 211).

[0003] MAOs exist in two subtypes, A / B, namely monoamine oxidase A (MAO-A) and MAO-B. Although the two have 80% sequence homology, they exhibit different substrate and inhibitor specificities. In the central nervous system and peripheral tissues, MAO-A mainly catalyzes the degradation of serotonin, tyramine, epinephrine and norepinephrine. It is worth noting that when the human body consumes a large amount of tyramine-rich foods, beverages and amine-containing drugs, inhibition, especially irreversible inhibition of MAO-A, can lead to severe hypertensive effects (commonly known as the "cheese effect") (J Neural Transm. 2018, 125, 1735). In the central nervous system, MAO-B is more involved in catalyzing the oxidative degradation of dopamine. With age, the expression and activity of MAO-B in glial cells gradually increase, about 4 times higher than normal, which will lead to more dopamine degradation and produce high levels of hydrogen peroxide, forming oxidative stress, causing apoptosis of dopaminergic neurons in the glial cells (J Parkinsons Dis. 2022, 12, 477), which is considered to be one of the important events in the pathogenesis of Parkinson's disease. In addition, MAO-B can oxidize and metabolize 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to produce a neurotoxin 1-methyl-4-phenyl-pyridinium ion (MPP + ), further exacerbating the pathological progression of Parkinson's disease models (Neurosci Biobehav Rev. 2022, 140, 104792). Therefore, inhibiting MAO-B activity can increase dopamine levels in the body and produce neuroprotective effects.

[0004] Clinically, inhibiting MAOs activity increases the levels of neurotransmitters such as nodopamine, norepinephrine, and serotonin, increasing the content of effective transmitters in the synaptic cleft, thereby achieving the purpose of treating central nervous system diseases such as Parkinson's disease, Alzheimer's disease, and depression. In particular, selective MAO-B inhibitors can not only inhibit the degradation of dopamine in the central nervous system and reduce the production of neurotoxic byproducts such as hydrogen peroxide and aldehydes, but also reduce the adverse reactions caused by inhibiting MAO-A production (ExpertOpin Drug Discov. 2019, 14, 995). Therefore, selective inhibition of MAO-B is one of the effective strategies for the clinical treatment of neurodegenerative diseases such as Parkinson's disease and Alzheimer's disease. Selegiline, rasagiline, and safinamide are MAO-B inhibitors that have been marketed and are mainly used in the clinical treatment of Parkinson's disease. Selegiline is a first-generation, irreversible MAO-B inhibitor that can be used as a monotherapy for early-stage Parkinson's disease or in combination with levodopa to enhance the effects of levodopa, effectively alleviating tremors, muscle rigidity, and movement disorders in patients with advanced Parkinson's disease. However, selegiline has poor selectivity for MAO-A and is associated with adverse reactions such as nausea, hallucinations, and hypertension. It is also metabolized to amphetamine derivatives, which produce central nervous system stimulant effects, significantly limiting its clinical use (Mol Psychiatry. 2016, 21, 1499). Rasagiline, a second-generation, irreversible MAO-B inhibitor, is used clinically as a monotherapy or as an adjunct to levodopa for the treatment of Parkinson's disease, but it can still cause adverse reactions such as headache, nausea, dizziness, anxiety, hallucinations, and insomnia (CNSDrugs. 2014, 28, 1083). Safinamide is a highly selective, reversible, third-generation MAO-B inhibitor that not only increases extracellular dopamine levels in the striatum but also blocks voltage-dependent sodium channels on neurons, regulating glutamate release. Clinically, safinamide is used in combination with levodopa / carbidopa to treat mid-to-late stage Parkinson's disease, but monotherapy is ineffective and has limitations (CNSDrugs. 2017, 31, 169). Consequently, irreversible MAO-B inhibitors have persistent inhibitory effects on the enzyme, leading to potential immune antigenic effects and the potential for adverse reactions. Highly selective, reversible MAO-B inhibitors, on the other hand, restore enzyme activity upon discontinuation and have fewer toxic side effects, but their clinical applicability as monotherapy is limited. Therefore, the search for novel, highly effective, selective, and reversible MAO-B inhibitors is a hot topic in the field of Parkinson's disease treatment.

[0005] β-carboline alkaloids are one of the main active ingredients of many medicinal plants such as Peganum harmala, Kapi wood, and Polygala tenuifolia, and have the potential to treat central nervous system diseases such as Parkinson's disease, Alzheimer's disease, and depression (Eur J Pharmacol. 2021, 93, 173837). It is worth noting that MAOs is one of the important targets for β-carboline alkaloids to exert the above-mentioned central nervous system activity, but problems such as weak target activity and poor selectivity seriously limit the drug development process of such compounds. In recent years, medicinal chemists have made some progress in the development of selective β-carboline MAOs inhibitors, but only submicromolar MAO-A or MAO-B inhibitors have been obtained (J Med Chem, 2021, 64, 1392). Therefore, the development of highly active and highly selective β-carboline MAO-B inhibitors for the treatment of neurodegenerative diseases such as Parkinson's disease and Alzheimer's disease has important research value. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide β-carboline compounds or pharmaceutically acceptable salts thereof with highly effective, highly selective, and reversible MAO-B inhibitory activity. The β-carboline compounds or pharmaceutically acceptable salts of the present invention significantly enhance their selectivity for MAO-B and exhibit highly selective MAO-B inhibition. The compounds exhibit significantly superior in vitro MAO-B inhibitory activity and selectivity to those of safinamide. Furthermore, the compounds exhibit reversible MAO-B inhibition and can be used to treat neurodegenerative diseases such as Parkinson's disease and Alzheimer's disease.

[0007] In order to solve the above technical problems, the technical solution proposed by the present invention is:

[0008] A β-carboline compound or a pharmaceutically acceptable salt thereof having a structure as shown in formula (I):

[0009]

[0010] Among them, R 1 is halogen; X is O or NH; n is 1 or 2; Ring A is a 3- to 6-membered cycloalkyl, a 4- to 6-membered heterocyclic group, an aryl group, or a heteroaryl group; R 2 is H, halogen, cyano, nitro, C1-C3 alkyl, C2-C3 alkenyl or C2-C3 alkynyl.

[0011] Preferably, R 1is F, Cl or Br; X is O or NH; n is 1 or 2; Ring A is cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, oxetanyl, tetrahydrofuranyl, tetrahydrothienyl, pyrrolidinyl, tetrahydropyranyl, piperidinyl, phenyl, furanyl, thienyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, triazolyl, tetrazolyl, pyridinyl, pyrimidinyl, pyrazinyl or pyridazinyl; when Ring A is phenyl, R 2 It is H, meta- or para-substituted F, Cl, Br or cyano.

[0012] Preferably, R 1 is F; X is NH; n is 1; Ring A is cyclopentyl, cyclohexyl, phenyl, triazolyl, tetrazolyl, pyridyl, pyrimidinyl or pyridazinyl; when Ring A is phenyl, R 2 It is H, meta- or para-substituted F, meta-substituted Cl or Br.

[0013] Most preferably, R 1 is F; X is NH; n is 1; Ring A is cyclohexyl or phenyl; when Ring A is phenyl, R 2 It is H, F substituted at the meta position.

[0014] The halogens described herein are F, Cl, Br, and I. The C1-C3 alkyl groups are selected from methyl, ethyl, n-propyl, and isopropyl. The C2-C3 alkenyl groups are selected from vinyl, propenyl, and allyl. The C2-C3 alkynyl groups are selected from ethynyl, propynyl, and propargyl.

[0015] Specifically, the β-carboline compound is selected from the following compounds:

[0016]

[0017]

[0018] The chemical names corresponding to the above compounds are:

[0019] N-(Cyclopropylmethyl)-7-fluoro-9H-pyrido[3,4-b]indole-1-carboxamide; N-(Cyclobutylmethyl)-7-fluoro-9H-pyrido[3,4-b]indole-1-carboxamide; N-(Cyclopentylmethyl)-7-fluoro-9H-pyrido[3,4-b]indole-1-carboxamide; N-(Cyclohexylmethyl)-7-fluoro-9H-pyrido[3,4-b]indole-1-carboxamide; 7-Fluoro-N-(oxetan-3-ylmethyl)-9H-pyrido[3,4-b]indole-1-carboxamide; 7-Fluoro-N-((tetrahydrofuran-3-yl)methyl)-9H-pyrido[3,4-b]indole-1-carboxamide; 7-Fluoro-N-((tetrahydrothiophen-3-yl)methyl)-9H-pyrido[3,4-b]indole-1-carboxamide )methyl)-9H-pyrido[3,4-b]indole-1-carboxamide; 7-Fluoro-N-(pyrrolidin-3-ylmethyl)-9H-pyrido[3,4-b]indole-1-carboxamide; 7-Fluoro-N-((tetrahydro-2H-pyran-4-yl)methyl)-9H-pyrido[3,4-b]indole-1-carboxamide; 7-Fluoro-N-(piperidin-4-ylmethyl)-9H-pyrido[3,4-b]indole-1-carboxamide; N-benzyl-7-fluoro-9H-pyrido[3,4-b]indole-1-carboxamide; 7-Fluoro-N-(3-fluorobenzyl)-9H-pyrido[3,4-b]indole-1-carboxamide; 7-Fluoro-N-(4-fluorobenzyl)-9H-pyrido[3,4-b ]indole-1-carboxamide; 7-Fluoro-N-(3-chlorobenzyl)-9H-pyrido[3,4-b]indole-1-carboxamide; 7-Fluoro-N-(4-chlorobenzyl)-9H-pyrido[3,4-b]indole-1-carboxamide; 7-Fluoro-N-(3-bromobenzyl)-9H-pyrido[3,4-b]indole-1-carboxamide; 7-Fluoro-N-(4-bromobenzyl)-9H-pyrido[3,4-b]indole-1-carboxamide; 7-Fluoro-N-(furan-3-ylmethyl)-9H-pyrido[3,4-b]indole-1-carboxamide; 7-Fluoro-N-(thiophen-3-ylmethyl)-9H-pyrido[3,4-b]indole-1-carboxamide; N-(1H-pyrrol-3-yl) methyl)-7-fluoro-9H-pyrido[3,4-b]indole-1-carboxamide; 7-fluoro-N-(oxazol-4-ylmethyl)-9H-pyrido[3,4-b]indole-1-carboxamide; 7-fluoro-N-(thiazol-4-ylmethyl)-9H-pyrido[3,4-b]indole-1-carboxamide; N-((1H-imidazol-4-yl)methyl)-7-fluoro-9H-pyrido[3,4-b]indole-1-carboxamide; N-((1H-1,2,3-triazol-4-yl)methyl)-7-fluoro-9H-pyrido[3,4-b]indole-1-carboxamide; N-((1H-tetrazol-5-yl)methyl)-7-fluoro-9H-pyrido[3,4-b]indole-1-carboxamide;7-Fluoro-N-(pyridin-4-ylmethyl)-9H-pyrido[3,4-b]indole-1-carboxamide; 7-Fluoro-N-(pyrimidin-4-ylmethyl)-9H-pyrido[3,4-b]indole-1-carboxamide; 7-Fluoro-N-(pyrazin-2-ylmethyl)-9H-pyrido[3,4-b]indole-1-carboxamide; 7-Fluoro-N-(pyridazin-4-ylmethyl)-9H-pyrido[3,4-b]indole-1-carboxamide.

[0020] The pharmaceutically acceptable salt of the β-carboline compound is hydrochloride, hydrobromide, sulfate, acetate, maleate or methanesulfonate, preferably hydrochloride.

[0021] Another object of the present invention is to provide the use of the β-carboline compound or a pharmaceutically acceptable salt thereof in the preparation of a highly effective, highly selective, and reversible MAO-B inhibitor.

[0022] Another object of the present invention is to provide the use of the β-carboline compounds or pharmaceutically acceptable salts thereof in the preparation of drugs for treating and / or preventing diseases regulated by monoamine oxidase B inhibitors.

[0023] The diseases regulated by monoamine oxidase B inhibitors are neurodegenerative diseases, and the neurodegenerative diseases are Parkinson's disease, Alzheimer's disease, etc.

[0024] Another object of the present invention is to provide the use of the β-carboline compound or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating and / or preventing neurodegenerative diseases such as Parkinson's disease and Alzheimer's disease.

[0025] Another object of the present invention is to provide a pharmaceutical composition comprising the β-carboline compound or a pharmaceutically acceptable salt thereof as an active ingredient and a pharmaceutically acceptable carrier in the form of tablets, capsules, granules, powders, microcapsules, pills, aerosols, suspensions, or oral solutions. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 These are the results of the reversibility test analysis of the inhibition of human recombinant MAO-B enzyme activity in vitro by compounds I-11 and I-12.

[0027] Figure 2 The blood-brain barrier penetration test results of compound I-12.

[0028] Figure 3 This is a typical diagram of the effect of compound I-12 on the total movement distance and movement speed of Parkinson's disease model zebrafish; wherein, the black line represents slow movement, the green line represents medium-speed movement, and the red line represents fast movement.

[0029] Figure 4 The effect of compound I-12 on the total movement distance of Parkinson's disease model zebrafish; compared with the model control group, *p<0.05, ***p<0.001.

[0030] Figure 5 The effect of compound I-12 on the movement speed of Parkinson's disease model zebrafish; compared with the model control group, *p<0.05, ***p<0.001.

[0031] Figure 6 This is the effect of compound I-12 on the body weight of Parkinson's disease model mice.

[0032] Figure 7 The effect of compound I-12 on spontaneous movement in Parkinson's disease model mice.

[0033] Figure 8 The effect of compound I-12 on the motor ability of Parkinson's disease model mice; compared with the model control group, *p<0.05, **p<0.01, ***p<0.001. DETAILED DESCRIPTION

[0034] In order to further illustrate the present invention, a series of examples are given below. These examples can enable those skilled in the art to fully understand the present invention, but should not be considered as limiting the scope of the present invention.

[0035] Example 1

[0036] Preparation of N-(cyclopropylmethyl)-7-fluoro-9H-pyrido[3,4-b]indole-1-carboxamide (Compound I-1)

[0037]

[0038] In a 100 mL round-bottom flask, 6-fluoroindoleethylamine (compound 1, 1.78 g, 10 mmol) was dissolved in DCM (20 mL), and ethyl glyoxylate (compound 2, 1.53 g, 15 mmol) was added. After stirring at room temperature for 10 min, TFA (3.07 g, 27 mmol) was added; the mixture was stirred at room temperature overnight, at which point the starting material disappeared as detected by TLC; the pH of the reaction solution was adjusted to 8 with 1 M saturated sodium carbonate solution, and the mixture was extracted with ethyl acetate. The organic layers were combined, washed with saturated brine, and dried over anhydrous sodium sulfate; the solvent was evaporated under reduced pressure to give a light yellow solid (compound 3, 2.1 g, 80% yield).

[0039] In a 100 mL round-bottom flask, compound 3 (1.5 g, 5.3 mmol) was added and dissolved in DMF (10 mL). 10% Pd / C (300 mg) was added with stirring at room temperature and the reaction was heated to 140°C. After completion of the reaction as monitored by TLC, the mixture was filtered and the filtrate was collected and concentrated by vacuum distillation to give a yellow crude product. The product was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate = 5 / 1 to 2 / 1, V / V) to give a light yellow solid (compound 4, 0.89 g, 65% yield).

[0040] In a 50 mL round-bottom flask, compound 4 (0.5 g, 1.9 mmol) was dissolved in EtOH (10 mL), cyclopropylmethylamine (0.165 g, 2.3 mmol) was added, and the reaction was stirred at 65 ° C. and monitored by TLC until the reaction was completed (); the reaction solution was concentrated in vacuo, an appropriate amount of water was added, and extracted with ethyl acetate; the organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by silica gel column chromatography (petroleum ether / ethyl acetate = 20:1 to 10:1, V / V) to give a light yellow solid (compound I-1, 0.47 g, yield 85%).

[0041] 1 H NMR (400MHz, DMSO-d6) δ11.81(s,1H),8.95(t,J=6.2Hz,1H),8.38(d,J=5.2Hz,1H),8.30(d,J=4.6Hz,2H),7.48(d,J=10.1Hz HRMS calcd for C 16 H 15 FN3O[M+H] + m / z 284.11937, found 284.11957.

[0042] Example 2

[0043] Preparation of N-(cyclobutylmethyl)-7-fluoro-9H-pyrido[3,4-b]indole-1-carboxamide (Compound I-2)

[0044]

[0045] Referring to the preparation method of compound I-1, 6-fluoroindoleethylamine and ethyl glyoxylate were used as starting materials to prepare intermediate 4, and cyclopropylmethylamine was replaced by an equal amount of cyclobutylmethylamine. Intermediate 4 reacted with cyclobutylmethylamine to prepare compound I-2.

[0046] 1 H NMR (400MHz, DMSO-d6) δ11.80(s,1H),8.85(t,J=6.2Hz,1H),8.36(d,J=5.0Hz,1H),8.27(dd,J=9.4,5.3Hz,2H),7.48(dd,J=10.0 HRMS calcd for C 17 H 17 FN3O[M+H] + m / z 298.13502,found 298.13584.

[0047] Example 3

[0048] Preparation of N-(cyclopentylmethyl)-7-fluoro-9H-pyrido[3,4-b]indole-1-carboxamide (Compound I-3)

[0049]

[0050] Referring to the preparation method of compound I-1, 6-fluoroindoleethylamine and ethyl glyoxylate were used as starting materials to prepare intermediate 4, and cyclopentylmethylamine was used to replace cyclopropylmethylamine in an equal amount. Intermediate 4 reacted with cyclopentylmethylamine to prepare compound I-3.

[0051] 1 H NMR (400MHz, DMSO-d6) δ11.82(s,1H),8.88(t,J=6.3Hz,1H),8.35(d,J=5.0Hz,1H),8.25(dd,J=9.5,5.4Hz,2H),7.48(dd,J=10.1,2.3Hz,1H),7.13 -6.98(m,1H),3.29(t,J=6.9Hz,2H),2.19(p,J=7.3Hz,1H),1.69-1.57(m, 2H),1.54(t,J=5.7Hz,2H),1.44(t,J=6.0Hz,2H),1.33-1.20(m,2H); HRMS calcd for C 18 H 19 FN3O[M+H] + m / z312.15067,found312.15095.

[0052] Example 4

[0053] Preparation of N-(cyclohexylmethyl)-7-fluoro-9H-pyrido[3,4-b]indole-1-carboxamide (Compound I-4)

[0054]

[0055] Referring to the preparation method of compound I-1, 6-fluoroindoleethylamine and ethyl glyoxylate were used as starting materials to prepare intermediate 4, and cyclohexylmethylamine was used to replace cyclopropylmethylamine in an equal amount. Intermediate 4 reacted with cyclohexylmethylamine to prepare compound I-4.

[0056] 1 H NMR (400MHz, DMSO-d6) δ11.81(s,1H),8.85(t,J=6.3Hz,1H),8.36(d,J=5.2Hz,1H),8.27(dd,J=9.5,5.4Hz,2H),7.47(dd,J=1 HRMScalcd for C 19 H 21 FN3O[M+H] + m / z 326.16632, found 326.16648.

[0057] Example 5

[0058] Preparation of 7-fluoro-N-(oxetane-3-ylmethyl)-9H-pyrido[3,4-b]indole-1-carboxamide (Compound I-5)

[0059]

[0060] Referring to the preparation method of compound I-1, 6-fluoroindoleethylamine and ethyl glyoxylate were used as starting materials to prepare intermediate 4, and cyclopropylmethylamine was replaced by an equal amount of 3-aminomethyloxetane. Intermediate 4 reacted with 3-aminomethyloxetane to prepare compound I-5.

[0061] 1H NMR (400MHz, DMSO-d6) δ11.81(s,1H),9.08(t,J=6.2Hz,1H),8.37(d,J=5.0Hz,1H),8.33-8.23(m,2H),7.48(dd,J=10.1,2.4H z,1H),7.09(dd,J=9.2,2.4Hz,1H),4.77-4.64(m,2H),4.62-4.54(m,2H),3.37(t,J=6.7Hz,1H),2.61-2.50(m,1H); HRMScalcd for C 16 H 15 FN3O2[M+H] + m / z 300.11483,found 300.10712.

[0062] Example 6

[0063] Preparation of 7-fluoro-N-((tetrahydrofuran-3-yl)methyl)-9H-pyrido[3,4-b]indole-1-carboxamide (Compound I-6)

[0064]

[0065] Referring to the preparation method of compound I-1, 6-fluoroindoleethylamine and ethyl glyoxylate were used as starting materials to prepare intermediate 4, and cyclopropylmethylamine was replaced with an equal amount of (tetrahydrofuran-3-yl)methylamine. Intermediate 4 was reacted with (tetrahydrofuran-3-yl)methylamine to prepare compound I-6.

[0066] 1 H NMR (400MHz, DMSO-d6) δ11.81(s,1H),9.08(t,J=6.2Hz,1H),8.37(d,J=5.0Hz,1H),8.33-8.23(m,2H),7.48(dd,J=10.1,2.4Hz,1H ),7.09(dd,J=9.2,2.4Hz,1H),3.77-3.64(m,2H),3.62-3.54(m,2H),3.37-3.16(m,2H),2.61-2.50(m,1H),1.98-1.61(m,2H); HRMS calcd for C 17 H 17 FN3O2[M+H] + m / z 314.12993, found 314.13035.

[0067] Example 7

[0068] Preparation of 7-fluoro-N-((tetrahydrothiophen-3-yl)methyl)-9H-pyrido[3,4-b]indole-1-carboxamide (Compound I-7)

[0069]

[0070] Referring to the preparation method of compound I-1, intermediate 4 was prepared using 6-fluoroindoleethylamine and ethyl glyoxylate as starting materials. Cyclopropylmethylamine was replaced with an equal amount of (tetrahydrothiophen-3-yl)methylamine, and intermediate 4 was reacted with (tetrahydrothiophen-3-yl)methylamine to prepare compound I-7.

[0071] 1 H NMR (400MHz, DMSO-d6) δ11.81(s,1H),9.08(t,J=6.2Hz,1H),8.37(d,J=5.0Hz,1H),8.33-8.23(m,2H),7.48(dd,J=10.1,2.4Hz,1H),7.09(dd,J=9. 2,2.4Hz,1H),3.77-3.64(m,2H),3.62-3.54(m,1H),3.40(dd,J=8.5,5.2 Hz,1H),3.27(t,J=6.7Hz,2H),2.51-2.40(m,1H),1.96-1.60(m,2H); HRMS calcd for C 17 H 17 FN3OS[M+H] + m / z 330.10764,found330.10970.

[0072] Example 8

[0073] Preparation of 7-fluoro-N-(pyrrolidin-3-ylmethyl)-9H-pyrido[3,4-b]indole-1-carboxamide (Compound I-8)

[0074]

[0075] Referring to the preparation method of compound I-1, 6-fluoroindoleethylamine and ethyl glyoxylate were used as starting materials to prepare intermediate 4, and cyclopropylmethylamine was replaced by an equal amount of pyrrolidine-3-methylamine. Intermediate 4 reacted with pyrrolidine-3-methylamine to prepare compound I-8.

[0076] 1H NMR (400MHz, DMSO-d6) δ11.81(s,1H),9.09(t,J=6.2Hz,1H),8.37(d,J=5.0Hz,1H),8.32-8.23(m,2H),7.48(dd,J=10.1,2.4Hz,1H),7.09(dd,J=9. 2,2.4Hz,1H),3.77-3.64(m,2H),3.52-3.44(m,1H),3.20(dd,J=8.5,5.2 Hz,1H),3.15(t,J=6.7Hz,2H),2.49-2.40(m,1H),1.94-1.56(m,2H); HRMS calcd for C 17 H 18 FN4O[M+H] + m / z 313.14646,found313.14874.

[0077] Example 9

[0078] Preparation of 7-fluoro-N-((tetrahydro-2H-pyran-4-yl)methyl)-9H-pyrido[3,4-b]indole-1-carboxamide (Compound I-9)

[0079]

[0080] Referring to the preparation method of compound I-1, 6-fluoroindoleethylamine and ethyl glyoxylate were used as starting materials to prepare intermediate 4, and cyclopropylmethylamine was replaced by an equal amount of 4-aminomethyltetrahydropyran. Intermediate 4 reacted with 4-aminomethyltetrahydropyran to prepare compound I-9.

[0081] 1 H NMR (400MHz, DMSO-d6) δ8.36(q,J=5.3Hz,2H),8.27(dd,J=8.7,5.5Hz,1H),7.45(dd,J=9.7,2.4Hz,1H),7.14-7.03(m HRMS calcd forC 18 H 19 FN3O2[M+H] + m / z 328.14558, found 328.14686.

[0082] Example 10

[0083] Preparation of 7-fluoro-N-(piperidin-4-ylmethyl)-9H-pyrido[3,4-b]indole-1-carboxamide (Compound I-10)

[0084]

[0085] Referring to the preparation method of compound I-1, 6-fluoroindoleethylamine and ethyl glyoxylate were used as starting materials to prepare intermediate 4, and cyclopropylmethylamine was replaced by an equal amount of 4-(aminomethyl)piperidine. Intermediate 4 reacted with 4-(aminomethyl)piperidine to prepare compound I-10.

[0086] 1 H NMR (400MHz, DMSO-d6) δ9.00(t,J=6.4Hz,1H),8.36(d,J=5.1Hz,1H),8.26(dd,J=12.8,5.0Hz,2H),7.46(dd,J=10.1,2.5Hz,1 H),7.13-7.02(m,1H),3.29(t,J=6.3Hz,2H),3.24-3.16(m,2H),2.84-2.69(m,2H),1.93-1.69(m,3H),1.46-1.29(m,2H); HRMS calcd for C 18 H 20 FN4O[M+H] + m / z 327.16157, found 327.16168.

[0087] Example 11

[0088] Preparation of N-benzyl-7-fluoro-9H-pyrido[3,4-b]indole-1-carboxamide (Compound I-11)

[0089]

[0090] Referring to the preparation method of compound I-1, 6-fluoroindoleethylamine and ethyl glyoxylate were used as starting materials to prepare intermediate 4, and cyclopropylmethylamine was replaced by an equal amount of benzylamine. The intermediate 4 reacted with benzylamine to prepare compound I-11. 1H NMR (400MHz, DMSO-d6) δ11.84(s,1H),9.49(t,J=6.4Hz,1H),8.39(d,J=5.0Hz,1H),8.34-8.25(m,2H),7.46(dd,J=10.1,2. HRMS calcd forC 19 H 14 FN3ONa[M+Na] + m / z 342.10131, found 342.10134.

[0091] Example 12

[0092] Preparation of 7-fluoro-N-(3-fluorobenzyl)-9H-pyrido[3,4-b]indole-1-carboxamide (Compound I-12)

[0093]

[0094] Referring to the preparation method of compound I-1, 6-fluoroindoleethylamine and ethyl glyoxylate were used as starting materials to prepare intermediate 4, and cyclopropylmethylamine was replaced by an equal amount of 3-fluorobenzylamine. Intermediate 4 reacted with 3-fluorobenzylamine to prepare compound I-12.

[0095] 1 H NMR (400MHz, DMSO-d6) δ11.83(s,1H),9.57(t,J=6.4Hz,1H),8.39(d,J=5.1Hz,1H),8.35-8.24(m,2H),7.46(dd, J=10.1,2.3Hz,1H),7.33(q,J=7.5Hz,1H),7.19(t,J=9.7Hz,2H),7.12-6.96(m,2H),4.55(d,J=6.4Hz,2H); HRMS calcd forC 19 H 13 F2N3ONa[M+Na] + m / z 360.09189, found 360.09185.

[0096] Example 13

[0097] Preparation of 7-fluoro-N-(4-fluorobenzyl)-9H-pyrido[3,4-b]indole-1-carboxamide (Compound I-13)

[0098]

[0099] Referring to the preparation method of compound I-1, 6-fluoroindoleethylamine and ethyl glyoxylate were used as starting materials to prepare intermediate 4, and cyclopropylmethylamine was replaced by an equal amount of 4-fluorobenzylamine. Intermediate 4 reacted with 4-fluorobenzylamine to prepare compound I-13.

[0100] 1 H NMR (400MHz, DMSO-d6) δ11.83(s,1H),9.51(t,J=6.4Hz,1H),8.38(d,J=5.0Hz,1H), 8.33-8.23(m,2H),7.53-7.37(m,3H),7.15-7.03(m,3H),4.52(d,J=6.4Hz,2H); HRMS calcd for C 19 H 13 F2N3ONa[M+Na] + m / z 360.09189, found 360.09177.

[0101] Example 14

[0102] Preparation of 7-fluoro-N-(3-chlorobenzyl)-9H-pyrido[3,4-b]indole-1-carboxamide (Compound I-14)

[0103]

[0104] Referring to the preparation method of compound I-1, 6-fluoroindoleethylamine and ethyl glyoxylate were used as starting materials to prepare intermediate 4. Cyclopropylmethylamine was replaced by an equal amount of 3-chlorobenzylamine, and intermediate 4 reacted with 3-chlorobenzylamine to prepare compound I-14.

[0105] 1 H NMR (400MHz, DMSO-d6) δ11.83(s,1H),9.59(t,J=6.4Hz,1H),8.39(d,J=5.0Hz,1H),8.34-8.2 5(m,2H),7.49-7.40(m,2H),7.36-7.23(m,3H),7.13-7.04(m,1H),4.54(d,J=6.4Hz,2H); HRMS calcd for C 19 H 13 ClFN3ONa[M+Na] + m / z 376.06234,found376.06210.

[0106] Example 15

[0107] Preparation of 7-fluoro-N-(4-chlorobenzyl)-9H-pyrido[3,4-b]indole-1-carboxamide (Compound I-15)

[0108]

[0109] Referring to the preparation method of compound I-1, 6-fluoroindoleethylamine and ethyl glyoxylate were used as starting materials to prepare intermediate 4, and cyclopropylmethylamine was replaced by an equal amount of 4-chlorobenzylamine. Intermediate 4 reacted with 4-chlorobenzylamine to prepare compound I-15.

[0110] 1 H NMR (400MHz, DMSO-d6) δ11.83(s,1H),9.55(t,J=6.5Hz,1H),8.38(d,J=5.0Hz,1H),8.34-8.23(m,2H ),7.46(dd,J=10.1,2.4Hz,1H),7.36(q,J=8.6Hz,4H),7.12-7.05(m,1H),4.53(d,J=6.3Hz,2H); HRMS calcd for C 19 H 13 ClFN3ONa[M+Na] + m / z376.06234,found376.06230.

[0111] Example 16

[0112] Preparation of 7-fluoro-N-(3-bromobenzyl)-9H-pyrido[3,4-b]indole-1-carboxamide (Compound I-16)

[0113]

[0114] Referring to the preparation method of compound I-1, 6-fluoroindoleethylamine and ethyl glyoxylate were used as starting materials to prepare intermediate 4. Cyclopropylmethylamine was replaced with an equal amount of 3-bromobenzylamine, and intermediate 4 reacted with 3-bromobenzylamine to prepare compound I-16.

[0115] 1H NMR (400MHz, DMSO-d6) δ11.83(s,1H),9.59(t,J=6.5Hz,1H),8.39(d,J=5.0Hz,1H),8.34-8.24(m,2H),7.57(t,J=2.0Hz,1H),7 HRMS calcd for C 19 H 13 BrFN3ONa[M+Na] + m / z 420.01182, found 422.00987.

[0116] Example 17

[0117] Preparation of 7-fluoro-N-(4-bromobenzyl)-9H-pyrido[3,4-b]indole-1-carboxamide (Compound I-17)

[0118]

[0119] Referring to the preparation method of compound I-1, 6-fluoroindoleethylamine and ethyl glyoxylate were used as starting materials to prepare intermediate 4, and cyclopropylmethylamine was replaced by an equal amount of 4-bromobenzylamine. Intermediate 4 reacted with 4-bromobenzylamine to prepare compound I-17.

[0120] 1 H NMR (400MHz, DMSO-d6) δ11.82(s,1H),9.55(t,J=6.4Hz,1H),8.39(d,J=5.0Hz,1H),8.34-8.23( HRMS calcd for C 19 H 13 BrFN3ONa[M+Na] + m / z 420.01182,found422.00110.

[0121] Example 18

[0122] Preparation of 7-fluoro-N-(furan-3-ylmethyl)-9H-pyrido[3,4-b]indole-1-carboxamide (Compound I-18)

[0123]

[0124] Referring to the preparation method of compound I-1, intermediate 4 was prepared using 6-fluoroindoleethylamine and ethyl glyoxylate as starting materials. Cyclopropylmethylamine was replaced with an equal amount of 3-furylmethylamine, and intermediate 4 was reacted with 3-furylmethylamine to prepare compound I-18.

[0125] 1 H NMR (400MHz, DMSO-d6) δ11.78(s,1H),9.49(t,J=6.4Hz,1H),8.39(d,J=5.0Hz,1H),8.34-8.25(m,2H),7.46(dd, J=10.1,2.5Hz,1H),7.37(d,J=7.2Hz,2H),7.20(t,J=7.3Hz,1H),6.20-6.10(m,1H),4.55(d,J=6.3Hz,2H); HRMS calcd for C 17 H 13 FN3O2[M+H] + m / z 310.09135, found 310.09155.

[0126] Example 19

[0127] Preparation of 7-fluoro-N-(thiophen-3-ylmethyl)-9H-pyrido[3,4-b]indole-1-carboxamide (Compound I-19)

[0128]

[0129] Referring to the preparation method of compound I-1, intermediate 4 was prepared using 6-fluoroindoleethylamine and ethyl glyoxylate as starting materials. Cyclopropylmethylamine was replaced with an equal amount of 3-aminomethylthiophene, and intermediate 4 reacted with 3-aminomethylthiophene to prepare compound I-19.

[0130] 1 H NMR (400MHz, DMSO-d6) δ11.78(s,1H),9.50(t,J=6.4Hz,1H),8.49(d,J=5.0Hz,1H),8.36-8.25(m,2H),7.47(dd, J=10.1,2.5Hz,1H),7.39(d,J=7.2Hz,2H),7.23(t,J=7.3Hz,1H),6.60-6.50(m,1H),4.55(d,J=6.3Hz,2H); HRMS calcd for C 17 H 13 FN3OS[M+H] +m / z 326.06851,found 326.06881.

[0131] Example 20

[0132] Preparation of N-((1H-pyrrol-3-yl)methyl)-7-fluoro-9H-pyrido[3,4-b]indole-1-carboxamide (Compound I-20)

[0133]

[0134] Referring to the preparation method of compound I-1, intermediate 4 was prepared using 6-fluoroindoleethylamine and ethyl glyoxylate as starting materials. Cyclopropylmethylamine was replaced with an equal amount of (1H-pyrrol-3-yl)methylamine, and intermediate 4 was reacted with (1H-pyrrol-3-yl)methylamine to prepare compound I-20.

[0135] 1 H NMR (400MHz, DMSO-d6) δ11.78(s,1H),9.60(t,J=6.4Hz,1H),9.50(t,J=6.4Hz,1H),8.49(d,J=5.0Hz,1H),8.36-8.25(m,2H),7.47(dd, HRMS calcd for C 17 H 14 FN4O[M+H] + m / z309.10734, found 309.10764.

[0136] Example 21

[0137] Preparation of 7-fluoro-N-(oxazol-4-ylmethyl)-9H-pyrido[3,4-b]indole-1-carboxamide (Compound I-21)

[0138]

[0139] Referring to the preparation method of compound I-1, 6-fluoroindoleethylamine and ethyl glyoxylate were used as starting materials to prepare intermediate 4, and cyclopropylmethylamine was replaced by an equal amount of 4-oxazolemethylamine. Intermediate 4 reacted with 4-oxazolemethylamine to prepare compound I-21.

[0140] 1H NMR (400MHz, DMSO-d6) δ11.78(s,1H),9.60(t,J=6.4Hz,1H),9.50(t,J=6.4Hz,1H),8.49(d,J=5.0Hz,1H), 8.36-8.25(m,2H),7.86-7.75(m,1H),7.67-7.60(m,1H),7.39(d,J=7.2Hz,1H),4.02(d,J=6.3Hz,2H); HRMS calcd for C 16 H 12 FN4O2[M+H] + m / z311.08660, found 311.08690.

[0141] Example 22

[0142] Preparation of 7-fluoro-N-(thiazol-4-ylmethyl)-9H-pyrido[3,4-b]indole-1-carboxamide (Compound I-22)

[0143]

[0144] Referring to the preparation method of compound I-1, intermediate 4 was prepared using 6-fluoroindoleethylamine and ethyl glyoxylate as starting materials. Cyclopropylmethylamine was replaced by an equal amount of 4-thiazolemethylamine, and intermediate 4 was reacted with 4-thiazolemethylamine to prepare compound I-22.

[0145] 1 H NMR (400MHz, DMSO-d6) δ11.79(s,1H),9.60(t,J=6.4Hz,1H),9.50(t,J=6.4Hz,1H),8.49(d,J=5.0Hz,1H), 8.36-8.25(m,2H),7.86-7.75(m,1H),7.60-7.50(m,1H),7.29(d,J=7.2Hz,1H),4.12(d,J=6.3Hz,2H); HRMS calcd for C 16 H 13 FN4OS[M+H] + m / z327.06376, found 327.06426.

[0146] Example 23

[0147] Preparation of N-((1H-imidazol-4-yl)methyl)-7-fluoro-9H-pyrido[3,4-b]indole-1-carboxamide (Compound I-23)

[0148]

[0149] Referring to the preparation method of compound I-1, intermediate 4 was prepared using 6-fluoroindoleethylamine and ethyl glyoxylate as starting materials. Cyclopropylmethylamine was replaced with an equal amount of (1H-imidazol-4-yl)methylamine, and intermediate 4 was reacted with (1H-imidazol-4-yl)methylamine to prepare compound I-23.

[0150] 1 H NMR (400MHz, DMSO-d6) δ12.69(s,1H),11.79(s,1H),9.60(t,J=6.4Hz,1H),9.50(t,J=6.4Hz,1H),8.49(d,J=5.0H z,1H),8.36-8.25(m,2H),7.84-7.75(m,1H),7.59-7.50(m,1H),7.26(d,J=7.2Hz,1H),4.12(d,J=6.3Hz,2H); HRMS calcd for C 16 H 13 FN5O[M+H] + m / z 310.10259, found 310.10359.

[0151] Example 24

[0152] Preparation of N-((1H-1,2,3-triazol-4-yl)methyl)-7-fluoro-9H-pyrido[3,4-b]indole-1-carboxamide (Compound I-24)

[0153]

[0154] Referring to the preparation method of compound I-1, intermediate 4 was prepared using 6-fluoroindoleethylamine and ethyl glyoxylate as starting materials. Cyclopropylmethylamine was replaced with an equal amount of 1H-1,2,3-triazol-4-ylmethylamine, and intermediate 4 was reacted with 1H-1,2,3-triazol-4-ylmethylamine to prepare compound I-24.

[0155] 1 H NMR (400MHz, DMSO-d6) δ11.99(s,1H),11.79(s,1H),9.60(t,J=6.4Hz,1H),8.70-8.58(m,2H),8.4 9(m,1H),7.66-7.55(m,1H),7.59-7.50(m,1H),7.26(d,J=7.2Hz,1H),4.12(d,J=6.3Hz,2H); HRMS calcd for C 15 H 12 FN6O[M+H]+ m / z 311.09784,found311.09584.

[0156] Example 25

[0157] Preparation of N-((1H-tetrazol-5-yl)methyl)-7-fluoro-9H-pyrido[3,4-b]indole-1-carboxamide (Compound I-25)

[0158]

[0159] Referring to the preparation method of compound I-1, intermediate 4 was prepared using 6-fluoroindoleethylamine and ethyl glyoxylate as starting materials. Cyclopropylmethylamine was replaced by an equal amount of 5-aminomethyltetrazole, and intermediate 4 was reacted with 5-aminomethyltetrazole to prepare compound I-25.

[0160] 1 H NMR (400MHz, DMSO-d6) δ11.99 (s, 1H), 11.79 (s, 1H), 9.60 (t, J = 6.4Hz, 1H), 8.70-8.58 (m, 2H), 8.49(m,1H),7.59-7.50(m,1H),7.26(d,J=7.2Hz,1H),6.25(s,1H),4.12(d,J=6.3Hz,2H); HRMS calcd for C 14 H 11 FN7O[M+H] + m / z 312.09319,found312.09584.

[0161] Example 26

[0162] Preparation of 7-fluoro-N-(pyridin-4-ylmethyl)-9H-pyrido[3,4-b]indole-1-carboxamide (Compound I-26)

[0163]

[0164] Referring to the preparation method of compound I-1, intermediate 4 was prepared using 6-fluoroindoleethylamine and ethyl glyoxylate as starting materials. Cyclopropylmethylamine was replaced by an equal amount of 4-(aminomethyl)pyridine, and intermediate 4 was reacted with 4-(aminomethyl)pyridine to prepare compound I-26.

[0165] 1H NMR (400MHz, DMSO-d6) δ11.84(s,1H),9.49(t,J=6.4Hz,1H),8.39(d,J=5.0Hz,1H),8.34-8.25(m,1H),7.46(dd,J=10.1,2.5Hz ,1H),7.37(d,J=7.2Hz,2H),7.29(t,J=7.5Hz,2H),7.20(t,J=7.3Hz,1H),7.13-7.04(m,1H),4.55(d,J=6.3Hz,2H); HRMScalcd for C 18 H 14 FN4O[M+H] + m / z 321.10734, found 321.10534.

[0166] Example 27

[0167] Preparation of 7-fluoro-N-(pyrimidin-4-ylmethyl)-9H-pyrido[3,4-b]indole-1-carboxamide (Compound I-27)

[0168]

[0169] Referring to the preparation method of compound I-1, intermediate 4 was prepared using 6-fluoroindoleethylamine and ethyl glyoxylate as starting materials. Cyclopropylmethylamine was replaced by an equal amount of 4-aminomethylpyrimidine, and intermediate 4 reacted with 4-aminomethylpyrimidine to prepare compound I-27.

[0170] 1 H NMR (400MHz, DMSO-d6) δ11.84(s,1H),9.49(t,J=6.4Hz,1H),8.39(d,J=5.0Hz,1H),8.34-8.25(m,1H),7.37( d,J=7.2Hz,2H),7.29(t,J=7.5Hz,2H),7.20(t,J=7.3Hz,1H),7.13-7.04(m,1H),4.55(d,J=6.3Hz,2H); HRMS calcd for C 17 H 13 FN5O[M+H] + m / z322.10259, found 322.10759.

[0171] Example 28

[0172] Preparation of 7-fluoro-N-(pyrazin-2-ylmethyl)-9H-pyrido[3,4-b]indole-1-carboxamide (Compound I-28)

[0173]

[0174] Referring to the preparation method of compound I-1, intermediate 4 was prepared using 6-fluoroindoleethylamine and ethyl glyoxylate as starting materials. Cyclopropylmethylamine was replaced by an equal amount of 2-aminomethylpyrazine, and intermediate 4 was reacted with 2-aminomethylpyrazine to prepare compound I-28.

[0175] 1 H NMR (400MHz, DMSO-d6) δ11.87(s,1H),9.49(t,J=6.4Hz,1H),8.39(d,J=5.0Hz,1H),8.34-8.25(m,1H),7.37( d,J=7.2Hz,2H),7.39(t,J=7.5Hz,2H),7.25(t,J=7.3Hz,1H),7.13-7.04(m,1H),4.55(d,J=6.3Hz,2H); HRMS calcd for C 17 H 13 FN5O[M+H] + m / z322.10259, found 322.10959.

[0176] Example 29

[0177] Preparation of 7-fluoro-N-(pyridazin-4-ylmethyl)-9H-pyrido[3,4-b]indole-1-carboxamide (Compound I-29)

[0178]

[0179] Referring to the preparation method of compound I-1, intermediate 4 was prepared using 6-fluoroindoleethylamine and ethyl glyoxylate as starting materials. Cyclopropylmethylamine was replaced by an equal amount of 4-aminomethylpyridazine, and intermediate 4 was reacted with 4-aminomethylpyridazine to prepare compound I-29.

[0180] 1 H NMR (400MHz, DMSO-d6) δ11.85(s,1H),9.49(t,J=6.4Hz,1H),8.39(d,J=5.0Hz,1H),8.35-8.25(m,1H),7.38( d,J=7.2Hz,2H),7.29(t,J=7.5Hz,2H),7.20(t,J=7.3Hz,1H),7.13-7.04(m,1H),4.45(d,J=6.3Hz,2H); HRMS calcd for C 17 H 13 FN5O[M+H] +m / z322.10259, found 322.10759.

[0181] Example 30

[0182] In vitro assay analysis of human recombinant MAOs enzyme inhibitory activity

[0183] (1) Experimental principle

[0184]

[0185] MAOs (monoamine oxidative deamination) enzymes in the human body catalyze the oxidative deamination of monoamines. Kynurenine is a nonspecific substrate for MAOs and can be converted to 4-hydroxyquinoline by MAOs. The inventors have developed an in vitro assay for MAOs inhibitory activity. Using fluorescence spectrophotometry, the 4-hydroxyquinoline content is measured to determine the degree of MAOs inhibition by the test compound.

[0186] (2) Experimental materials and main instruments

[0187] Experimental materials: human recombinant MAO-A and MAO-B proteins, kynurenine dihydrobromide (molecular weight 326.03), 4-hydroxyquinoline (molecular weight 145.16), safinamide, tripotassium phosphate, DMSO, 2N hydrochloric acid, ultrapure water, PCR tubes and cryovials.

[0188] Main instruments: Bio-Tek microplate reader, centrifuge, constant temperature shaking incubator, vortexer, pH meter.

[0189] (3) Experimental steps

[0190] The reaction volume per well was 200 μL: 180 μL enzyme working solution + 10 μL inhibitor + 10 μL kynurenine stock solution (50 μM). Finally, 80 μL NaOH solution (2N) was added to terminate the reaction.

[0191] Preparation of 100mM buffer: To a 200mL cryovial, add 160mL of ultrapure water and 4.246g of tripotassium phosphate, sequentially. Adjust the pH to 7.4 with 2N hydrochloric acid, and then dilute to 200mL with ultrapure water. The concentration of tripotassium phosphate in this buffer is 100mM. Use this buffer for all preparations unless otherwise noted.

[0192] 1) Preparation of experimental mother solution:

[0193] Preparation of kynurenine stock solution: Weigh 3.26 mg of kynurenine dihydrobromide and dissolve it in 10 mL of buffer to obtain a kynurenine dihydrobromide stock solution with a concentration of 1 mM.

[0194] Preparation of inhibitor stock solution: The positive control drug Safinamide and the test compounds (Compounds I-1 to I-29, Harmane, Norharmane) were prepared with DMSO to prepare an inhibitor stock solution with a concentration of 1 mM. The inhibitor stock solution was then diluted 2 to 3 times its volume with DMSO to obtain 8 concentrations for each inhibitor (Safinamide, test compound).

[0195] Prepare 4-hydroxyquinoline solution: weigh 1.4516 mg and dissolve in 10 mL of buffer to obtain a 1 mM 4-hydroxyquinoline solution; dilute with buffer to 100 μM, then dilute the solution by 2-fold by volume to 12 concentrations starting from 100 μM.

[0196] 2) Preparation of enzyme working solution:

[0197] Preparation of MAO-A working solution: 12.6 mL buffer + 21 μL MAO-A stock solution.

[0198] Preparation of MAO-B working solution: 12.6 mL buffer + 42 μL MAO-B stock solution.

[0199] The final concentrations of MAO-A and MAO-B enzymes in the reaction system (200 μL) were 7.5 μg / mL and 15 μg / mL, respectively.

[0200] 3) Determination of standard curve:

[0201] The fluorescence was detected using the 4-hydroxyquinoline solution prepared above at an excitation wavelength of 310 nm and an emission wavelength of 400 nm. A standard linear curve was drawn with the concentration of 4-hydroxyquinoline as the abscissa and the fluorescence intensity as the ordinate.

[0202] 4) Enzymatic reaction:

[0203] One blank control group (no enzyme or inhibitor, except for the inhibitor solution in 4% DMSO buffer) was used, with all other conditions replicated. One negative control group (no inhibitor, except for the inhibitor solution in 4% DMSO buffer) was used, with all other conditions replicated. One positive control group and 31 test compound groups were used. All procedures except heating and incubation were performed at 0°C. Two replicates were used for each group.

[0204] Positive control group or test compound group: Add 180 μL of enzyme working solution to each PCR tube, add 10 μL of the positive control drug (Safinamide) or the test compound (Compound I-3 to Compound I-29, Harmane, Norharmane) stock solution, vortex mix; add 10 μL of kynurenine stock solution, vortex mix, transfer to 37°C, and incubate for 0.5 h. Return to room temperature (25°C), add 80 μL of sodium hydroxide solution (2N, i.e., 2g NaOH dissolved in 25mL buffer, prepared immediately before use) to each PCR tube, centrifuge at 9000 rpm for 3 min to remove the precipitate, transfer 200 μL of the supernatant from each tube to a 96-well plate, and measure the fluorescence intensity using a microplate reader at an excitation wavelength of 310 nm and an emission wavelength of 400 nm. Calculate the MAOs enzyme inhibition rate (%) of the test compound (concentration 50 μM).

[0205] MAOs enzyme inhibition rate (%) = [(negative group intensity - test group intensity) / (negative group intensity - blank group intensity)] × 100

[0206] At this concentration, if the MAOs enzyme inhibition rate of the test compound is greater than 70%, the test compound is diluted according to the "preparation of inhibitor stock solution" to obtain 8 concentrations, and its inhibition rate on MAO enzyme at different concentrations is detected. The dose-effect curve is fitted using GraphPad software to calculate its half-maximal inhibitory concentration IC 50 value.

[0207] (4) Experimental results

[0208] As shown in Table 1, most of the compounds of the present invention have a highly selective inhibitory effect on MAO-B, but a weak inhibitory activity on MAO-A. Among them, compounds I-3, I-4, I-11, I-12, I-13, I-14, I-16, I-24, I-25, I-26, I-27 and I-29 have better in vitro inhibitory activity than the third-generation MAO-B inhibitor safinamide (IC 50 =0.044±0.0015 μM). In particular, the in vitro MAO-B inhibitory activities of compounds I-4, I-11, and I-12 were IC 50 The value reached 10 nM, which is more than 4 times the activity of safinamide, and the selectivity ratio for MAO-B / MAO-A was as high as more than 5000. The above results show that the in vitro MAO-B inhibitory activity and selectivity of compounds I-4, I-11 and I-12 of the present invention are significantly better than those of safinamide.

[0209] Table 1. Test data of in vitro human recombinant MAOs enzyme inhibition activity

[0210]

[0211]

[0212] Example 31

[0213] Reversibility test analysis of compound I-11 and I-12 on inhibition of human recombinant MAO-B enzyme activity in vitro

[0214] The reversibility test of the inhibition of MAO-B activity in vitro was performed on compounds I-11 and I-12 using the dilution method to evaluate the type of inhibition of MAO-B by the compounds of the present invention.

[0215] The preparation of enzyme working solution, test compound and kynurenine stock solution refers to Example 30. Set up 4 test groups, 1 blank group and 1 negative group. Test groups: Rasagiline, Safinamide, Compounds I-11 and I-12, respectively, and other conditions are parallel. Blank group: does not contain enzyme and inhibitor, the inhibitor solution is changed to a 4% DMSO buffer solution, and other conditions are parallel. Negative group: does not contain inhibitor, the inhibitor solution is changed to a 4% DMSO buffer solution, and other conditions are parallel. Except for the heating and incubation process, other operations are carried out at 0°C.

[0216] The test was carried out in a PCR tube (1 mL). 900 μL of enzyme working solution was added to the PCR tube, and 50 μL of the stock solution of the test compound (rasagiline, safinamide, compound I-11 or compound I-12) was added and vortexed to mix. The blank group and the negative control group were operated at the same time; the tube was transferred to 37°C and incubated for 15 minutes, and then returned to room temperature (25°C). The mixed solution after incubation was taken, and the kynuramine stock solution was added to dilute the test compound to 1 μM, 0.1 μM, and 0.01 μM, respectively. The final volume was 400 μL, and the tube was transferred to 37°C and incubated for 15 minutes. Return to room temperature (25°C), add 80 μL of sodium hydroxide solution (2N, i.e., 2 g NaOH dissolved in 25 mL buffer, prepared immediately); centrifuge at 9000 rpm for 3 min to remove the precipitate, transfer 200 μL of the supernatant to a 96-well plate, and measure the fluorescence intensity using a microplate reader at an excitation wavelength of 310 nm and an emission wavelength of 400 nm. Calculate the MAO-B enzyme inhibition rate (%) of the compound at concentrations of 1 μM, 0.1 μM, and 0.01 μM.

[0217] MAO-B enzyme inhibition rate (%) = [(negative group intensity - test group intensity) / (negative group intensity - blank group intensity)] × 100

[0218] The experimental results are as follows Figure 1As shown, compounds I-11 and I-12 of the present invention have reversible inhibitory effects on MAO-B, and their pharmacodynamic behavior is the same as that of the third-generation selective, reversible MAO-B inhibitor safinamide. This shows that all compounds of the present invention are reversible MAO-B inhibitors.

[0219] Example 32

[0220] Pharmacokinetics of Compound I-12

[0221] Male C57BL / 6J mice (n=3 for each dosing route) were administered compound I-12 (20 mg / kg, solvent: DMSO, Solutol (polyethylene glycol-15 hydroxystearate), and Saline, 5:10:85, by volume) via both intravenous (IV) and oral (PO) administration. Blood samples were collected at 0.083, 0.25, 0.5, 1, 2, 4, 8, and 24 h after IV administration or 0.25, 0.5, 1, 2, 4, 6, 8, and 24 h after oral administration. Blood samples were placed in K2-EDTA-containing centrifuge tubes, stored on ice, and centrifuged at 6800 rpm for 6 min at 2-8°C. The supernatant plasma was temporarily stored at approximately -80°C until LC-MS analysis. Pharmacokinetic properties were calculated using Phoenix WinNonlin 7.0.

[0222] Bioavailability (F) (%) = (AUC po / AUC iv) × (dose iv / dose po) × 100

[0223] The experimental results are shown in Table 2. Compound I-12 has good pharmacokinetic parameters, including an oral half-life of T 1 / 2 =2.91h, oral bioavailability F(%)=34.5%.

[0224] Table 2. Pharmacokinetic parameters of compound I-12

[0225]

[0226] Example 33

[0227] Blood-brain barrier permeability test of compound I-12

[0228] Nine male C57BL / 6J mice received a single oral dose of compound I-12 (20 mg / kg, solvent: DMSO, Solutol, and Saline in a volume ratio of 5:10:85). Blood samples and brain tissue were collected from three mice at each time point 0.5, 1, and 2 hours after administration. Blood was collected via the submandibular vein or other appropriate methods, with approximately 30 μL of blood collected per time point. The blood was anticoagulated with K2-EDTA and placed on ice after collection. The animals were euthanized with CO2, and brain tissue was then collected. Blood samples were centrifuged within 2 hours of collection to separate plasma (centrifugation conditions: 2-8°C, 6800 rpm for 6 minutes). The collected plasma samples and brain tissue were stored in a -80°C freezer until LC-MS analysis.

[0229] The experimental results are as follows Figure 2 As shown, compound I-12 can effectively penetrate the blood-brain barrier of mice, the drug content ratio in brain tissue / plasma is stable, and it is gradually metabolized and cleared over time.

[0230] Example 34

[0231] Pharmacodynamic evaluation of compound I-12 in improving the motor ability of zebrafish model of Parkinson's disease

[0232] (1) Experimental materials and main instruments

[0233] 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine hydrochloride (MPTP), DMSO, safinamide, nomifenac, dissecting microscope (SZX7), CCD camera (VertA1), precision electronic balance (CP214), 6-well plate, 96-well plate, behavioral analyzer (Zebra Lab 3.11).

[0234] (2) Experimental animals

[0235] Wild-type AB zebrafish (Hangzhou Huante Biotechnology Co., Ltd.), 4 days post-fertilization (dpf), were obtained under license number SYXK(Zhejiang)2022-0004. Their husbandry and management conform to AAALAC accreditation (certification number 001458). Zebrafish were maintained at 28°C in aquaculture water (water quality: 200 mg of instant sea salt per liter of reverse osmosis water, conductivity of 450–550 μS / cm, pH of 6.5–8.5, and hardness of 50–100 mg / L CaCO₃).

[0236] (3) Sample preparation

[0237] Preparation of Safinamide Stock Solution: Safinamide was prepared with DMSO to a stock solution with a concentration of 2.50 mg / mL. The Safinamide stock solution was diluted with DMSO as needed and stored at -20°C.

[0238] Preparation of Compound I-12 mother solution: Compound I-12 was prepared into a mother solution with a concentration of 2.50 mg / mL using DMSO; the mother solution of Compound I-12 was diluted with DMSO as needed and stored at -20°C.

[0239] Preparation of positive control drug stock solution: Nomifen maleate or safinamide was prepared into a positive control drug stock solution with a concentration of 50.0 mg / mL using DMSO and stored at -20°C.

[0240] (4) Detection method

[0241] 1) Determination of the maximum detectable concentration (MTC) for Parkinson's disease prevention

[0242] Experimental, normal, and model control groups were established, with 30 zebrafish treated in each group. Wild-type AB zebrafish (4 dpf) were randomly selected and plated in 6-well plates with a volume of 3 mL per well. Zebrafish in the normal control group were maintained in aquaculture water without MPTP and compound I-12. Zebrafish in the experimental group were simultaneously administered with water-soluble MPTP (MPTP concentration of 25.0 μg / mL) and compound I-12 (concentrations shown in Table 3) at 28°C for 48 hours. Zebrafish in the model control group were administered with water-soluble MPTP (MPTP concentration of 25.0 μg / mL) to establish a zebrafish Parkinson's disease model. Zebrafish in the experimental and model control groups were treated at 28°C for 48 hours, and the MTC of the samples was measured.

[0243] Table 3. Concentration exploration experiment for evaluating the efficacy of compound I-12 in preventing Parkinson's disease (n=30)

[0244]

[0245]

[0246] The experimental results are shown in Table 3, which show that the MTC value of compound I-12 for Parkinson's disease model zebrafish is 1.56 μg / mL.

[0247] 2) Evaluation of efficacy in preventing Parkinson's disease

[0248] Wild-type AB zebrafish (4 dpf) were randomly selected and placed in 6-well plates with a 3 mL volume per well. Thirty zebrafish were treated in each well. The zebrafish were divided into a normal control group, an experimental group, a positive control group (nomifensine and safinamide), and a model control group. The normal control group was treated with water without MPTP and compound I-12. The model control group was treated with MPTP (MPTP concentration: 25.0 μg / mL) dissolved in water. Both the experimental and positive control groups were treated with MPTP dissolved in water (concentrations shown in Table 4), and MPTP (final concentration: 25.0 μg / mL, prepared in ultrapure water) was administered simultaneously with the treatment. After 48 hours of treatment at 28°C, ten zebrafish were randomly selected from each group and placed in a 96-well plate, one per well, with 200 μL of untreated water per well. The total distance and speed of the zebrafish were measured using a behavioral analyzer over a 1-hour period. Statistical analysis of these indicators was used to evaluate the efficacy of the samples in preventing Parkinson's disease. Statistical results are expressed as mean ± SE. Statistical analysis was performed using SPSS 26.0 software, and p < 0.05 indicated that the difference was statistically significant.

[0249] Table 4. Evaluation of the efficacy of compound I-12 in preventing Parkinson's disease (n=10)

[0250]

[0251] Note: Compared with the model control group, *p<0.05, ***p<0.001.

[0252] In the zebrafish model of Parkinson's disease, the efficacy of compound I-12 and safinamide in preventing Parkinson's disease is shown in Table 4. Figure 3 、 Figure 4 and Figure 5 . The experimental results showed that compound I-12 significantly improved the total movement distance and movement speed of zebrafish. It is worth noting that the efficacy of compound I-12 in preventing Parkinson's disease at a concentration of 1.56μg / mL is comparable to that of safinamide at a concentration of 3.12μg / mL, and a low concentration of compound I-12 (0.781μg / mL) still has the efficacy of preventing Parkinson's disease. This shows that compound I-12 has the efficacy of preventing Parkinson's disease. However, safinamide did not show the efficacy of preventing Parkinson's disease at a lower concentration of 1.56μg / mL.

[0253] The above results show that in the zebrafish model of Parkinson's disease, the compound I-12 of the present invention has the efficacy of preventing Parkinson's disease, and its activity is better than that of the third-generation MAO-B inhibitor safinamide.

[0254] Example 35

[0255] Pharmacodynamic evaluation of compound I-12 in improving motor function in Parkinson's disease model mice

[0256] Experimental plan

[0257] 80 C57BL / 6J mice were tested by rotarod (rotation speed of 10 r / min, 5 min, for 2 consecutive days). Mice with poor motor ability were screened out, and 60 mice with good motor ability were randomly divided into 6 groups: normal control group, model group, safinamide group, low-dose group of compound I-12, medium-dose group of compound I-12, and high-dose group of compound I-12. Mice in the safinamide group, low-dose group of compound I-12, medium-dose group of compound I-12, and high-dose group of compound I-12 were given orally every day (safinamide 2.5 mg / kg / days; Compound I-12 was administered at 1.25 mg / kg / day, 2.5 mg / kg / day, and 5 mg / kg / day, respectively; the normal control group and the model group were gavaged with the same volume of blank solvent every day; the solvent for safinamide and Compound I-12 was PBS containing 0.1% DMSO). After 3 days of pre-administration, the mice in the model group, safinamide group, and three dose groups of Compound I-12 were intraperitoneally injected with MPTP (dissolved in physiological saline, 20 mg / kg / time, 4 times a day, with an interval of 2 hours), and then the administration was stopped. The behavioral changes of the mice were detected after 7 days.

[0258] (1) Spontaneous activity counting experiment: The mice were placed in a 40×40×40 cm voluntary activity box. The mice were first adapted to the box for 5 min (open field test). Then, the crawling trajectory and movement speed of the mice were observed and recorded within 10 min to evaluate their voluntary activity ability.

[0259] (2) Rotarod test: The mice were placed on a rotarod at a speed of 40 r / min. The time from the start of the rotarod rotation to the time the mice left the rotarod was defined as the rotarod latency. The test lasted 5 min and was repeated 3 times with a 1-hour interval. The mice were trained for 2 consecutive days before the test, at a speed of 10 r / min for 5 min, once daily.

[0260] (3) Suspension test: Two metal rods were fixed upright and connected with a 1 mm diameter wire, kept horizontal and 30 cm above the ground. The mice were held by their tails, with their heads down and their tails up, on the wires. The training lasted for 3 days, once a day, and the test was performed on the 4th day, for a total of 3 tests. The scoring criteria were as follows: 4 points for mice grasping the wires with both hind paws, 3 points for mice grasping the wires with only one hind paw, 2 points for mice grasping the wires with both forepaws, 1 point for mice grasping the wires with one forepaw, and 0 points for mice falling.

[0261] (4) Pole climbing test: A 25cm diameter cork ball was fixed to the top of a 50cm long and 1cm thick wooden pole. Gauze was wrapped around the pole to prevent slipping. The mouse was then placed on the ball and its performance was observed and scored. The scoring criteria were as follows: 5 points if the mouse slid down step by step using all four limbs; 4 points if the mouse slid down step by step using all four limbs but with some hind limbs sliding; 3 points if the mouse slid down after climbing half the distance but was able to hold the pole tightly; 2 points if the mouse slid before climbing half the distance; 1 point if the mouse fell down after climbing half the distance; 0 points if the mouse fell down before climbing half the distance. Each mouse was tested twice and the average value was taken.

[0262] Experimental results

[0263] (1) Weight change trend

[0264] like Figure 6 As shown, acute MPTP injection caused changes in mouse body weight. The body weight of mice in the model group and the three-dose intervention groups of compound I-12 decreased slightly compared with the normal control group; however, the body weight loss of mice in the three-dose intervention groups of compound I-12 was not significantly different from that in the model group.

[0265] (2) General status observation

[0266] A few minutes after MPTP injection, mice exhibited a series of acute behavioral changes, including tremors, decreased movement, spinal stiffness, and salivation. These symptoms disappeared within 0.5 to 1 hour, with no significant differences among the groups.

[0267] (3) Evaluation of autonomous activity ability

[0268] The open field test was used to evaluate the autonomous activity ability of mice. The changes in the autonomous activity ability and spontaneous exploration ability of mice were evaluated by three parameters: total distance moved within a fixed time (total distance), percentage of distance moved in the center area of ​​the mice to the total distance (distance in center), and percentage of time spent in the center area of ​​the mice to the total time (time in center). Figure 7 The results showed that the autonomous activity and spontaneous exploration abilities of mice in the acute MPTP-induced model did not change significantly. Compound I-12 also did not significantly affect the autonomous activity and spontaneous exploration abilities of the model mice.

[0269] (4) Evaluation of athletic ability based on the rotarod test

[0270] The rotarod test was used to evaluate the motor ability of mice. The changes in the mice's motor ability were evaluated by the changes in the time it took for the mice to fall from the rotarod rotating at a certain speed. Figure 8As shown, the ability of mice in the acute MPTP-induced model to maintain balance on a rotarod at a higher speed (40 r / min) was significantly weaker than that of the normal control group, and the time they maintained balance on the rotarod was significantly reduced. The medium and high dose groups of compound I-12 were able to effectively improve the above-mentioned movement disorder behaviors, with statistically significant differences compared with the model group.

[0271] (5) Evaluation of athletic ability based on the suspension test

[0272] The mice's motor ability was evaluated by using a suspension test. The changes in the mice's ability to use their hind limbs to balance and grasp the bar during the upper limb suspension process were used to evaluate the changes in the mice's motor ability. Figure 8 As shown, in the acute MPTP-induced model, the ability of mice to stabilize their hind limbs and grasp the wire while suspended was significantly reduced, manifested as only being able to grasp the wire with one hind paw, or even having difficulty grasping the wire with one hind paw, and a significant decrease in scores. The groups treated with compound I-12 showed significant recovery in this evaluation item, reaching statistical significance compared to the model group.

[0273] (6) Evaluation of athletic ability based on pole climbing experiment

[0274] The pole climbing test was used to evaluate the motor ability of mice. The changes in the mice's motor ability were evaluated by the changes in the mice's crawling down from a high wooden pole covered with gauze. Figure 8 As shown, in the acute MPTP-induced model, mice experienced hind paw slips during downward crawling, accompanied by sliding behavior, and their motor coordination scores decreased significantly. All groups treated with compound I-12 showed some recovery in this evaluation item, with the scores of mice in the medium and high dose groups showing statistically significant differences compared to the model group.

[0275] The above experimental results show that MPTP modeling leads to a decrease in the limb movement coordination ability of mice, and the intervention of compound I-12 can improve the above movement disorder behavior.

[0276] Example 36

[0277] Preparation of Compound I-12 Hydrochloride

[0278] Weigh 10g of compound I-12 into a 250mL three-necked flask, dissolve in 150mL of 95% ethanol, and stir at room temperature. Turn on the condenser and control the temperature at 0-5°C. Slowly add 18mL of hydrogen chloride-ethanol solution (2.0mol / L) dropwise to the reaction mixture. After the addition is complete, incubate at 0-5°C for 2h to allow white crystals to precipitate. Filter under reduced pressure and dry under vacuum at 50°C to obtain the hydrochloride salt of compound I-12.

[0279] Example 37

[0280] Preparation of Compound I-12 Tablets

[0281] Prescription (based on the prescription quantity of 500 tablets): 50g of pure compound I-12, 30g of sucrose, 40g of corn starch, and 1g of magnesium stearate.

[0282] Preparation: Active ingredient Compound I-12 is mixed with sucrose and corn starch, moistened with water, stirred, dried, crushed, and sieved. Magnesium stearate is added, mixed, and compressed into tablets. The average tablet weight is 242 mg per tablet, and the active ingredient content is 100 mg.

[0283] The present invention is not limited to the specific technical solutions described in the above embodiments, and all technical solutions formed by equivalent replacement are within the protection scope required by the present invention.

Claims

1. A β-carboline compound having a structure as shown in formula (I) or a pharmaceutically acceptable salt thereof: in, R 1 is halogen; X is O or NH; n is 1 or 2; Ring A is cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, oxetanyl, tetrahydrofuranyl, tetrahydrothienyl, pyrrolidinyl, tetrahydropyranyl, piperidinyl, phenyl, furanyl, thienyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, triazolyl, tetrazolyl, pyridinyl, pyrimidinyl, pyrazinyl or pyridazinyl; R 2 It is H, halogen, cyano, nitro, or C1-C3 alkyl.

2. The β-carboline compound or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: R 1 is F, Cl or Br; X is O or NH; n is 1 or 2; when ring A is phenyl, R 2 It is H, meta- or para-substituted F, Cl, Br or cyano.

3. The β-carboline compound or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: R 1 is F; X is NH; n is 1; Ring A is cyclopentyl, cyclohexyl, phenyl, triazolyl, tetrazolyl, pyridyl, pyrimidinyl or pyridazinyl; when Ring A is phenyl, R 2 It is H, meta- or para-substituted F, meta-substituted Cl or Br.

4. The β-carboline compound or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: R 1 is F; X is NH; n is 1; Ring A is cyclohexyl or phenyl; when Ring A is phenyl, R 2 It is H, F substituted at the meta position.

5. A β-carboline compound or a pharmaceutically acceptable salt thereof as shown in the following structure:

6. The β-carboline compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 5, characterized in that: The pharmaceutically acceptable salt of the β-carboline compound is hydrochloride, hydrobromide, sulfate, acetate, maleate or methanesulfonate.

7. Use of the β-carboline compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 5 in the preparation of an MAO-B inhibitor.

8. Use of the β-carboline compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 5 in the preparation of a medicament for treating and / or preventing diseases modulated by monoamine oxidase B inhibitors.

9. The use according to claim 8, characterized in that: The disease regulated by the monoamine oxidase B inhibitor is a neurodegenerative disease, and the neurodegenerative disease is Parkinson's disease and Alzheimer's disease.

10. A pharmaceutical composition, characterized in that: The pharmaceutical composition is a tablet, capsule, granule, powder, microcapsule, pill, aerosol, suspension or oral solution prepared with the β-carboline compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 5 as an active ingredient and a pharmaceutically acceptable carrier.

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