A monoamine oxidase b inhibitor and pharmaceutical uses thereof

Abstract

The application discloses a monoamine oxidase B inhibitor and a pharmaceutical application thereof. A beta-carboline compound or a pharmaceutically acceptable salt thereof is shown in formula (I): R 1 is H or halogen; R 2 is H or halogen; X is O or NH; R 3 is,,,,,,,,,, or. The application discloses a use of the beta-carboline compound or the pharmaceutically acceptable salt thereof in preparation of a high-efficiency, high-selectivity and reversible MAO-B inhibitor. The application discloses a use of the beta-carboline compound or the pharmaceutically acceptable salt thereof in preparation of a drug for treating and / or preventing a monoamine oxidase B-mediated disease.

Description

Technical Field

[0001] This invention belongs to the pharmaceutical field and relates to a monoamine oxidase B inhibitor and its use in the preparation of medicaments for treating and / or preventing monoamine oxidase B-mediated diseases. Specifically, it relates to β-carboline compounds or pharmaceutically acceptable salts thereof, and their use as monoamine oxidase B inhibitors in the preparation of medicaments for treating and / or preventing monoamine oxidase B-mediated diseases. Background Technology

[0002] Monoamine oxidases (MAOs) are a class of flavin adenine dinucleotide (FAD)-dependent enzymes widely distributed on the outer mitochondrial membrane of mammalian cells. MAOs play a crucial "gating" role in regulating the homeostasis of bioactive amines in the central nervous system (CNS) and peripheral tissues. Although MAO-mediated oxidative deamination is a normal physiological metabolic pathway for monoamine neurotransmitters, its byproducts—hydrogen peroxide (H₂O₂), ammonia (NH₃), and reactive aldehydes—have significant cytotoxic effects. Hydrogen peroxide, in particular, generates highly oxidizing hydroxyl radicals (·OH) via the Fenton reaction in the brain with the participation of iron ions, inducing mitochondrial dysfunction, lipid peroxidation, and protein denaturation. Long-term, chronic oxidative stress can lead to irreversible neuronal apoptosis. Numerous clinical and pathological studies have confirmed that abnormal upregulation of MAOs activity is highly correlated with the occurrence and development of neurodegenerative diseases such as Parkinson's disease (PD), Alzheimer's disease (AD), and amyotrophic lateral sclerosis (ALS) (Expert Opin Ther Pat. 2018, 28, 211).

[0003] The human body contains two isoenzymes encoded by different genes: MAO-A and MAO-B. MAO-A is mainly distributed in the intestines, liver, and catecholamine terminals of neurons, preferentially catalyzing serotonin (5-HT), norepinephrine (NE), and tyramine. Clinically, non-selective inhibition of MAO-A leads to the inability to timely degrade exogenous tyramine, which enters the systemic circulation in large quantities, prompting the release of NE from sympathetic nerve endings and inducing extremely dangerous paroxysmal hypertension, known as the "cheese effect." MAO-B is mainly located in glial cells in the human brain, preferentially catalyzing dopamine (DA), phenylethylamine (PEA), and benzylamine. Its expression level increases significantly with age and the progression of Parkinson's disease (PD) (up to several times that of youth). Excessive enhancement of MAO-B activity not only leads to pathological depletion of striatal dopamine levels but also becomes a major source of reactive oxygen species (ROS) in the brain. In addition, MAO-B can convert the environmental protoxin MPTP into the neurotoxin MPP. +It specifically damages dopaminergic neurons in the substantia nigra (J Parkinsons Dis. 2022, 12, 477). Therefore, developing selective MAO-B inhibitors can ensure the safety of clinical use and achieve the dual goals of "symptomatic treatment" and "neuroprotection" by increasing endogenous DA levels and reducing oxidative damage.

[0004] Currently, all clinically used MAO-B inhibitors have varying degrees of pharmacological defects. (1) Irreversible inhibitors (selegiline, rasagiline) are permanently bound to enzymes via covalent bonds. After discontinuation of these drugs, the recovery of enzyme activity depends on the biosynthesis of new enzymes, which takes a long time and leads to potential cumulative toxicity and immune antigen reactions. In addition, selegiline is metabolized in vivo into amphetamines, which have central excitatory and potential cardiovascular toxicity, and can easily cause insomnia, anxiety and hallucinations (MolPsychiatry. 2016, 21, 1499). (2) Reversible inhibitors (safenamide) avoid the risk of covalent binding, but their inhibitory effect on MAO-B is not strong. Clinically, they are only used as adjunctive therapy for levodopa, and their broad-spectrum monotherapy for PD is limited (CNS Drugs. 2017, 31, 169). Therefore, it is of great research significance to find reversible MAO-B inhibitors with novel chemical structures, strong inhibitory effects and high selectivity.

[0005] Modern pharmacological studies have found that β-carboline derivatives have a wide range of pharmacological activities, especially in neuroprotection, anti-neuroinflammatory and antidepressant effects, showing great development value (Eur J Pharmacol. 2021, 93, 173837). However, existing research on β-carboline MAO inhibitors still faces significant challenges (J Med Chem. 2021, 64, 1392). (1) Poor subtype selectivity. MAO-A and MAO-B have similar catalytic pocket structures, and most reported β-carboline derivatives exhibit dual inhibitory activity, leading to a high risk of side effects. (2) Weak inhibitory effect. The inhibitory activity of most β-carboline compounds against MAO-B is still at the micromolar or submicromolar level, which cannot meet the needs of drug development. Therefore, improving the target specificity and bioactivity of β-carboline compounds through precise structural modification remains a bottleneck in this field. Summary of the Invention

[0006] The purpose of this invention is to provide a highly efficient, selective, and reversible MAO-B inhibitor. This novel MAO-B inhibitor can be used to treat neurodegenerative diseases such as Parkinson's disease and Alzheimer's disease.

[0007] The objective of this invention is achieved through the following technical solution:

[0008] β-carboline compounds or pharmaceutically acceptable salts thereof with structures as shown in formula (I):

[0009] ;

[0010] Among them, R 1 H or halogen; R 2 H or halogen; X is O or NH; R 3 for , , , , , , , , , , , , , or .

[0011] Preferred, R 1 For F, Cl, or Br; for O or NH; for R 3 for , , , , , , , , , , , , , or .

[0012] Preferred, R 1 F is F; X is O; R is R 3 for , , , , , , , , , , , , , or .

[0013] The optimal choice, R 1 F is F; X is O; R is R3 for or .

[0014] The halogens mentioned in this invention are F, Cl, Br, and I.

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

[0016]

[0017]

[0018]

[0019]

[0020]

[0021] .

[0022] The chemical names of the above β-carboline compounds are:

[0023] (1S,4R)-bicyclo[2.2.1]hept-2-yl-6-fluoro-9H-pyrido[3,4-b]indole-1-carboxylate; (1R,4S)-bicyclo[2.2.1]hept-2-yl-6-fluoro-9H-pyrido[3,4-b]indole-1-carboxylate; (1R,4R)-bicyclo[2.2.1]hept-5-en-2-yl-6-fluoro-9H-pyrido[3,4-b]indole-1-carboxylate; (1S,4S)-bicyclo[2.2.1]hept-5-en-2-yl-6-fluoro-9H-pyrido[3,4-b]indole-1-carboxylate; (1R,4R)-1,7,7-trimethylbicyclo[2.2.1]hept-2-yl-6-fluoro-9H-pyrido[3,4-b]indole-1-carboxylate; (1R,4R)-1,7,7-trimethylbicyclo[2.2.1]hept-2-yl-6-fluoro-9H-pyrido[3,4-b]indole-1-carboxylate (1S,4S)-1,7,7-trimethylbicyclo[2.2.1]hept-2-yl-6-fluoro-9H-pyrido[3,4-b]indole-1-carboxylate; (1R,4S)-1,3,3-trimethylbicyclo[2.2.1]hept-2-yl-6-fluoro-9H-pyrido[3,4-b]indole-1-carboxylate; (1S,4R)-1,3,3-trimethylbicyclo[2.2.1]hept-2-yl-6-fluoro-9H-pyrido[3,4-b]indole-1-carboxylate; (1R,4R)-1,2,7,7-tetramethylbicyclo[2.2.1]heptane-2-yl-6-fluoro-9H-pyrido[3,4-b]indole-1-carboxylate; (1S,4S)-1,2,7,7-Tetramethylbicyclo[2.2.1]heptane-2-yl-6-fluoro-9H-pyrido[3,4-b]indole-1-carboxylate; adamantane-1-yl-6-fluoro-9H-pyrido[3,4-b]indole-1-carboxylate; adamantane-2-yl-6-fluoro-9H-pyrido[3,4-b]indole-1-carboxylate; 1-adamantylmethyl-6-fluoro-9H-pyrido[3,4-b]indole-1-carboxylate; ((1S,4R)-bicyclo[2.2.1]heptane-2-yl)methyl-6-fluoro-9H-pyrido[3,4-b]indole-1-carboxylate; ((1R,4S)-bicyclo[2.2.1]heptane-2-yl)methyl-6-fluoro-9H-pyrido[3,4-b]indole-1-carboxylate; H-pyrido[3,4-b]indole-1-carboxylic acid ester; (1S,4R)-bicyclo[2.2.1]hept-2-yl-7-fluoro-9H-pyrido[3,4-b]indole-1-carboxylic acid ester; (1R,4S)-bicyclo[2.2.1]hept-2-yl-7-fluoro-9H-pyrido[3,4-b]indole-1-carboxylic acid ester; (1R,4R)-bicyclo[2.2.1]hept-5-en-2-yl-7-fluoro-9H-pyrido[3,4-b]indole-1-carboxylic acid ester; (1S,4S)-bicyclo[2.2.1]hept-5-en-2-yl-7-fluoro-9H-pyrido[3,4-b]indole-1-carboxylic acid ester; (1R,4R)-1,7,7-trimethylbicyclo[2.2.1]1] Hept-2-yl-7-fluoro-9H-pyrido[3,4-b]indole-1-carboxylate; (1S,4S)-1,7,7-trimethylbicyclo[2.2.1]hept-2-yl-7-fluoro-9H-pyrido[3,4-b]indole-1-carboxylate; (1R,4S)-1,3,3-trimethylbicyclo[2.2.1]hept-2-yl-7-fluoro-9H-pyrido [3,4-b]indole-1-carboxylate; (1S,4R)-1,3,3-trimethylbicyclo[2.2.1]hept-2-yl-7-fluoro-9H-pyrido[3,4-b]indole-1-carboxylate; (1R,4R)-1,2,7,7-tetramethylbicyclo[2.2.1]heptane-2-yl-7-fluoro-9H-pyrido[3,4-b]indole-1-carboxylate; (1S,4S)-1,2,7,7-Tetramethylbicyclo[2.2.1]heptane-2-yl-7-fluoro-9H-pyrido[3,4-b]indole-1-carboxylate; adamantane-1-yl-7-fluoro-9H-pyrido[3,4-b]indole-1-carboxylate; adamantane-2-yl-7-fluoro-9H-pyrido[3,4-b]indole-1-carboxylate; 1-adamantyl Methyl-7-fluoro-9H-pyrido[3,4-b]indole-1-carboxylic acid ester; ((1S,4R)-bicyclo[2.2.1]hept-2-yl)methyl-7-fluoro-9H-pyrido[3,4-b]indole-1-carboxylic acid ester; ((1R,4S)-bicyclo[2.2.1]hept-2-yl)methyl-7-fluoro-9H-pyrido[3,4-b]indole-1-carboxylic acid ester.

[0024] The pharmaceutically acceptable salts of the β-carboline compounds are hydrochloride, hydrobromide, sulfate, acetate, maleate, methanesulfonate, or p-toluenesulfonate, preferably methanesulfonate.

[0025] Another object of the present invention is to provide the use of the aforementioned β-carboline compounds or pharmaceutically acceptable salts thereof in the preparation of highly effective, highly selective, and reversible MAO-B inhibitors.

[0026] Another object of the present invention is to provide the use of the aforementioned β-carboline compounds or pharmaceutically acceptable salts thereof in the preparation of medicaments for the treatment and / or prevention of monoamine oxidase B-mediated diseases.

[0027] The monoamine oxidase B-mediated diseases mentioned above are neurodegenerative diseases, including Parkinson's syndrome, Alzheimer's disease, etc.

[0028] 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 medicaments for the treatment and / or prevention of neurodegenerative diseases such as Parkinson's syndrome and Alzheimer's disease.

[0029] The aforementioned Parkinson's syndrome presents with motor and / or non-motor symptoms of Parkinson's disease; the non-motor symptoms include: depression, anxiety, pain, decreased sense of smell, sleep disturbances, and cognitive impairment.

[0030] Specifically, the Parkinson's syndrome mentioned refers to Parkinson's disease and Parkinson's disease with depression.

[0031] Another object of the present invention is to provide a pharmaceutical composition, wherein the pharmaceutical composition is prepared in the form of tablets, capsules, granules, powders, microcapsules, drops, aerosols, suspensions or oral liquids, using the β-carboline compound or a pharmaceutically acceptable salt thereof as the active ingredient and a pharmaceutically acceptable carrier. Attached Figure Description

[0032] Figure 1 The results are analyzed for the reversibility of the inhibition of recombinant human MAO-B enzyme activity by compounds I-20 and I-29 in vitro.

[0033] Figure 2 The results are from the blood-brain barrier permeability test of compound I-20.

[0034] Figure 3 The effect of compound I-20 on the total distance traveled in a zebrafish model of Parkinson's disease was investigated; compared with the model control group, *p < 0.05, ***p < 0.001.

[0035] Figure 4 The effect of compound I-20 on the movement speed of zebrafish, a model of Parkinson's disease; compared with the model control group, *p < 0.05, ***p < 0.001.

[0036] Figure 5 The effect of compound I-20 on the proportion of light field movement time in a zebrafish model of Parkinson's disease with depression was investigated; compared with the model control group, *p < 0.05, ***p < 0.001.

[0037] Figure 6 The effect of compound I-20 on body weight in a mouse model of Parkinson's disease.

[0038] Figure 7 The effect of compound I-20 on spontaneous movement in a mouse model of Parkinson's disease.

[0039] Figure 8 The effect of compound I-20 on motor function in a mouse model of Parkinson's disease was investigated; compared with the control group, *p < 0.05, **p < 0.01, ***p < 0.001.

[0040] Figure 9The effect of compound I-20 on depressive symptoms in a mouse model of depression was investigated; compared with the model group, *p < 0.05, **p < 0.01, ***p < 0.001. Detailed Implementation

[0041] To further illustrate the present invention, a series of embodiments are given below. These embodiments are intended to enable those skilled in the art to fully understand the present invention, but should not be considered as limiting the scope of the invention.

[0042] Example 1

[0043] Preparation of (1S,4R)-bicyclo[2.2.1]hept-2-yl-6-fluoro-9H-pyrido[3,4-b]indole-1-carboxylate (compound I-1)

[0044]

[0045] 1) At 0 °C, ethyl glyoxylate (13.5 g, 132 mmol) and trifluoroacetic acid (16.8 mL, 220 mmol) were added to a dichloromethane (150 mL) solution of compound 1 (19.6 g, 110 mmol). The mixture was stirred at room temperature (25 °C) for 12 h. The reaction was monitored by TLC until it was complete. The mixture was quenched with water, and the pH of the aqueous layer was adjusted to 8 with saturated NaHCO3 solution. The mixture was extracted with dichloromethane (50 mL × 3). The organic layer was washed with saturated brine, dried over anhydrous Na2SO4, filtered, and the solvent was removed by vacuum distillation to obtain 26 g of pale yellow crude compound 2.

[0046] 2) Iodophenyl diacetate (63.8 g, 198 mmol) was added to a solution of crude compound 2 (26 g) in N,N-dimethylformamide (80 mL). The mixture was heated at 100 °C for 3 hours. The reaction was monitored by TLC until it was complete. The mixture was quenched with saturated NaHCO3 solution, extracted with ethyl acetate (50 mL × 3), washed with saturated brine, dried over anhydrous Na2SO4, filtered, and the solvent was removed by vacuum distillation to obtain compound 3 (pale yellow solid, 25.0 g, 88% overall yield of the two steps). 1H NMR (400 MHz, CDCl3) δ 8.81 (d, J = 4.8 Hz, 1H), 8.35 (d, J = 4.8 Hz, 1H), 7.96 (dd, J =12.1, 2.9 Hz, 1H), 7.55 (dd, J = 7.2, 4.6 Hz, 1H), 7.32–7.25 (m, 1H), 4.37(q, J = 6.4 Hz, 3H), 1.36 (d, J = 12.8 Hz, 2H); HRMS calcd for C 14 H 12 FN2O2 [M +H] + m / z 259.08048, found 259.08053.

[0047] 3) At 0 °C, AlCl3 (13.3 g, 100 mmol) was added to a tetrahydrofuran (150 mL) solution of compound 3 (25.8 g, 100 mmol), and stirred for 10 min. Then (1S,4R)-bicyclo[2.2.1]heptan-2-ol (56.1 g, 500 mmol) was added, and the mixture was stirred at 60 °C for 12 h. The reaction was monitored by TLC until it was complete. The solvent was evaporated under reduced pressure, and the mixture was extracted with ethyl acetate (50 mL × 3). The organic layer was washed with saturated brine, dried over anhydrous Na2SO4, filtered, and the solvent was removed by vacuum distillation. A 2 M hydrochloric acid-ethyl acetate solution was added, and the mixture was cooled at 0 °C to crystallize. The crystals were filtered, washed with ethyl acetate, and dried to obtain the hydrochloride salt of the target compound I-1 (bright yellow crystals, 31.8 g, yield 88%). 1H NMR (400 MHz, CDCl3) δ 8.78 (d, J = 4.8 Hz, 1H), 8.27 (d, J = 4.8 Hz, 1H), 7.96 (dd, J = 12.1, 2.9 Hz, 1H), 7.55 (dd, J = 7.2,4.6 Hz, 1H), 7.32–7.25 (m, 1H), 5.23 (dt, J = 4.5, 3.7 Hz, 1H), 2.16 (ttd, J= 6.7, 3.3, 2.0 Hz, 1H), 2.05 (tq, J = 4.5, 2.9 Hz, 1H), 1.95 (ddd, J = 13.4,5.5, 3.8 Hz, 1H), HRMS calcdfor C 19 H 18 FN2O2 [M + H] + m / z 325.13523, found 325.13519.

[0048] Example 2

[0049] Preparation of (1R,4S)-bicyclo[2.2.1]hept-2-yl-6-fluoro-9H-pyrido[3,4-b]indole-1-carboxylate (compound I-2)

[0050]

[0051] Following the preparation method of compound I-1, (1S,4R)-bicyclo[2.2.1]hep-2-ol was replaced with an equimolar amount of (1R,4S)-bicyclo[2.2.1]hep-2-ol, and compound 3 was reacted with (1R,4S)-bicyclo[2.2.1]hep-2-ol to prepare compound I-2. 1H NMR (400 MHz, CDCl3) δ 8.80 (d, J = 4.8 Hz, 1H), 8.24 (d, J = 4.8 Hz, 1H), 7.96 (dd, J = 12.1, 2.9 Hz, 1H), 7.53 (dd, J = 7.2, 4.6 Hz, 1H), 7.32 –7.22 (m, 1H), 5.23 (dt, J = 4.5, 3.7 Hz, 1H), 2.16 (ttd, J = 6.7, 3.3, 2.0Hz, 1H), 2.05 (tq, J = 4.5, 2.9 Hz, 1H), 1.94 (ddd, J = 13.4, 5.5, 3.8 Hz,1H), 1.82 – 1.71 (m, 2H), 1.70 – 1.64 (m, 1H), 1.58 (dddt, J = 20.5, 12.4,5.8, 4.1 Hz, 2H), 1.48 – 1.40 (m, 1H), 1.40 – 1.30 (m, 1H); HRMS calcd forC 19 H 18 FN2O2 [M + H] + m / z 325.13523, found 325.13520.

[0052] Example 3

[0053] Preparation of (1R,4R)-bicyclo[2.2.1]hept-5-en-2-yl-6-fluoro-9H-pyrido[3,4-b]indole-1-carboxylate (compound I-3)

[0054]

[0055] Following the preparation method of compound I-1, (1S,4R)-bicyclo[2.2.1]hep-5-en-2-ol was replaced with an equimolar amount of (1R,4R)-bicyclo[2.2.1]hep-5-en-2-ol, and compound I-3 was prepared by reacting compound 3 with (1R,4R)-bicyclo[2.2.1]hep-5-en-2-ol. 1H NMR (400 MHz, CDCl3) δ 8.78 (d, J = 4.7 Hz, 2H), 8.27 (d, J =4.7 Hz, 2H), 7.96 (dd, J = 12.0, 2.7 Hz, 2H), 7.55 (dd, J = 7.2, 4.7 Hz, 2H),7.32 – 7.25 (m, 2H), 6.04 (dddd, J = 7.4, 3.8, 1.8, 0.9 Hz, 2H), 5.94 (ttd, J= 6.4, 1.8, 0.9 Hz, 2H), 5.18 (qd, J = 4.2, 1.8 Hz, 2H), 2.86 (tdd, J = 5.2,3.9, HRMS calcd for C 19 H 16 FN2O2 [M + H] + m / z323.11958, found 323.11960.

[0056] Example 4

[0057] Preparation of (1S,4S)-bicyclo[2.2.1]hept-5-en-2-yl-6-fluoro-9H-pyrido[3,4-b]indole-1-carboxylate (compound I-4)

[0058]

[0059] Following the preparation method of compound I-1, compound I-4 was prepared by reacting compound 3 with (1S,4S)-bicyclo[2.2.1]hep-5-en-2-ol instead of (1S,4R)-bicyclo[2.2.1]hep-2-ol. 1H NMR (400 MHz, CDCl3) δ 8.76 (d, J = 4.7 Hz, 2H), 8.24 (d, J =4.7 Hz, 2H), 7.96 (dd, J = 12.0, 2.7 Hz, 2H), 7.56 (dd, J = 7.2, 4.7 Hz, 2H),7.32 – 7.24 (m, 2H), 6.04 (dddd, J = 7.4, 3.8, 1.8, 0.9 Hz, 2H), 5.94 (ttd, J= 6.4, 1.8, 0.9 Hz, 2H), 5.19 (qd, J = 4.2, 1.8 Hz, 2H), 2.85 (tdd, J = 5.2,3.9, HRMS calcd for C 19 H 16 FN2O2 [M + H] + m / z323.11958, found 323.11961.

[0060] Example 5

[0061] Preparation of (1R,4R)-1,7,7-trimethylbicyclo[2.2.1]hept-2-yl-6-fluoro-9H-pyrido[3,4-b]indole-1-carboxylate (compound I-5)

[0062]

[0063] Following the preparation method of compound I-1, compound I-5 was prepared by reacting compound 3 with (1R,4R)-1,7,7-trimethylbicyclo[2.2.1]heptane-2-ol instead of (1S,4R)-bicyclo[2.2.1]heptane-2-ol. 1H NMR (400 MHz, CDCl3) δ 8.78 (d, J = 4.8Hz, 1H), 8.27 (d, J = 4.8 Hz, 1H), 7.96 (dd, J = 12.1, 2.9 Hz, 1H), 7.55 (dd,J = 7.2, 4.6 Hz, 1H), 7.32 – 7.25 (m, 1H), 4.75 (tq, J = 4.8, 1.6 Hz, 1H), 2.03 (dt, J = 13.8, 5.0 Hz, 1H), 1.89 – 1.65 (m, 4H), 1.36 – 1.23 (m, 2H), 0.98 (d, J = 1.4 Hz, 3H), 0.91 (d, J = 4.0 Hz, 6H); HRMS calcd for C 22 H 24 FN2O2[M + H] + m / z 367.18218, found 367.18224.

[0064] Example 6

[0065] Preparation of (1S,4S)-1,7,7-trimethylbicyclo[2.2.1]hept-2-yl-6-fluoro-9H-pyrido[3,4-b]indole-1-carboxylate (compound I-6)

[0066]

[0067] Following the preparation method of compound I-1, compound I-6 was prepared by reacting compound 3 with (1S,4S)-1,7,7-trimethylbicyclo[2.2.1]heptane-2-ol instead of (1S,4R)-bicyclo[2.2.1]heptane-2-ol. 1H NMR (400 MHz, CDCl3) δ 8.78 (d, J = 4.8Hz, 1H), 8.27 (d, J = 4.8 Hz, 1H), 7.96 (dd, J = 12.1, 2.9 Hz, 1H), 7.55 (dd,J = 7.2, 4.6 Hz, 1H), 7.32 – 7.25 (m, 1H), 4.75 (tq, J = 4.8, 1.6 Hz, 1H), 2.03 (dt, J = 13.8, 5.0 Hz, 1H), 1.89 – 1.65 (m, 4H), 1.36 – 1.23 (m, 2H), 0.98 (d, J = 1.4 Hz, 3H), 0.91 (d, J = 2.9 Hz, 6H); HRMS calcd for C 22 H 24 FN2O2[M + H] + m / z 367.18218, found 367.18223.

[0068] Example 7

[0069] Preparation of (1R,4S)-1,3,3-trimethylbicyclo[2.2.1]hept-2-yl-6-fluoro-9H-pyrido[3,4-b]indole-1-carboxylate (compound I-7)

[0070]

[0071] Following the preparation method of compound I-1, compound I-7 was prepared by replacing (1S,4R)-bicyclo[2.2.1]heptan-2-ol with an equimolar amount of (1R,4S)-1,3,3-trimethylbicyclo[2.2.1]heptan-2-ol. 1H NMR (400 MHz, CDCl3) δ 8.78 (d, J = 4.8Hz, 1H), 8.27 (d, J = 4.8 Hz, 1H), 7.96 (dd, J = 12.1, 2.9 Hz, 1H), 7.55 (dd,J = 7.2, 4.6 Hz, 1H), 7.32 – 7.25 (m, 1H), 4.55 (dh, J = 2.9, 1.5 Hz, 1H), 1.82 (ddtq, J = 5.6, 4.0, 2.8, 1.4 Hz, 1H), 1.78 – 1.64 (m, 3H), 1.52 – 1.39(m, 3H), 1.04 – 0.94 (m, 8H); HRMS calcd for C 22 H 24 FN2O2 [M + H] + m / z367.18218, found 367.18222.

[0072] Example 8

[0073] Preparation of (1S,4R)-1,3,3-trimethylbicyclo[2.2.1]hept-2-yl-6-fluoro-9H-pyrido[3,4-b]indole-1-carboxylate (compound I-8)

[0074]

[0075] Following the preparation method of compound I-1, compound I-8 was prepared by reacting compound 3 with (1S,4R)-1,3,3-trimethylbicyclo[2.2.1]heptane-2-ol instead of (1S,4R)-bicyclo[2.2.1]heptane-2-ol. 1H NMR (400 MHz, CDCl3) δ 8.79 (d, J = 4.8Hz, 1H), 8.27 (d, J = 4.8 Hz, 1H), 7.94 (dd, J = 12.1, 2.9 Hz, 1H), 7.54 (dd,J = 7.2, 4.6 Hz, 1H), 7.32 – 7.24 (m, 1H), 4.55 (dh, J = 2.9, 1.5 Hz, 1H), 1.82 (ddtq, J = 5.6, 4.0, 2.8, 1.4 Hz, 1H), 1.80 – 1.64 (m, 3H), 1.53 – 1.39(m, 3H), 1.04 – 0.96 (m, 8H); HRMS calcd for C 22 H 24 FN2O2 [M + H] + m / z367.18218, found 367.18222.

[0076] Example 9

[0077] Preparation of (1R,4R)-1,2,7,7-tetramethylbicyclo[2.2.1]hept-2-yl-6-fluoro-9H-pyrido[3,4-b]indole-1-carboxylate (compound I-9)

[0078]

[0079] Following the preparation method of compound I-1, compound I-9 was prepared by replacing (1S,4R)-bicyclo[2.2.1]heptan-2-ol with an equimolar amount of (1R,4R)-1,2,7,7-tetramethylbicyclo[2.2.1]heptan-2-ol with (1R,4R)-1,2,7,7-tetramethylbicyclo[2.2.1]heptan-2-ol. 1H NMR (400 MHz, CDCl3) δ 8.78 (d, J =4.8 Hz, 1H), 8.27 (d, J = 4.8 Hz, 1H), 7.96 (dd, J = 12.1, 2.9 Hz, 1H), 7.55(dd, J = 7.2, 4.6 Hz, 1H), 7.32 – 7.25 (m, 1H), 2.18 (dd, J = 13.7, 4.2 Hz,1H), 1.96 (dd, J = 13.7, 6.1 Hz, 1H), 1.85 – 1.76 (m, 1H), 1.76 – 1.65 (m,2H), 1.47 – 1.35 (m, 2H), 1.33 (s, 2H), 1.07 (s, 2H), 1.00 (s, 2H), 0.96 (s,2H); HRMS calcd for C 23 H 26 FN2O2 [M + H] + m / z 381.19783, found 381.19792.

[0080] Example 10

[0081] Preparation of (1S,4S)-1,2,7,7-tetramethylbicyclo[2.2.1]hept-2-yl-6-fluoro-9H-pyrido[3,4-b]indole-1-carboxylate (compound I-10)

[0082]

[0083] Following the preparation method of compound I-1, compound I-10 was prepared by replacing (1S,4R)-bicyclo[2.2.1]heptan-2-ol with an equimolar amount of (1S,4S)-1,2,7,7-tetramethylbicyclo[2.2.1]heptan-2-ol with (1S,4S)-1,2,7,7-tetramethylbicyclo[2.2.1]heptan-2-ol. 1H NMR (400 MHz, CDCl3) δ 8.78 (d, J= 4.8 Hz, 1H), 8.27 (d, J = 4.8 Hz, 1H), 7.96 (dd, J = 12.1, 2.9 Hz, 1H), 7.55 (dd, J = 7.2, 4.6 Hz, 1H), 7.32 – 7.25 (m, 1H), 2.22 – 2.14 (m, 1H), 2.00 – 1.92 (m, 1H), 1.85 – 1.76 (m, 1H), 1.76 – 1.67 (m, 2H), 1.47 – 1.35(m, 2H), 1.07 (s, 2H), 1.00 (s, 2H), 0.96 (s, 2H); HRMS calcd for C 23 H 26 FN2O2[M + H] + m / z 381.19783, found 381.19790.

[0084] Example 11

[0085] Preparation of adamantane-1-yl-6-fluoro-9H-pyrido[3,4-b]indole-1-carboxylate (compound I-11)

[0086]

[0087] Following the preparation method of compound I-1, (1S,4R)-bicyclo[2.2.1]heptan-2-ol was replaced with an equal amount of 1-adamantanol, and compound 3 was reacted with 1-adamantanol to prepare compound I-11. 1 H NMR (400 MHz, CDCl3) δ8.78 (d, J = 4.8 Hz, 1H), 8.27 (d, J = 4.8 Hz, 1H), 7.96 (dd, J = 12.1, 2.9Hz, 1H), 7.55 (dd, J = 7.2, 4.6 Hz, 1H), 7.32 – 7.25 (m, 1H), 2.28 (s, 2H), 2.20 (qd, J = 5.5, 4.7 Hz, 3H), 1.76 – 1.63 (m, 6H); HRMS calcd for C 22 H 22 FN2O2[M + H] + m / z 365.16653, found 365.16650.

[0088] Example 12

[0089] Preparation of adamantane-2-yl-6-fluoro-9H-pyrido[3,4-b]indole-1-carboxylate (compound I-12)

[0090]

[0091] Following the preparation method of compound I-1, (1S,4R)-bicyclo[2.2.1]heptan-2-ol was replaced with an equal amount of 2-adamantanol, and compound 3 was reacted with 2-adamantanol to prepare compound I-12. 1 H NMR (400 MHz, CDCl3) δ8.78 (d, J = 4.8 Hz, 1H), 8.27 (d, J = 4.8 Hz, 1H), 7.96 (dd, J = 12.1, 2.9Hz, 1H), 7.55 (dd, J = 7.2, 4.6 Hz, 1H), 7.32 – 7.25 (m, 1H), 4.94 (t, J =5.8 Hz, 1H), 1.90 – 1.81 (m, 7H), 1.77 – 1.70 (m, 2H), 1.65 – 1.54 (m, 6H);HRMS calcd for C 22 H 22 FN2O2 [M + H] + m / z 365.16653, found 365.16649.

[0092] Example 13

[0093] Preparation of 1-adamantylmethyl-6-fluoro-9H-pyrido[3,4-b]indole-1-carboxylic acid ester (compound I-13)

[0094]

[0095] Following the preparation method of compound I-1, (1S,4R)-bicyclo[2.2.1]hep-2-ol was replaced with an equal amount of 1-adamantyl methanol, and compound 3 was reacted with 1-adamantyl methanol to prepare compound I-13. 1H NMR (400 MHz, CDCl3) δ 8.78 (d, J = 4.8 Hz, 1H), 8.27 (d, J = 4.8 Hz, 1H), 7.96 (dd, J =12.1, 2.9 Hz, 1H), 7.55 (dd, J = 7.2, 4.6 Hz, 1H), 7.32 – 7.25 (m, 1H), 4.28 (s, 2H), 2.03 (dqd, J = 10.6, 5.7, 4.9 Hz, 3H), 1.72 (dt, J = 13.0, 5.7 Hz, 3H), 1.57 (s, 3H), 1.55 (dt, J = 13.0, 5.7 Hz, 3H);HRMS calcd for C 23 H 24 FN2O2[M + H] + m / z 379.18218, found 379.18214.

[0096] Example 14

[0097] Preparation of ((1S,4R)-bicyclo[2.2.1]hept-2-yl)methyl-6-fluoro-9H-pyrido[3,4-b]indole-1-carboxylate (compound I-14)

[0098]

[0099] Following the preparation method of compound I-1, compound I-14 was prepared by reacting compound 3 with ((1S,4R)-bicyclo[2.2.1]hep-2-yl)methanol in an equal amount to replace (1S,4R)-bicyclo[2.2.1]hep-2-ol. 1H NMR (400 MHz, CDCl3) δ 8.78 (d, J = 4.7 Hz, 1H), 8.27 (d,J = 4.7 Hz, 1H), 7.96 (dd, J = 12.0, 2.7 Hz, 1H), 7.55 (dd, J = 7.2, 4.7 Hz,1H), 7.32 – 7.25 (m, 1H), 4.35 (dd, J = 11.3, 3.8 Hz, 1H), 4.13 (dd, J =11.3, 3.8 Hz, 1H), 2.10 – 1.97 (m, 2H), 1.85 (ddt, J = 6.1, 4.4, 2.2 Hz, 1H),1.68 (dt, J = 12.8, 5.1 Hz, 1H), 1.59 (ddd, J = 16.2, 14.9, 7.9, 4.3 Hz, 3H),1.44 – 1.29 (m, 5H);HRMS calcd for C 20 H 20 FN2O2 [M + H] + m / z 339.15088, found339.15083.

[0100] Example 15

[0101] Preparation of ((1R,4S)-bicyclo[2.2.1]hept-2-yl)methyl-6-fluoro-9H-pyrido[3,4-b]indole-1-carboxylate (compound I-15)

[0102]

[0103] Following the preparation method of compound I-1, compound I-15 was prepared by reacting compound 3 with ((1R,4S)-bicyclo[2.2.1]hep-2-yl)methanol in an equal amount to replace (1S,4R)-bicyclo[2.2.1]hep-2-ol. 1H NMR (400 MHz, CDCl3) δ 8.79 (d, J = 4.7 Hz, 1H), 8.27 (d,J = 4.7 Hz, 1H), 7.95 (dd, J = 12.0, 2.7 Hz, 1H), 7.55 (dd, J = 7.2, 4.7 Hz,1H), 7.34 – 7.25 (m, 1H), 4.35 (dd, J = 11.3, 3.8 Hz, 1H), 4.12 (dd, J =11.3, 3.8 Hz, 1H), 2.11 – 1.96 (m, 2H), 1.85 (ddt, J = 6.1, 4.4, 2.2 Hz, 1H),1.68 (dt, J = 12.8, 5.1 Hz, 1H), 1.60 (ddd, J = 16.2, 14.9, 7.9, 4.3 Hz, 3H),1.44 – 1.30 (m, 5H);HRMS calcd for C 20 H 20 FN2O2 [M + H] + m / z 339.15088, found339.15084.

[0104] Example 16

[0105] Preparation of (1S,4R)-bicyclo[2.2.1]hept-2-yl-7-fluoro-9H-pyrido[3,4-b]indole-1-carboxylate (compound I-16)

[0106]

[0107] Following the preparation method of compound I-1, compound 6 was prepared using compound 4 as the starting material. Compound 6 was then reacted with (1S,4R)-bicyclo[2.2.1]hepta-2-ol to prepare compound I-16. 1H NMR (400 MHz, CDCl3) δ 8.79 (d, J= 4.8 Hz, 1H), 8.31 (d, J = 4.8 Hz, 1H), 8.12 (dd, J = 8.2, 4.9 Hz, 1H), 7.22(dd, J = 12.1, 2.1 Hz, 1H), 7.11 – 7.04 (m, 1H), 5.23 (dt, J = 4.5, 3.7 Hz, 1H), 2.16 (dtq, J = 5.4, 3.8, 2.7 Hz, 1H), 2.05 (tq, J = 4.5, 2.9 Hz, 1H), 1.95 (ddd, J = 13.4, 5.5, 3.8 Hz, 1H), 1.82 – 1.70 (m, 2H), 1.70 – 1.63 (m,1H), 1.58 (dddt, J = 20.5, 12.4, 5.8, 4.1 Hz, 2H), 1.48 – 1.40 (m, 1H), 1.40– 1.32 (m, 1H);HRMS calcd for C 19 H 18 FN2O2 [M + H] + m / z 325.13523, found325.13521.

[0108] Example 17

[0109] Preparation of (1R,4S)-bicyclo[2.2.1]hept-2-yl-7-fluoro-9H-pyrido[3,4-b]indole-1-carboxylate (compound I-17)

[0110]

[0111] Following the preparation method of compound I-16, compound I-17 was prepared by reacting compound 6 with (1R,4S)-bicyclo[2.2.1]hep-2-ol in an equal amount. 1H NMR (400 MHz, CDCl3) δ 8.78 (d, J = 4.8 Hz, 1H), 8.30 (d, J = 4.8 Hz, 1H), 8.13 (dd, J = 8.2, 4.9 Hz, 1H), 7.22 (dd, J = 12.1, 2.1 Hz, 1H), 7.10 –7.02 (m, 1H), 5.24 (dt, J = 4.5, 3.7 Hz, 1H), 2.16 (dtq, J = 5.4, 3.8, 2.7Hz, 1H), 2.06 (tq, J = 4.5, 2.9 Hz, 1H), 1.95 (ddd, J = 13.4, 5.5, 3.8 Hz,1H), 1.82 – 1.71 (m, 2H), 1.70 – 1.62 (m, 1H), 1.58 (dddt, J = 20.5, 12.4,5.8, 4.1 Hz, 2H), 1.48 – 1.39 (m, 1H), 1.40 – 1.31 (m, 1H); HRMS calcd forC 19 H 18 FN2O2 [M + H] + m / z 325.13523, found 325.13520.

[0112] Example 18

[0113] Preparation of (1R,4R)-bicyclo[2.2.1]hept-5-en-2-yl-7-fluoro-9H-pyrido[3,4-b]indole-1-carboxylate (compound I-18)

[0114]

[0115] Following the preparation method of compound I-16, compound I-18 was prepared by replacing (1S,4R)-bicyclo[2.2.1]hep-5-en-2-ol with an equimolar amount of (1R,4R)-bicyclo[2.2.1]hep-5-en-2-ol with compound 6. 1H NMR (400 MHz, CDCl3) δ 8.79 (d, J = 4.7 Hz, 2H), 8.31 (d, J= 4.8 Hz, 2H), 8.12 (dd, J = 8.2, 4.9 Hz, 2H), 7.22 (dd, J = 12.1, 2.2 Hz, 2H), 7.11 – 7.04 (m, 2H), 6.04 (ddd, J = 7.5, 3.8, 1.8, 0.9 Hz, 2H), 5.94 (td, J = 6.5, 1.7, 0.9 Hz, 2H), 5.18 (qd, J = 4.2, 1.8 Hz, 2H), 2.86 (dd, J =6.7, 5.2, HRMS calcd for C 19 H 16 FN2O2 [M +H] + m / z 323.11958, found 323.11954.

[0116] Example 19

[0117] Preparation of (1S,4S)-bicyclo[2.2.1]hept-5-en-2-yl-7-fluoro-9H-pyrido[3,4-b]indole-1-carboxylate (compound I-19)

[0118]

[0119] Following the preparation method of compound I-16, compound I-19 was prepared by reacting compound 6 with (1S,4S)-bicyclo[2.2.1]hep-5-en-2-ol instead of (1S,4R)-bicyclo[2.2.1]hep-2-ol. 1H NMR (400 MHz, CDCl3) δ 8.78 (d, J = 4.7 Hz, 2H), 8.32 (d, J= 4.8 Hz, 2H), 8.13 (dd, J = 8.2, 4.9 Hz, 2H), 7.23 (dd, J = 12.1, 2.2 Hz, 2H), 7.11 – 7.04 (m, 2H), 6.04 (ddd, J = 7.5, 3.8, 1.8, 0.9 Hz, 2H), 5.94 (td, J = 6.5, 1.7, 0.9 Hz, 2H), 5.17 (qd, J = 4.2, 1.8 Hz, 2H), 2.86 (dd, J =6.7, 5.2, HRMS calcd for C 19 H 16 FN2O2 [M +H] + m / z 323.11958, found 323.11961.

[0120] Example 20

[0121] Preparation of (1R,4R)-1,7,7-trimethylbicyclo[2.2.1]hept-2-yl-7-fluoro-9H-pyrido[3,4-b]indole-1-carboxylate (compound I-20)

[0122]

[0123] Following the preparation method of compound I-16, compound I-20 was prepared by reacting compound 6 with (1R,4R)-1,7,7-trimethylbicyclo[2.2.1]heptane-2-ol instead of (1S,4R)-bicyclo[2.2.1]heptane-2-ol. 1H NMR (400 MHz, CDCl3) δ 8.79 (d, J =4.8 Hz, 1H), 8.31 (d, J = 4.8 Hz, 1H), 8.12 (dd, J = 8.2, 4.9 Hz, 1H), 7.25 –7.19 (m, 1H), 7.08 (ddd, J = 10.4, 8.1, 2.1 Hz, 1H), 4.75 (tq, J = 4.8, 1.6Hz, 1H), 2.03 (dt, J = 13.8, 5.0 Hz, 1H), 1.89 – 1.65 (m, 4H), 1.36 – 1.23(m, 2H), 0.98 (d, J = 1.4 Hz, 3H), 0.91 (d, J = 4.0 Hz, 6H);HRMS calcd forC 22 H 24 FN2O2 [M + H] + m / z 367.18218, found 367.18228.

[0124] Example 21

[0125] Preparation of (1S,4S)-1,7,7-trimethylbicyclo[2.2.1]hept-2-yl-7-fluoro-9H-pyrido[3,4-b]indole-1-carboxylate (compound I-21)

[0126]

[0127] Following the preparation method of compound I-16, compound I-21 was prepared by replacing (1S,4R)-bicyclo[2.2.1]heptan-2-ol with an equimolar amount of (1S,4S)-1,7,7-trimethylbicyclo[2.2.1]heptan-2-ol. 1H NMR (400 MHz, CDCl3) δ 8.79 (d, J =4.8 Hz, 1H), 8.31 (d, J = 4.8 Hz, 1H), 8.12 (dd, J = 8.2, 4.9 Hz, 1H), 7.25 –7.19 (m, 1H), 7.08 (ddd, J = 10.4, 8.1, 2.1 Hz, 1H), 4.75 (tq, J = 4.8, 1.6Hz, 1H), 2.03 (dt, J = 13.8, 5.0 Hz, 1H), 1.89 – 1.65 (m, 4H), 1.36 – 1.23(m, 2H), 0.98 (d, J = 1.4 Hz, 3H), 0.91 (d, J = 2.9 Hz, 6H);HRMS calcd forC 22 H 24 FN2O2 [M + H] + m / z 367.18218, found 367.18226.

[0128] Example 22

[0129] Preparation of (1R,4S)-1,3,3-trimethylbicyclo[2.2.1]hept-2-yl-7-fluoro-9H-pyrido[3,4-b]indole-1-carboxylate (compound I-22)

[0130]

[0131] Following the preparation method of compound I-16, compound I-22 was prepared by replacing (1S,4R)-bicyclo[2.2.1]heptan-2-ol with an equimolar amount of (1R,4S)-1,3,3-trimethylbicyclo[2.2.1]heptan-2-ol. 1H NMR (400 MHz, CDCl3) δ 8.79 (d, J =4.8 Hz, 1H), 8.31 (d, J = 4.8 Hz, 1H), 8.12 (dd, J = 8.2, 4.9 Hz, 1H), 7.25 –7.19 (m, 1H), 7.08 (ddd, J = 10.4, 8.2, 2.1 Hz, 1H), 4.55 (dh, J = 2.9, 1.5Hz, 1H), 1.82 (ddtq, J = 5.6, 4.0, 2.8, 1.4 Hz, 1H), 1.78 – 1.64 (m, 3H),1.52 – 1.39 (m, 3H), 1.04 – 0.94 (m, 8H);HRMS calcd for C 22 H 24 FN2O2 [M + H] + m / z 367.18218, found 367.18221.

[0132] Example 23

[0133] Preparation of (1S,4R)-1,3,3-trimethylbicyclo[2.2.1]hept-2-yl-7-fluoro-9H-pyrido[3,4-b]indole-1-carboxylate (compound I-23)

[0134]

[0135] Following the preparation method of compound I-16, compound I-23 was prepared by replacing (1S,4R)-bicyclo[2.2.1]heptan-2-ol with an equimolar amount of (1S,4R)-1,3,3-trimethylbicyclo[2.2.1]heptan-2-ol. 1H NMR (400 MHz, CDCl3) δ 8.80 (d, J =4.8 Hz, 1H), 8.30 (d, J = 4.8 Hz, 1H), 8.12 (dd, J = 8.2, 4.9 Hz, 1H), 7.26 –7.19 (m, 1H), 7.08 (ddd, J = 10.4, 8.2, 2.1 Hz, 1H), 4.55 (dh, J = 2.9, 1.5Hz, 1H), 1.82 (ddtq, J = 5.6, 4.0, 2.8, 1.4 Hz, 1H), 1.78 – 1.64 (m, 3H),1.52 – 1.40 (m, 3H), 1.04 – 0.95 (m, 8H);HRMS calcd for C 22 H 24 FN2O2 [M + H] + m / z 367.18218, found 367.18214.

[0136] Example 24

[0137] Preparation of (1R,4R)-1,2,7,7-tetramethylbicyclo[2.2.1]hept-2-yl-7-fluoro-9H-pyrido[3,4-b]indole-1-carboxylate (compound I-24)

[0138]

[0139] Following the preparation method of compound I-16, compound I-24 was prepared by replacing (1S,4R)-bicyclo[2.2.1]heptan-2-ol with an equimolar amount of (1R,4R)-1,2,7,7-tetramethylbicyclo[2.2.1]heptan-2-ol. 1H NMR (400 MHz, CDCl3) δ 8.79 (d, J= 4.8 Hz, 1H), 8.31 (d, J = 4.8 Hz, 1H), 8.12 (dd, J = 8.2, 4.9 Hz, 1H), 7.25– 7.19 (m, 1H), 7.08 (ddd, J = 10.4, 8.2, 2.1 Hz, 1H), 2.18 (dd, J = 13.7,4.2 Hz, 1H), 1.96 (dd, J = 13.7, 6.1 Hz, 1H), 1.85 – 1.76 (m, 1H), 1.76 –1.65 (m, 2H), 1.47 – 1.35 (m, 2H), 1.33 (s, 2H), 1.07 (s, 2H), 1.00 (s, 2H),0.96 (s, 2H);HRMS calcd for C 23 H 26 FN2O2 [M + H] + m / z 381.19783, found381.19787.

[0140] Example 25

[0141] Preparation of (1S,4S)-1,2,7,7-tetramethylbicyclo[2.2.1]hept-2-yl-7-fluoro-9H-pyrido[3,4-b]indole-1-carboxylate (compound I-25)

[0142]

[0143] Following the preparation method of compound I-16, compound I-25 was prepared by replacing (1S,4R)-bicyclo[2.2.1]heptan-2-ol with an equimolar amount of (1S,4S)-1,2,7,7-tetramethylbicyclo[2.2.1]heptan-2-ol. 1H NMR (400 MHz, CDCl3) δ 8.79 (d, J= 4.8 Hz, 1H), 8.31 (d, J = 4.8 Hz, 1H), 8.12 (dd, J = 8.2, 4.9 Hz, 1H), 7.22(dd, J = 12.1, 2.2 Hz, 1H), 7.12 – 7.04 (m, 1H), 2.22 – 2.14 (m, 1H), 2.00 –1.92 (m, 1H), 1.85 – 1.76 (m, 1H), 1.76 – 1.67 (m, 2H), 1.47 – 1.35 (m, 2H),1.33 (s, 2H), 1.07 (s, 2H), 1.00 (s, 2H), 0.96 (s, 2H);HRMS calcd forC 23 H 26 FN2O2 [M + H] + m / z 381.19783, found 381.19778.

[0144] Example 26

[0145] Preparation of adamantane-1-yl-7-fluoro-9H-pyrido[3,4-b]indole-1-carboxylate (compound I-26)

[0146]

[0147] Following the preparation method of compound I-16, (1S,4R)-bicyclo[2.2.1]hepta-2-ol was replaced with an equimolar amount of 1-adamantanol, and compound I-26 was prepared by reacting compound 6 with 1-adamantanol. 1 H NMR (400 MHz, CDCl3) δ8.79 (d, J = 4.8 Hz, 1H), 8.31 (d, J = 4.8 Hz, 1H), 8.12 (dd, J = 8.2, 4.9Hz, 1H), 7.25 – 7.19 (m, 1H), 7.08 (ddd, J = 10.4, 8.2, 2.1 Hz, 1H), 2.28 (s,2H), 2.20 (qd, J = 5.5, 4.7 Hz, 3H), 1.76 – 1.63 (m, 6H);HRMS calcd forC 22 H 22 FN2O2 [M + H] +m / z 365.16653, found 365.16648.

[0148] Example 27

[0149] Preparation of adamantane-2-yl-7-fluoro-9H-pyrido[3,4-b]indole-1-carboxylate (compound I-27)

[0150]

[0151] Following the preparation method of compound I-16, (1S,4R)-bicyclo[2.2.1]heptan-2-ol was replaced with an equimolar amount of 2-adamantanol, and compound I-27 was prepared by reacting compound 6 with 2-adamantanol. 1 H NMR (400 MHz, CDCl3) δ8.79 (d, J = 4.8 Hz, 1H), 8.31 (d, J = 4.8 Hz, 1H), 8.12 (dd, J = 8.2, 4.9Hz, 1H), 7.25 – 7.19 (m, 1H), 7.08 (ddd, J = HRMS calcd for C 22 H 22 FN2O2 [M + H] + m / z 365.16653, found365.16649.

[0152] Example 28

[0153] Preparation of 1-adamantylmethyl-7-fluoro-9H-pyrido[3,4-b]indole-1-carboxylic acid ester (compound I-28)

[0154]

[0155] Following the preparation method of compound I-16, (1S,4R)-bicyclo[2.2.1]hepta-2-ol was replaced with an equal amount of 1-adamantyl methanol, and compound I-28 was prepared by reacting compound 6 with 1-adamantyl methanol. 1H NMR (400 MHz, CDCl3) δ 8.79 (d, J = 4.8 Hz, 1H), 8.31 (d, J = 4.8 Hz, 1H), 8.12 (dd, J =8.2, 4.9 Hz, 1H), 7.25 – 7.19 (m, 1H), 7.08 (ddd, J = 10.4, 8.1, 2.1 Hz, 1H), 4.28 (s, 2H), 2.03 (dqd, J = 10.6, 5.7, 4.9 Hz, 3H), 1.72 (dt, J = 13.0, 5.7Hz, 3H), 1.57 (s, 3H), 1.55 (dt, J = 13.0, 5.7 Hz, 3H);HRMS calcd forC 23 H 24 FN2O2 [M + H] + m / z 379.18218, found 379.18212.

[0156] Example 29

[0157] Preparation of ((1S,4R)-bicyclo[2.2.1]hept-2-yl)methyl-7-fluoro-9H-pyrido[3,4-b]indole-1-carboxylate (compound I-29)

[0158]

[0159] Following the preparation method of compound I-16, compound I-29 was prepared by reacting compound 6 with ((1S,4R)-bicyclo[2.2.1]hep-2-yl)methanol in an equal amount to replace (1S,4R)-bicyclo[2.2.1]hep-2-ol. 1H NMR (400 MHz, CDCl3) δ 8.79 (d, J=4.7 Hz, 1H), 8.31 (d, J=4.8 Hz, 1H), 8.12 (dd, J=8.2, 4.9 Hz, 1H), 7.22 (dd, J=12.1, 2.2 Hz, 1H), 7.11–7.04 (m, 1H), 4.35 (dd, J = 11.3, 3.8 Hz, 1H), 4.13 (dd, J = 11.3, 3.8Hz, 1H), 2.10–1.97 (m, 2H), 1.85 (ddt, J = 6.1, 4.4, 2.2 Hz, 1H), 1.68 (dt, J= 12.8, 5.1 Hz, 1H), 1.59 (dddd, J = 16.2, 14.9, 7.9, 4.3 Hz, 3H), 1.44–1.29(m, 5H);HRMS calcd for C 20 H 20 FN2O2 [M + H] + m / z 339.15088, found 339.15085.

[0160] Example 30

[0161] Preparation of ((1R,4S)-bicyclo[2.2.1]hept-2-yl)methyl-7-fluoro-9H-pyrido[3,4-b]indole-1-carboxylate (compound I-30)

[0162]

[0163] Following the preparation method of compound I-16, compound I-30 was prepared by reacting compound 6 with ((1R,4S)-bicyclo[2.2.1]hep-2-yl)methanol in an equal amount to replace (1S,4R)-bicyclo[2.2.1]hep-2-ol. 1H NMR (400 MHz, CDCl3) δ 8.81 (d, J=4.7 Hz, 1H), 8.31 (d, J=4.8 Hz, 1H), 8.14 (dd, J=8.2, 4.9 Hz, 1H), 7.21 (dd, J=12.1, 2.2 Hz, 1H),7.11–7.04 (m, 1H), 4.35 (dd, J=11.3, 3.8 Hz, 1H), 4.16 (dd, J=11.3, 3.8 Hz,1H), 2.12–1.98 (m, 2H), 1.85 (ddt, J=6.1, 4.4, 2.2 Hz, 1H), 1.68 (dt, J=12.8,5.1 Hz, 1H), 1.59 (dddd, J=16.2, 14.9, 7.9, 4.3 Hz, 3H), 1.44–1.27 (m, 5H);HRMS calcd for C 20 H 20 FN2O2 [M + H] + m / z 339.15088, found 339.15082.

[0164] Example 31

[0165] In vitro assay and analysis of recombinant human MAOs enzyme inhibitory activity

[0166] (1) Experimental Principle

[0167]

[0168] MAOs enzymes in the human body can catalyze the oxidative deamination of monoamines. Kyrenuramine is a non-specific substrate of MAOs enzymes and can be catalyzed by MAOs to convert into 4-hydroxyquinoline. The inventors established an in vitro method for detecting MAOs enzyme inhibitory activity, using fluorescence spectrophotometry to detect the content of 4-hydroxyquinoline to determine the degree of inhibition of MAOs enzyme by the test compound.

[0169] (2) Experimental materials and main instruments

[0170] Experimental materials: recombinant human MAO-A and MAO-B proteins, kynurenamine dihydrobromide (molecular weight 326.03), 4-hydroxyquinoline (molecular weight 145.16), safinamide, tripotassium phosphate, DMSO, 2N hydrochloric acid, ultrapure water, PCR tubes and cryovials.

[0171] Main instruments: Bio-Tek microplate reader, centrifuge, constant temperature shaking incubator, vortex mixer, pH meter.

[0172] (3) Experimental steps

[0173] The reaction volume per well was 200 μL: 180 μL enzyme working solution + 10 μL inhibitor + 10 μL kynurenamine stock solution (50 μM). Finally, 80 μL NaOH solution (2N) was added to terminate the reaction.

[0174] Preparation of buffer solution (100 mM): Take a 200 mL cryovial, add 160 mL of ultrapure water and tripotassium phosphate (4.246 g) sequentially, adjust the pH of the system to 7.4 with 2N hydrochloric acid, and then bring the volume to 200 mL with ultrapure water. The concentration of tripotassium phosphate in this buffer solution is 100 mM. Unless otherwise specified, use this buffer solution to prepare all solutions.

[0175] 1) Preparation of the experimental stock solution:

[0176] Preparation of kynurenamine stock solution: Weigh 3.26 mg of kynurenamine dihydrobromide and dissolve it in 10 mL of buffer solution to obtain a 1 mM kynurenamine dihydrobromide stock solution.

[0177] Preparation of inhibitor stock solution: Safinamide (positive control) and test compounds (compounds I-1 to I-30, Harmane, Norharmane) were prepared into a 1 mM inhibitor stock solution using DMSO. The inhibitor stock solution was then diluted 2 to 3 times by volume with DMSO, resulting in 8 concentrations for each inhibitor (Safinamide, test compound).

[0178] Preparation of 4-hydroxyquinoline solution: Weigh 1.4516 mg of 4-hydroxyquinoline and dissolve it in 10 mL of buffer to obtain a 1 mM 4-hydroxyquinoline solution; dilute with buffer to 100 μM, and then use 100 μM as the starting concentration to dilute by volume in 2-fold increments to obtain 12 concentrations.

[0179] 2) Preparation of enzyme working solution:

[0180] Preparation of MAO-A working solution: 12.6 mL buffer solution + 21 μL MAO-A stock solution.

[0181] Preparation of MAO-B working solution: 12.6 mL buffer solution + 42 μL MAO-B stock solution.

[0182] 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.

[0183] 3) Determination of the standard curve:

[0184] The fluorescence of the 4-hydroxyquinoline solution prepared above was detected under the conditions of excitation wavelength of 310 nm and emission wavelength of 400 nm. A standard linear curve was plotted with the concentration of 4-hydroxyquinoline as the x-axis and the fluorescence intensity as the y-axis.

[0185] 4) Enzymatic reactions:

[0186] One blank control group: containing neither enzyme nor inhibitor; the inhibitor solution was replaced with a 4% DMSO buffer solution; all other conditions were replicated. One negative control group: containing no inhibitor; the inhibitor solution was replaced with a 4% DMSO buffer solution; all other conditions were replicated. One positive control group and 30 test compound groups were included. Except for heating and incubation, all operations were performed at 0 °C. Each group was repeated in duplicate.

[0187] Positive control group or test compound group: Add 180 μL of enzyme working solution to each PCR tube, add 10 μL of positive control drug (Safinamide) or test compound (compound I-1 to compound I-30, Harmane, Norharmane) stock solution, vortex to mix; add 10 μL of kynurenamine stock solution, vortex to mix, transfer to 37 ℃ and incubate for 0.5 h. After returning to room temperature (25 ℃), add 80 μL of sodium hydroxide solution (2N, i.e., 2 g NaOH dissolved in 25 mL buffer, freshly prepared) to each PCR tube, centrifuge at 9000 r / min for 3 min to remove precipitate, transfer 200 μL of supernatant from each tube to a 96-well plate, and detect 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).

[0188] MAOs enzyme inhibition rate (%) = [(Negative group intensity - Test group intensity) / (Negative group intensity - Blank group intensity)] × 100

[0189] At this concentration, if the MAOs enzyme inhibition rate of the test compound is >70%, the test compound is diluted to obtain 8 concentrations according to the "Preparation of Inhibitor Stock Solution". The inhibition rate of MAOs enzyme at different concentrations is detected, and the dose-response curve is fitted using GraphPad software to calculate its half-maximal inhibitory concentration (IC50). 50 Value; repeated at least twice at different time points.

[0190] (4) Experimental Results

[0191] As shown in Table 1, most of the compounds in this invention exhibit high selective inhibition of MAO-B, while showing weak inhibitory activity against MAO-A. Among them, compounds I-5, I-11, I-13, I-14, I-15, I-20, I-26, I-28, I-29, and I-30 demonstrate superior in vitro inhibitory activity compared to the third-generation MAO-B inhibitor safinamide (IC50). 50 = 0.074 ± 0.0054 μM). In particular, the in vitro MAO-B inhibitory activity IC50 of compounds I-20 and I-29 was 0.074 ± 0.0054 μM. 50 The activity reached 10 nM, which is more than 20 times that of safinamide, and the selectivity ratio for MAO-B / MAO-A was as high as 10,000 or more. These results indicate that compounds I-20 and I-29 of this invention exhibit significantly better in vitro MAO-B inhibitory activity and selectivity than safinamide.

[0192] Table 1. Test data on in vitro recombinant human MAOs enzyme inhibitory activity

[0193]

[0194] Example 32

[0195] Analysis of the reversibility of in vitro inhibition of recombinant human MAO-B enzyme activity by compounds I-20 and I-29

[0196] The reversibility of MAO-B activity inhibition of compounds I-20 and I-29 in vitro was analyzed by dilution method to evaluate the type of MAO-B inhibition by the compounds of the present invention.

[0197] The preparation of the enzyme working solution, the test compound, and the kynurenamine stock solution followed the procedures in Example 31. Four test groups, one blank group, and one negative group were set up. Test groups: rasagiline, safinamide, compounds I-20, and I-29, respectively; other conditions were paralleled. Blank group: Contains neither enzyme nor inhibitor; the inhibitor solution was replaced with a 4% DMSO buffer solution; other conditions were paralleled. Negative group: Contains no inhibitor; the inhibitor solution was replaced with a 4% DMSO buffer solution; other conditions were paralleled. Except for heating and incubation, all other operations were performed at 0°C.

[0198] The experiment was performed in PCR tubes (1 mL). 900 μL of enzyme working solution was added to the PCR tube, followed by 50 μL of the stock solution of the test compound (rasagiline, safenamide, compound I-20, or compound I-29). The mixture was vortexed and mixed. The blank and negative groups were also treated simultaneously. The mixture was then transferred to 37 °C and incubated for 15 min. After returning to room temperature (25 °C), the incubated mixture was taken and diluted with kynurenamine stock solution to 1 μM, 0.1 μM, and 0.01 μM, respectively, to a final volume of 400 μL. The mixture was then transferred to 37 °C and incubated for 15 min. After restoring to room temperature (25 °C), add 80 μL of sodium hydroxide solution (2N, i.e., 2g NaOH dissolved in 25 mL buffer, freshly prepared); centrifuge at 9000 r / min for 3 min to remove the precipitate, transfer 200 μL of the supernatant to a 96-well plate, and detect 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.

[0199] MAO-B enzyme inhibition rate (%) = [(negative group intensity - test group intensity) / (negative group intensity - blank group intensity)] × 100.

[0200] Experimental results are as follows Figure 1 As shown, compounds I-20 and I-29 exhibit reversible inhibition of MAO-B, and their pharmacodynamic behavior is similar to that of safinamide, a third-generation selective and reversible MAO-B inhibitor. This indicates that all compounds of this invention are reversible MAO-B inhibitors.

[0201] Example 33

[0202] Pharmacokinetics of Compound I-20

[0203] Three male C57BL / 6J mice (n=3 for each administration route) were administered compound I-20 (20 mg / kg, solvent: DMSO, Solutol (polyethylene glycol-15-hydroxystearate), and Saline in a volume ratio of 5:10:85) via 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 at 0.25, 0.5, 1, 2, 4, 6, 8, and 24 h after oral administration. Blood samples were placed in centrifuge tubes containing K2-EDTA, stored on ice, and centrifuged at 6800 rpm for 6 min at 2–8 °C. The resulting supernatant plasma was temporarily stored at approximately -80 °C until LC-MS analysis. Relevant pharmacokinetic properties were calculated using Phoenix WinNonlin 7.0.

[0204] Bioavailability (F) (%) = (AUC po / AUC iv) × (dosei.v. / dose po) × 100.

[0205] The experimental results are shown in Table 2. Compound I-20 has good pharmacokinetic parameters, including an oral half-life T0. 1 / 2 = 4.91 h, oral bioavailability F (%) = 74.5%.

[0206] Table 2. Pharmacokinetic parameters of compound I-20 (mean ± SD)

[0207]

[0208] Example 34

[0209] Blood-brain barrier permeability test of compound I-20

[0210] Nine male C57BL / 6J mice were orally administered compound I-20 once (dosage: 20 mg / kg, solvent: DMSO, Solutol and Saline in a volume ratio of 5:10:85). At 0.5 h, 1 h, and 2 h after administration, 3 mice were randomly selected at each time point to collect blood samples and brain tissues. Blood was collected via the submandibular vein or other appropriate methods, and approximately 30 μL of blood was collected per sample per time point. The blood samples were anticoagulated with K2-EDTA and placed on ice. After the animals were euthanized with CO2, the brain tissues were then collected. After collecting the blood samples, plasma was separated by centrifugation within 2 h (centrifugation conditions: 6800 rpm for 6 min at 2-8 °C). The collected plasma samples and brain tissues were stored at -80 ℃ in a freezer until LC-MS analysis.

[0211] The experimental results are as Figure 2 shown. Compound I-20 can effectively cross the blood-brain barrier of mice. The drug content ratio in brain / plasma (Brain / Plasma Ratio) is stable and is gradually metabolized and cleared over time.

[0212] Example 35

[0213] Pharmacodynamic evaluation of compound I-20 on improving the motor ability of zebrafish with Parkinson's disease model

[0214] (1) Experimental materials and main instruments

[0215] 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine hydrochloride (MPTP), DMSO, safinamide, nomifensine, dissecting microscope (SZX7), CCD camera (VertA1), precision electronic balance (CP214), 6-well plates, 96-well plates, behavior analyzer (Zebra Lab 3.11).

[0216] (2) Experimental animals

[0217] Wild-type AB strain zebrafish (Hangzhou Huante Biotechnology Co., Ltd.), 4 days post-fertilization (4 dpf) in age. The use license number is: SYXK(Zhe)2022-0004, and the feeding management meets the requirements of international AAALAC accreditation (accreditation number: 001458). The zebrafish were all raised in fish-raising water at 28 °C (water quality: 200 mg of instant sea salt was added to every 1 L of reverse osmosis water, the conductivity was 450-550 μS / cm, the pH was 6.5-8.5, and the hardness was 50-100 mg / L CaCO3).

[0218] (3) Sample preparation

[0219] Preparation of safenamide stock solution: Safenamide is prepared into a stock solution with a concentration of 2.50 mg / mL using DMSO; the safenamide stock solution is diluted with DMSO as needed and stored at -20℃.

[0220] Preparation of stock solution of compound I-20: Compound I-20 was prepared into a stock solution with a concentration of 2.50 mg / mL using DMSO; the stock solution of compound I-20 was diluted with DMSO as needed and stored at -20℃.

[0221] Preparation of positive control stock solution: Nomiphene neomaleate or safenamide were prepared into positive control stock solutions with a concentration of 50.0 mg / mL using DMSO and stored at -20℃.

[0222] (4) Detection method

[0223] 1) Determination of the maximum detectable concentration (MTC) for preventing Parkinson's disease

[0224] An experimental group, a normal control group, and a model control group were set up, with 30 zebrafish treated in each group. Wild-type AB strain zebrafish (4 dpf) were randomly selected and placed in 6-well plates (3 mL per well). The normal control group zebrafish were placed in fish tank water without MPTP and compound I-20. The experimental group zebrafish were simultaneously treated with water-soluble MPTP (MPTP concentration 25.0 μg / mL) and water-soluble compound I-20 (concentration shown in Table 3). The model control group zebrafish were treated with water-soluble MPTP (MPTP concentration 25.0 μg / mL) to establish a zebrafish Parkinson's disease model. Both the experimental and model control groups were treated at 28℃ for 48 h, and the MTC of the samples was measured.

[0225] Table 3. Concentration Exploration Experiment for Efficacy Evaluation of Compound I-20 in Preventing Parkinson's Disease (n = 30)

[0226]

[0227] The experimental results are shown in Table 3, which shows that the MTC value of compound I-20 in zebrafish, a model of Parkinson's disease, is 1.56 μg / mL.

[0228] 2) Evaluation of efficacy in preventing Parkinson's disease

[0229] Wild-type AB strain zebrafish (4 dpf) were randomly selected and placed in 6-well plates, with a volume of 3 mL per well, and 30 zebrafish were treated per well. They were divided into a normal control group, an experimental group, a positive control group (nomifencin and safenamide), and a model control group. The normal control group zebrafish were placed in fish tank water without MPTP and compound I-20. The model control group zebrafish were given water-soluble MPTP (MPTP concentration of 25.0 μg / mL). The experimental and positive control groups were given water-soluble MPTP (compound I-20 concentration shown in Table 4), and MPTP (final concentration 25.0 μg / mL, prepared in ultrapure water) concurrently with the drug administration. After treatment at 28℃ for 48 h, 10 zebrafish from each group were randomly selected and placed in 96-well plates, one zebrafish per well, using fish tank water without drugs, with a volume of 200 μL per well. The total movement distance and movement speed of the zebrafish over 1 h were measured using a behavioral analyzer. The statistical analysis results of these indicators were 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 was considered statistically significant.

[0230] Table 4. Efficacy evaluation of compound I-20 in preventing Parkinson's disease (n = 10)

[0231]

[0232] Note: Compared with the model control group, *p < 0.05, ***p < 0.001.

[0233] The efficacy of compound I-20 and safinamide in preventing Parkinson's disease in a zebrafish model is shown in Table 4. Figure 3 and Figure 4 Experimental results showed that compound I-20 significantly improved the total movement distance and speed of zebrafish, exhibiting excellent dose-dependent effects. Notably, compound I-20 at a very low concentration of 0.391 μg / mL demonstrated superior preventive efficacy against Parkinson's disease compared to safinamide at a concentration of 3.12 μg / mL. In contrast, safinamide at a lower concentration of 1.56 μg / mL failed to show statistically significant preventive efficacy against Parkinson's disease. These experimental results fully demonstrate that compound I-20 possesses extremely strong preventive efficacy against Parkinson's disease in a zebrafish model of Parkinson's disease, with significantly better efficacy than the currently available third-generation MAO-B inhibitor safinamide, indicating excellent potential for drug development.

[0234] Example 36

[0235] Pharmacodynamic evaluation of compound I-20 in improving depressive symptoms in zebrafish model of Parkinson's disease with depression

[0236] Experimental animals: Zebrafish were all raised in fish culture water at 28 °C (Water quality: 200 mg of instant sea salt was added to every 1 L of reverse osmosis water, with a conductivity of 450 - 550 μS / cm; pH of 6.5 - 8.5; hardness of 50 - 100 mg / L, calculated as CaCO3). The license number for the use of experimental animals is: SYXK (Zhe) 2022 - 0004. The feeding management conforms to the requirements of international AAALAC accreditation (Accreditation number: 001458), and the IACUC ethical review number is: IACUC - 2024 - 9085 - 01.

[0237] Instruments, consumables and reagents: Dissecting microscope (OLYMPUS, model: SZX7), CCD camera (Shanghai Tusen Vision Technology Co., Ltd., model: VertA1), precision electronic balance (OHAUS, model: CP214); 6 - well plates (Zhejiang Beilanbo Biotechnology Co., Ltd.), 24 - well plates (Nest Biotech), 96 - well plates (Nest Biotech), behavior analyzer (ViewPoint, model Zebra Lab 3.11), multi - functional microplate reader (TECAN, model: SPARK); LPS (Sigma, batch number: 1001164401), 1 - methyl - 4 - phenyl - 1,2,3,6 - tetrahydropyridine hydrochloride (MPTP; Shanghai Aladdin Biochemical Technology Co., Ltd., batch number: J2210583), dimethyl sulfoxide (DMSO; Sigma), Zebrafish 5 - HT Elisa Kit (Shanghai Enzyme - linked Biotechnology Co., Ltd., batch number: 202403), Zebrafish DA Elisa Kit (Shanghai Enzyme - linked Biotechnology Co., Ltd., batch number: 202403).

[0238] Sample preparation information: Compound I - 20, with water as the solvent. The positive control drug is nomifensine maleate (hereinafter referred to as nomifensine; Sigma, batch number: 029M4053V), with water as the solvent.

[0239] Detection Method: Wild-type AB strain zebrafish (4 dpf) were randomly selected and placed in 6-well plates, with 30 zebrafish treated in each well (experimental group). The samples (compound I-20 concentrations are shown in Table 3) were administered in water-soluble form. The positive control, nomiphene, was administered at a concentration of 1.50 μg / mL. A normal control group and a model control group were also included, with a volume of 3 mL per well. Except for the normal control group, both the experimental and model control groups received LPS (50.0 ng / zebrafish) injected into the yolk sac and MPTP (25 μM) in water-soluble form to establish a zebrafish model of Parkinson's disease with depression. After treatment at 28℃ for 48 h, 10 zebrafish from each group were randomly selected and placed in 24-well plates, one zebrafish per well, with a volume of 1 mL per well. The proportion of light field movement time in the zebrafish was measured using a behavioral analyzer to evaluate the efficacy of the samples in improving depressive symptoms in the zebrafish model of Parkinson's disease with depression. Statistical results are expressed as mean ± SE. Statistical analysis was performed using SPSS 26.0 software. A p < 0.05 indicated that the difference was statistically significant.

[0240] The results are detailed in Table 5 and... Figure 5 Compound I-20 can improve depressive symptoms in a zebrafish model of Parkinson's disease with depression, specifically by increasing the proportion of light field movement time in zebrafish.

[0241] Table 5. Pharmacological evaluation results of compound I-20 in improving depressive symptoms (n = 10)

[0242]

[0243] Note: Compared with the model control group, *p < 0.05, ***p < 0.001.

[0244] Example 37

[0245] Pharmacodynamic evaluation of compound I-20 in improving motor function in a mouse model of Parkinson's disease

[0246] Experimental protocol

[0247] Eighty C57BL / 6J mice were used. Rotand testing (10 r / min, 5 min, for 2 consecutive days) was employed to screen out mice with poor motor function. The remaining 60 mice with good motor function were randomly divided into 6 groups: normal control group, model group, safenamide group, low-dose compound I-20 group, medium-dose compound I-20 group, high-dose compound I-20 group, and the safenamide group, low-dose compound I-20 group, medium-dose compound I-20 group, and high-dose compound I-20 group were administered the following medications daily by gavage: safenamide 2.5 mg / kg / day, prepared with a blank solvent; compound I-20 1.25 mg / kg / day, 2.5 mg / kg / day, and 5 mg / kg / day, respectively, prepared with a blank solvent; the normal control group and model group were administered the same volume of blank solvent daily by gavage. The blank solvent contained 0.1... Three days after pre-administration (using %DMSO in PBS), mice in the model group, safenamide group, and three dosage groups of compound I-20 were intraperitoneally injected with MPTP (dissolved in physiological saline, 20 mg / kg / time, 4 times a day, 2 h apart). After that, the administration was stopped, and behavioral changes in the mice were detected 7 days later.

[0248] (1) Autonomous activity counting experiment: The mice were placed in an autonomous activity box (40×40×40 cm). The mice adapted to the autonomous activity box for 5 minutes (open field experiment). Then, the crawling trajectory and movement speed of the mice were observed and recorded within 10 minutes to evaluate their autonomous activity ability.

[0249] (2) Rotary bar test: The mice were placed on a rotary bar at a speed of 40 r / min. The time from when the mouse started rotating to when it left the rotary bar was taken as the mouse's rotary bar latency. The test time was 5 min, repeated 3 times with an interval of 1 hour. The mice were trained for 2 consecutive days before the test. During the training, the speed was 10 r / min and the training time was 5 min, once a day.

[0250] (3) Suspension test: Two metal rods were selected and fixed upright, connected by a 1 mm diameter metal wire, kept horizontal and 30 cm above the ground; the mouse was held by its tail and placed head down and tail up on the metal wire; training was conducted for 3 days, once a day, and the test was performed on the 4th day, for a total of 3 tests. The scoring criteria are as follows: 4 points for the mouse grasping the metal wire with both hind paws, 3 points for the mouse grasping the metal wire with only one hind paw, 2 points for the mouse grasping the metal wire with both front paws, 1 point for the mouse grasping the metal wire with one front paw, and 0 points for the mouse falling.

[0251] (4) Pole Climbing Experiment: A cork ball with a diameter of 25 cm was fixed to the top of a wooden pole 50 cm long and 1 cm thick. Gauze was wrapped around the pole to prevent slipping. The test mice were then placed on the ball, and their performance was observed and scored. The scoring criteria were as follows: 5 points for using all four limbs and sliding down step by step in a coordinated manner; 4 points for using all four limbs and crawling down step by step, but with some hind limb slipping; 3 points for sliding down after climbing half the distance but being able to hold onto the pole; 2 points for sliding before climbing half the distance; 1 point for falling after climbing half the distance but being unable to hold onto the pole; and 0 points for falling before climbing half the distance. Each mouse was tested twice, and the average value was taken.

[0252] Experimental results

[0253] (1) Weight change trend

[0254] 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 dosage groups of compound I-20 was lower than that of mice in the normal control group; however, the body weight loss of mice in the three dosage groups of compound I-20 was not significantly different from that in the model group.

[0255] (2) General condition observation

[0256] Minutes after MPTP injection, a series of acute behavioral changes were observed in mice, including tremors, reduced movement, spinal rigidity, and drooling. These symptoms disappeared within 0.5 to 1 hour, with no significant differences between groups.

[0257] (3) Assessment of independent activity ability

[0258] The open field test was used to evaluate the spontaneous activity ability of mice. Changes in the mice's spontaneous activity and exploration abilities were assessed using three parameters: total distance traveled within a fixed time period, distance in the center as a percentage of the total distance traveled, and time in the center as a percentage of the total time spent in the center. Figure 7 As shown in the figure. The results indicate that the spontaneous activity and exploration abilities of mice in the MPTP-induced acute model were not significantly altered. Compound I-20 also did not have a significant effect on the spontaneous activity and exploration abilities of the model mice.

[0259] (4) Evaluation of motor ability based on the rotator bar test

[0260] The rotundus test was used to evaluate the motor abilities of mice. Changes in motor ability were assessed by measuring the time it took for mice to fall from a rotundus rotating at a certain speed. Results are as follows: Figure 8As shown, MPTP-induced acute model mice exhibited significantly weaker balance maintenance on a rotarod at a higher rotation speed (40 r / min) compared to the normal control group, with a significantly shorter maintenance time on the rotarod. The aforementioned motor dysfunction behaviors were effectively improved in mice at all doses of compound I-20, showing statistically significant differences compared to the model group.

[0261] (5) Evaluation of motor ability based on suspension test

[0262] The motor abilities of mice were evaluated using a suspension test. Changes in the mice's ability to use their hind limbs to grasp a balance bar during upper limb suspension were used to assess changes in their motor abilities. Results are as follows: Figure 8 As shown, in the MPTP-induced acute model, mice exhibited a significant decrease in their ability to stabilize their bodies and grasp metal wires while suspended, manifesting as an inability to grasp the wires even with only one hind paw, or even difficulty grasping the wires with just one hind paw, resulting in a significant drop in scores. Mice in each dose group of compound I-20 showed significant recovery in this evaluation item, achieving statistical differences compared to the model group.

[0263] (6) Evaluation of athletic ability based on pole climbing experiment

[0264] The motor skills of mice were evaluated using a pole-climbing test. Changes in the mice's motor skills were assessed by observing their climbing motion downwards from a height on a wooden pole covered with gauze for slip resistance. Results are as follows: Figure 8 As shown, in the MPTP-induced acute model, mice experienced hind paw slippage during downward crawling, accompanied by sliding behavior, and a significant decrease in motor coordination scores. Mice in each dose group of compound I-20 showed good recovery in this evaluation item, with statistically significant differences compared to the model group.

[0265] The above experimental results indicate that MPTP modeling leads to a decrease in limb motor coordination in mice, and intervention with compound I-20 can improve the aforementioned motor dysfunction.

[0266] Example 38

[0267] Pharmacodynamic evaluation of compound I-20 in improving depressive symptoms in a mouse model of depression

[0268] Experimental protocol

[0269] Forty C57BL / 6J mice were housed with free access to water and food at 22±2℃ under a 12 / 12-hour reverse light-dark cycle. Rotand testing (10 r / min, 5 min, repeated for 2 days) was used to screen out mice with poor motor function. The remaining 30 mice with good motor function were randomly divided into three groups: a control group, a model group (LPS), and a compound I-20 treatment group (LPS+I-20). LPS (2 mg / kg, Sigma, St. Louis, MO, USA) was stereotactically injected into the substantia nigra region of the midbrain of mice in the model group and the compound I-20 treatment group to simulate microglial cell activation and neuronal damage, thereby inducing a Parkinson's disease model with depression. Compound I-20 was dissolved in a mixture of DMSO (1%), Tween 20 (2%), and physiological saline (97%) and administered intraperitoneally at a dose of 2.5 mg / kg / day once daily for 2 days, starting before or on the same day as LPS injection. Following LPS injection, compound I-20 was administered according to the same protocol until the experimental results were obtained. Behavioral changes in mice were assessed 3 days later.

[0270] (1) Tail suspension test: Mice were suspended by their tails at a height of 50 cm above the table for 10 minutes. Behavior of each mouse was recorded. During the test, observers unaware of the treatment group recorded behavior as stillness or struggling. Stillness time was calculated during the 10-minute test.

[0271] (2) Forced swimming test: Mice were placed individually in a glass cylinder (20 cm high, 15 cm in diameter) at a depth of 12 cm and a water temperature of 25°C. The total resting time during the 5-minute test was recorded according to the method described by Porsolt et al. (1977). The resting state of each mouse was determined by Smart 3.0 software (Panlab, Spain).

[0272] Experimental results

[0273] (1) Evaluation of the ability of the tail suspension test to improve depressive symptoms

[0274] The ability of compound I-20 to improve depressive symptoms in mice was evaluated using a tail suspension test, and the improvement in depressive symptoms was assessed by changes in the resting time of the mice. Results are as follows: Figure 9 As shown, the resting time in LPS-induced acute depression-like model mice was significantly longer than that in the normal control group. Compound I-20 effectively improved the aforementioned acute depression-like behaviors, showing a statistically significant difference compared to the model group.

[0275] (2) Evaluation of the ability to improve depressive symptoms based on the forced swimming test

[0276] The ability of compound I-20 to improve depressive symptoms in mice was evaluated using a forced swimming test, and the improvement in depressive symptoms was assessed by changes in the mice's resting time. Results are as follows: Figure 9 As shown, the resting time in LPS-induced acute depression-like model mice was significantly longer than that in the normal control group. Compound I-20 effectively improved the aforementioned acute depression-like behaviors, showing a statistically significant difference compared to the model group.

[0277] Example 39

[0278] Preparation of compound I-20 methanesulfonate

[0279] Weigh 10 g of compound I-20 and place it in a 250 mL three-necked flask. Dissolve it in 150 mL of ethyl acetate and stir at room temperature. Turn on the condenser and control the temperature at 0–5 °C. Weigh an equivalent amount of methanesulfonic acid and add it to the reaction solution. Keep the temperature at 0–5 °C for 2 h, and white crystals will gradually precipitate. Filter under reduced pressure and dry under vacuum at 50 °C to obtain the methanesulfonate of compound I-20.

[0280] Example 40

[0281] Preparation of Compound I-20 Tablets

[0282] Prescription (based on a prescription of 500 tablets): 50 g of pure compound I-20, 30 g of sucrose, 40 g of corn starch, and 1 g of magnesium stearate.

[0283] Preparation method: The active ingredient compound I-20 is mixed with sucrose and corn starch, moistened with water, stirred evenly, dried, pulverized and sieved, magnesium stearate is added, mixed evenly, and compressed into tablets. The average tablet weight is 242 mg / tablet, and the active ingredient content is 100 mg.

[0284] This invention is not limited to the specific technical solutions described in the above embodiments. All technical solutions formed by equivalent substitutions are within the scope of protection claimed by this invention.

Claims

1. β-carboline compounds with structures as shown in formula (I) or pharmaceutically acceptable salts thereof: ； in, R 1 H or halogen; R 2 H or halogen; X is O or NH; R 3 for , , , , , , , , , , , , , or .

2. The β-carboline compound or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: R 1 For H; R 2 For F, Cl, or Br; for X, O or NH; for R 3 for , , , , , , , , , , , , , or ; R 1 For F, Cl, or Br; R 2 H is H; X is O or NH; R is O; 3 for , , , , , , , , , , , , , or .

3. The β-carboline compound or a pharmaceutically acceptable salt thereof according to claim 2, characterized in that: R 1 For H; R 2 F is F; X is O; R is R 3 for , , , , , , , , , , , , , or ; R 1 For F; R 2 H is H; X is O; R is O. 3 for , , , , , , , , , , , , , or .

4. β-carboline compounds or their pharmaceutically acceptable salts, as shown in the following formula: ； ； ； ； ； ； ； 。 5. The β-carboline compound or a pharmaceutically acceptable salt thereof according to any one of claims 1-4, characterized in that: The pharmaceutically acceptable salts of the β-carboline compounds are hydrochloride, hydrobromide, sulfate, acetate, maleate, methanesulfonate, or p-toluenesulfonate.

6. Use of the β-carboline compound of any one of claims 1-4 or a pharmaceutically acceptable salt thereof in the preparation of a monoamine oxidase B inhibitor.

7. Use of the β-carboline compound of any one of claims 1-4 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating and / or preventing monoamine oxidase B-mediated diseases.

8. The use according to claim 7, characterized in that: The monoamine oxidase B-mediated diseases mentioned above are neurodegenerative diseases, including Parkinson's syndrome and Alzheimer's disease.

9. The use according to claim 8, characterized in that: The aforementioned Parkinson's syndrome presents with motor and / or non-motor symptoms of Parkinson's disease; the non-motor symptoms include: depression, anxiety, pain, decreased sense of smell, sleep disturbances, and cognitive impairment.

10. A pharmaceutical composition, characterized in that: The pharmaceutical composition described herein is prepared as tablets, capsules, granules, powders, microcapsules, drops, aerosols, suspensions, or oral liquids using a β-carboline compound or a pharmaceutically acceptable salt as the active ingredient and a pharmaceutically acceptable carrier.

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