CN111689901A

CN111689901A - Compound with TDO and IDO1 dual inhibitory activity and application thereof in preparing medicament for treating neurodegenerative diseases

Info

Publication number: CN111689901A
Application number: CN201910190615.8A
Authority: CN
Inventors: 钱珊; 曹治兴; 徐世军; 李玉芝; 王周玉; 李国菠; 杨羚羚
Original assignee: Xihua University; Chengdu University of Traditional Chinese Medicine
Current assignee: Xihua University; Chengdu University of Traditional Chinese Medicine
Priority date: 2019-03-13
Filing date: 2019-03-13
Publication date: 2020-09-22

Abstract

The invention provides a compound shown as a formula I, or a pharmaceutically acceptable salt thereof, or a solvate thereof, which can selectively inhibit TDO and IDO1 and has a remarkable inhibitory effect on TDO and/or IDO 1. In addition, the compound prepared by the invention has obvious anti-tumor effect, has certain treatment effect on Parkinson's disease and Alzheimer's disease, and has good application prospect in the field of medicine preparation.

Description

Compound with TDO and IDO1 dual inhibitory activity and application thereof in preparing medicament for treating neurodegenerative diseases

Technical Field

The invention belongs to the field of chemical medicines, and particularly relates to an indazole derivative with double inhibitory activities of TDO and IDO1 and application thereof in preparing medicines for treating neurodegenerative diseases.

Background

Indoleamine 2,3-dioxygenase 1 (indolamine 2,3-dioxygenase 1, IDO1) and tryptophan 2,3-dioxygenase (tryptophan 2,3-dioxygenase, TDO) are rate-limiting enzymes that catalyze the epoxidation of indole in Indoleamine molecules such as tryptophan and enable the indole to be catabolized according to the canine uric acid pathway.

TDO and IDO1 play important roles in tumor immune-. Normally, TDO and IDO1 are expressed at low level in vivo, and most tumor cells form high-expression TDO and IDO1, L-tryptophan is converted into N-formyl kynurenine, the tryptophan concentration in the cell microenvironment is reduced, the tryptophan-dependent T cell synthesis is stopped at the G1 stage, and the T cell proliferation is inhibited, so that the killing effect of the immune system of the body on tumor tissues is inhibited. Meanwhile, the tryptophan metabolite under the action of TDO and IDO1 has cytotoxicity, and can directly dissolve T cells.

Therefore, inhibiting the activity of TDO and/or IDO1 can effectively prevent the degradation of tryptophan around tumor cells and promote the proliferation of T cells, thereby enhancing the body's ability to attack tumor cells. In addition, TDO and/or IDO1 inhibitors may also be used in combination with chemotherapeutic agents to reduce drug resistance in tumor cells, thereby enhancing the anti-tumor activity of conventional cytotoxic therapies. Co-administration of TDO and/or IDO1 inhibitors may also improve the therapeutic efficacy of therapeutic vaccines for cancer patients.

In addition, studies have shown that neurodegenerative diseases are closely related to Tryptophan (TRP) and its active metabolites. More than 95% of TRP is degraded by kynurenine pathway as essential amino acid for human body, and metabolites 3-hydroxykynurenine (3-HK), quinolinic acid (QUIN), etc. have neurotoxic effect, and can remarkably promote the development of neurodegenerative diseases. TDO and IDO1 catalyze the initiation and speed limiting steps of this pathway. Research shows that in patients or animal models with Alzheimer's Disease (AD) and Parkinson's Disease (PD), the expression of TDO and IDO1 in related nerve regions is increased, so that neurotoxicity 3-HK and QUIN catalyzed by the TDO and the IDO1 are remarkably increased, and in contrast, the kynurenic acid (KYNA) level with neuroprotection is reduced, and finally the neurodegenerative progress is promoted. Recent studies have shown that TDO and IDO1 are effective targets for the treatment of PD and AD.

Besides, TDO and IDO1 mediated TRP metabolism is also closely related to brain glioma, depression, schizophrenia, nervous system inflammation, diseases related to cellular immune activation, and the like.

Therefore, the compound capable of effectively inhibiting TDO and IDO1 is researched, and the compound has a wide clinical application prospect.

Disclosure of Invention

It is an object of the present invention to provide a compound which is effective in inhibiting TDO and/or IDO 1.

The invention provides a compound shown as a formula I, or a pharmaceutically acceptable salt or solvate thereof:

wherein, A ring is selected from

Wherein: r_a、R_b、R_cEach independently selected from H, C1-4 alkyl, -L₁-R_x，L₁Selected from C ═ O, 1-4 methylene, R_xSelected from substituted or unsubstituted saturated carbocycle or heterocycle, substituted or unsubstituted unsaturated carbocycle or heterocycle, the substituent is selected from hydroxyl, nitro, carboxyl, halogen, halogenated or unhalogenated C1-4 alkyl, C1-4 alkoxy; y is selected from O or S;

R₆、R₁、R₃、R₄each independently selected from H, halogen, halo C1-4 alkyl, halo C1-4 alkoxy, cyano, amino, -COOR_f、

-NH₂Wherein: r_fSelected from C1-4 alkyl; x is selected from NH and O; e is selected from C ═ O, 0-4 methylene groups; r2 is selected from substituted or unsubstituted saturated carbocyclic or heterocyclic ring, substituted or unsubstituted unsaturated carbocyclic or heterocyclic ring, substituted or unsubstituted fused ring,

The substituent on R2 is selected from hydroxyl, nitryl, carboxyl, halogen, halogenated or non-halogenated C1-4 alkyl, C1-4 alkoxy,

Wherein R is_dSelected from H, C1-4 alkoxy, R_eSelected from C1-4 alkyl;

and when ring A is

R₃、R₄Is H, R₁When it is bromine, R₆Is not:

when ring A is

R₃、R₄Is H, R₁When it is methyl, R₆Is not:

when ring A is

R₁、R₃、R₄When is H, R₆Is not:

further, the air conditioner is provided with a fan,

in a compound of formula I:

a ring is selected from

Wherein: r_a、R_b、R_cEach independently selected from H, C1-4 alkyl, -L₁-R_x，L₁Selected from C ═ O, 1-2 methylene, R_xSelected from substituted or unsubstituted 4-6 membered saturated carbocyclic or heterocyclic ring, substituted or unsubstituted 4-6 membered unsaturated carbocyclic or heterocyclic ring, said substituents being selected from hydroxy, nitro, carboxy, halo, halogenated or non-halogenated C1-4 alkyl, C1-4 alkoxy; y is selected from O or S;

R₆is selected from

-NH₂Wherein: x is selected from NH and O; e is selected from C ═ O, 0-2 methylene groups; r₂Selected from substituted or unsubstituted 4-6 membered saturated carbocyclic or heterocyclic ring, substituted or unsubstituted 4-6 membered unsaturated carbocyclic or heterocyclic ring, substituted or unsubstituted fused ring,

Wherein R is_dSelected from H, C1-4 alkoxy, R_eSelected from C1-4 alkyl;

R₁、R₃、R₄each independently selected from H, halogen, halo C1-4 alkyl, halo C1-4 alkoxy, cyano, amino, -COOR_fWherein R is_fSelected from C1-4 alkyl.

Further, the air conditioner is provided with a fan,

in a compound of formula I:

a ring is selected from

Wherein R is_aSelected from H, C1-3 alkyl, -L₁-R_x，L₁Selected from C ═ O, 1-2 methylene, R_xSelected from substituted or unsubstituted 6-membered saturated carbocycle, substituted or unsubstituted 6-membered saturated nitrogen ring, substituted or unsubstituted benzene ring, the substituent is selected from hydroxyl, nitro; rb and Rc are H;

R₆is selected from

-NH₂Wherein X is selected from NH and O; e is selected from C ═ O, 0-2 methylene groups; r₂Selected from substituted or unsubstituted 4-6 membered saturated nitrogen heterocycle, 4-6 membered unsaturated nitrogen heterocycle, substituted or unsubstituted 4-6 membered saturated carbon heterocycle, substituted or unsubstituted benzene ring, andindole group,

The R is₂The substituent on the (B) is selected from hydroxyl, nitryl, carboxyl, halogen, halogenated or non-halogenated C1-3 alkyl, C1-3 alkoxy,

Wherein R is_dSelected from H, C1-3 alkoxy, R_eSelected from C1-3 alkyl;

R₁、R₃、R₄each independently selected from H, halogen, halogenated C1-3 alkyl, cyano, amino, -COOR_fWherein R is_fSelected from C1-3 alkyl.

Further, the air conditioner is provided with a fan,

the structure of the compound is shown as a formula I-1:

wherein E is selected from 0-2 methylene groups;

R₁selected from H, halogen, halogenated C1 alkyl, cyano, amino, -COOR_fWherein R is_fIs selected from methyl; r₄、R₃

Each independently selected from H, halogen;

R₂selected from substituted or unsubstituted 4-6 membered saturated nitrogen heterocycle, 6 membered unsaturated nitrogen heterocycle, substituted or unsubstituted 6 membered saturated carbon heterocycle, substituted or unsubstituted benzene ring, substituted or unsubstituted indolyl,

The substituent on R2 is selected from hydroxyl, nitryl, carboxyl, halogen, halogenated or non-halogenated C1-3 alkyl, C1-3 alkoxy,

Wherein R is_dSelected from H, C1-3 alkoxy, R_eSelected from C1-3 alkyl.

Further, the air conditioner is provided with a fan,

a compound of formula I-1:

e is selected from 0-1 methylene;

R₁selected from H, halogen, halomethyl, cyano, amino, -COOR_fWherein R is_fIs selected from methyl; r₄、R₃Each independently selected from H, halogen;

R₂is selected from 4-6 membered saturated nitrogen heterocycle, 6 membered unsaturated nitrogen heterocycle, substituted or unsubstituted 6 membered saturated carbon cycle, substituted or unsubstituted benzene ring, wherein, the substituent on the 6 membered saturated carbon cycle is selected from hydroxyl, the substituent on the benzene ring is selected from hydroxyl, nitryl, carboxyl, halogen, halogenated or non-halogenated methyl, methoxyl, methyl, ethyl, propyl, butyl, ethyl,

Wherein R is_dSelected from H, methoxy, R_eSelected from methyl.

Further, in the compound represented by the formula I-1:

e is selected from 0-1 methylene;

R₁selected from H, halomethyl, halogen, preferably H, trifluoromethane, bromine;

R₃、R₄selected from H, halogen, preferably H, bromine;

R₂selected from the group consisting of a hydroxy-substituted 6-membered saturated carbocyclic ring, a nitro-substituted benzene ring, and an unsubstituted 6-membered nitrogen heterocycle.

Further, the structure of the compound is shown as formula I-2:

wherein X is selected from O, E is selected from methylene; or, X is selected from NH and E is selected from C ═ O;

R₂is selected from substituted or unsubstituted benzene rings, and the substituent is selected from nitro;

R₁、R₃、R₄each independently selected from H, halogen, preferably H, bromine.

Further, the structure of the compound is shown as formula I-3:

wherein R is_aSelected from C1-3 alkyl, -L₁-R_x，L₁Selected from C ═ O, 1-2 methylene, R_xSelected from substituted or unsubstituted 6-membered saturated carbocycle, substituted or unsubstituted 6-membered saturated nitrogen ring, substituted or unsubstituted benzene ring, the substituent is selected from hydroxyl, nitro;

R₆selected from amino,

R₂Is selected from substituted or unsubstituted benzene rings, and the substituent is selected from nitro; r₃、R₄Is selected from H;

R₁selected from halogens, preferably bromine.

Further, in the compounds of formula I:

a ring is selected from

R₆Is selected from

R2 is selected from substituted or unsubstituted benzene ring, and the substituent is selected from hydroxyl and nitro; r₁、R₃、R₄Each independently selected from H.

Further, the structure of the compound is:

the invention also provides the use of an indazole derivative for preparing TDO and/or IDO1 inhibitors, which is characterized in that: the indazole derivative is

Or a compound according to any one of claims 1 to 10, or a pharmaceutically acceptable salt thereof, or a solvate thereof.

Further, the inhibitor is a drug for treating neurodegenerative diseases, tumors, infectious diseases associated with cellular immune activation.

Further, the neurodegenerative disease is preferably parkinson disease, alzheimer disease, depression; the tumor is malignant tumor, preferably colon cancer and brain glioma.

Experiments prove that the compound shown in the formula I, or pharmaceutically acceptable salt or solvate thereof prepared by the invention can selectively inhibit TDO and IDO1, and has obvious inhibitory effect on TDO and/or IDO 1. In addition, the compound prepared by the invention has obvious anti-tumor effect, has certain treatment effect on Parkinson's disease and Alzheimer's disease, and has good application prospect in the field of medicine preparation.

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

The minimum and maximum carbon atom content of the hydrocarbon groups are indicated by a prefix, e.g., C1-4 alkyl indicates any alkyl group containing 1-4 carbon atoms.

Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.

The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.

Drawings

FIG. 1 is a graph showing the antitumor activity of groups of CT26 colon cancer mice.

Figure 2 is a graph of the combined synergistic activity of groups in CT26 colon cancer mice.

Fig. 3 is a tumor weight statistic for each group of mice treated with CT26 colon cancer.

FIG. 4 is a photograph of tumors from various groups of mice treated with CT26 colon cancer.

FIG. 5 is a graph showing the trend of the directional cruise escape latency of the groups of D-galactose induced AD model mice.

FIG. 6 shows the results of space exploration experiments on D-galactose induced AD model mice for each group.

FIG. 7 shows the results of the dark avoidance experiments on D-galactose induced AD model mice in each group.

FIG. 8 shows the results of T-SOD and MDA kit experiments on the remaining brain tissue after separation of hippocampus and cortex from D-galactose induced AD model mice for each group.

FIG. 9 is a flow chart of the experiment for evaluating the protection effect of MPTP-induced C57BL/6 Parkinson model mice for each group.

FIG. 10 shows the results of open field experiments on MPTP-induced C57BL/6 Parkinson model mice for each group.

FIG. 11 shows the results of experiments on the autonomic activity of MPTP-induced C57BL/6 Parkinson model mice in each group.

FIG. 12 shows the results of a pole climbing experiment on MPTP-induced C57BL/6 Parkinson model mice.

FIG. 13 shows the results of suspension experiments on MPTP-induced C57BL/6 Parkinson model mice for each group.

Figure 14 is the results of pharmacokinetic testing of the mice for each group.

FIG. 15 shows experimental procedures for groups of D-galactose induced AD model mice.

Note: in the figures of the present invention, P <0.05, P <0.01, P <0.001, compared to the model group.

Detailed Description

A. Synthesis of the desired intermediate

(a) First class intermediate backbones

The synthesis of TB-2-1 and TB-38-1 is carried out according to the patent CN108689936A, and the synthesis of the compounds TA-19-1 and TB-11-1 is carried out according to the patent CN 108689936A.

(b) Synthesis of intermediate skeleton of the second type

1 preparation of intermediate A2

Compound A1(5.0g,22.1mmol) was dissolved in 30mL of toluene, DPPA (7.9g,28.7mmol) and Et3N (6.2mL,44.2mmol) were added with stirring, and after stirring for 10min, benzyl alcohol (6.9mL,66.2mmol) was added. After the addition, the temperature is raised to 110 ℃ for reflux. After 3h, the reaction is completed, the reaction solution is removed by decompression and concentration, the residue is diluted by water, EA extraction is carried out for 3 times, organic layers are combined, the mixture is washed by saturated saline solution, dried by anhydrous sodium sulfate, filtered and concentrated, and a crude product is obtained. The crude product was purified by column chromatography to give a pale yellow solid a2(6.22g,18.8mmol) in 85% yield.

2 preparation of intermediate A3

A2(100mg,0.3mmol) was dissolved in the mixed solvent (methanol: dioxane ═ 1: 1), and iron powder (51mg,0.9mmol) and 10N concentrated hydrochloric acid (120 μ L,1.21mmol) were added alternately under ice-cooling. After the addition, the temperature is raised to 80 ℃, the reaction is completely stirred for 1h, the reaction is filtered while the reaction is hot, the concentration is carried out under reduced pressure, and the column chromatography purification is carried out, so as to obtain yellow solid A3(50mg,0.17mmol) with the yield of 55%.

3 preparation of intermediate A4

Dissolving compound A3(2.38g,7.9mmol) in glacial acetic acid, cooling to 10 ℃, slowly adding NaNO2(0.66g,9.5mmol), stirring at the same temperature for 10min, raising the temperature to room temperature, stirring for 30min, pouring the reaction solution into a large amount of ice water, separating out yellow solid, filtering by a Buchner funnel, and drying in vacuum to obtain light yellow solid A4(2.3g,7.4mmol) with the yield of 93%.

4 preparation of intermediate A5

Compound a4(2.4g,7.9mmol) was dissolved in a mixed solvent (ethanol: water ═ 2: 1), iron powder (2.21g,39.5mmol) and NH were added cross-wise under ice-bath₄Cl (221mg,3.95 mmol). After the addition, the temperature is raised to 80 ℃, the reaction is completely carried out for 15min by stirring, the hot reaction solution is filtered, a filter cake is washed for multiple times, the filtrate is dried by spinning, and the yellow solid A5(1.7g,6.03mmol) is obtained by column chromatography purification, wherein the yield is 76%.

(c) Synthesis of the third type of intermediate skeleton

1 Synthesis of intermediate B1

A1(2.0g,8.8mmol) and sulfonamide (2.5g,26.5mmol) were dissolved in sulfolane (15mL), phosphorus oxychloride (4.ImL,44.2mmol) was added in portions, and after the addition was completed, the reaction was stirred at 120 ℃ and was completed after 4 h. Adding a large amount of water to precipitate a white solid, stirring at-5 ℃ for 1h, and filtering. The solid was dried under vacuum to give the product as a pale yellow solid (1.47g,7.13mmol) in 88% yield

2 Synthesis of intermediate B2

Compound B2 was prepared as a yellow solid in 60% yield from compound B1 according to the synthesis method for compound A3.

3 Synthesis of intermediate B3

Compound B3 was prepared as a yellow solid in 80% yield from compound B2 according to the synthesis method for compound a 4.

4 Synthesis of intermediate B4

Compound B4 was obtained as a yellow solid with a yield of 81% by reference to the synthesis method of compound a5, starting from compound B3.

(d) Synthesis of intermediate skeleton of the fourth type

1 Synthesis of intermediate C2

Compound C1(2.0g,13.2mmol) was dissolved in acetonitrile, cooled to 0 deg.C, NBS (2.57g,14.5mmol) was added in portions, and after completion of the addition, the mixture was stirred at the same temperature for 30min to complete the reaction, spin-dried, and purified by column chromatography as a bright yellow solid (2.68g,11.6mmol), yield 88%.

2 Synthesis of intermediate C3

Compound C3 was obtained as a yellow solid with a yield of 80% by reference to the synthesis method of compound a4, starting from compound C2.

Synthesis of 3 intermediate C4

Compound C4 was obtained as a yellow solid with a yield of 81% by reference to the synthesis method of compound a5, starting from compound C3.

(e) Synthesis of intermediate skeleton of the fifth type

Taking a first intermediate 4-aminoindazole as a raw material, dissolving 4-aminoindazole (0.4g,3.01mmol) in acetonitrile, cooling to 0 ℃, adding NBS (1.07g,6.02mmol) in batches, stirring at the same temperature for 5min after the addition is finished, completely reacting, concentrating under reduced pressure, and purifying by column chromatography to obtain a bright yellow solid D3(0.5g,1.7mmol) with the yield of 57%.

(f) Synthesis of intermediate skeleton of the sixth type

1 Synthesis of Compounds 1-2

Accurately weighing compound 1-1(2g, 8.849mmol), dissolving in 100ml volumetric flask containing 15ml of LEOH, heating to 60 deg.C, adding 30% Na₂S₂O₄(3.85g, 22.123mmol) of aqueous solution, stirring at the same temperature for 3h after the addition of the raw materials, finding that the raw materials are not completely reacted, continuing the reaction for a period of time without obvious change, and stopping the reaction. Concentrating the reaction solution, adding water to dissolve the reaction solution, adjusting the pH value to about 3 by using a 3N HCl solution, extracting the reaction solution for 3 times by using ethyl acetate, adding saturated saline solution into an extracted ethyl acetate layer to remove water, then adding anhydrous magnesium sulfate into the ethyl acetate layer after the water removal to remove the water, collecting the ethyl acetate layer, and concentrating the ethyl acetate layer in a spin-drying manner to obtain a crude product. Separating the crude product by column chromatographyThe crude product was subjected to column chromatography to give 0.81g (yield: 46.7%) of a white solid powder.

2 Synthesis of Compounds 1-3

Compound 1-2(0.68g, 3.47mmol) was dissolved in 25ml of glacial acetic acid, and sodium nitrite (288mg, 4.16mmol) was slowly added under ice bath, and after completion of addition, the reaction was warmed to room temperature. After 20 minutes the reaction was substantially complete and stopped. The reaction solution was poured into a beaker containing a large amount of ice water, and the solid precipitated and was filtered off and dried (if not completely, the solid could be dissolved in methanol and then dried by spinning) to yield a pale yellow solid powder of 0.53g with a yield of 73.8%.

3 Synthesis of Compounds 1-5

Accurately weighing compound 1-3(300mg, 1.45mmol) and dissolving in 10 mM NaOH, slowly adding SOCl dropwise at 0 deg.C₂(0.86ml, 7, 7.25mmol), and after the addition, the temperature was raised to 40 ℃ for reaction. After 1h, the raw materials have reacted, the reaction is continued for 0.5h, the reactants are obviously changed, and the reaction is stopped. Removing methanol from the reaction solution by rotary evaporation, adding water and solid NaHCO₃Adjusting pH to neutral, extracting with ethyl acetate for 3 times, spin drying organic layer to obtain crude product, and purifying by column chromatography to obtain dark yellow solid powder 281mg with yield 87.8%.

Synthesis of 4 Compounds 1-6

Compounds 1-5(280mg, 1.27mmol) were dissolved in 12mL EtOH: h₂O is 2: 1, Fe (255mg, 6.33mmol) and NH were slowly added₄Cl (33mg, 0.61mmol), after addition, was heated to 80 ℃ for reflux reaction. After 1.5h, the reaction was complete and stopped. Filtering the reaction solution by using a suction filter funnel and diatomite, repeatedly washing iron mud by using MeOH for three times, concentrating the filtrate under reduced pressure to obtain a crude product, and purifying the crude product by using column chromatography to obtain light yellow solid powder 225mg with the yield of 93%.

(g) Synthesis of first class of intermediate starting materials

1 Synthesis of Compound TA-16-2

Compound TA-16-1(200mg,1.515mmol) was dissolved in DCM (8mL) and placed in an ice bath, and the oxidant DESS-Malin (2.568g,6.060mmol) was added thereto, and after 2 hours of reaction with stirring at room temperature, DCM (10mL) was added to the reaction solution to dilute it, and it was washed twice with saturated sodium bicarbonate (15mL), and the organic phase was dried over anhydrous magnesium sulfate, concentrated under reduced pressure, and filtered to give 167.4mg of a white liquid with a special flavor, with a yield of 86.3%.

2 Synthesis of Compound TA-16-3

Dissolving the compound TA-16-2(120mg,0.939mmol) in DCM (6mL), adding D-proline (10.8mg,0.0939mmol), and stirring at room temperature for 2h until the raw materials react completely. Vacuum concentrating, and performing column chromatography to obtain white liquid 99.5mg with yield of 82.9%.

(h) Synthesis of a second class of intermediate starting materials

The synthesis was performed with reference to the synthesis of the first class of intermediate starting materials.

(i) Synthesis of a third class of intermediate starting materials

1 Synthesis of Compound TB-31-2:

accurately weighing a compound TB-31-1(1000mg, 5.52mmol), adding the compound TB-31-1 into a reaction bottle, dissolving the compound TB-31-1 with 15mL of MeOH, slowly dropping thionyl chloride (2mL, 18.05mmol) into the reaction bottle in a fume hood under ice bath, carrying out reflux reaction at 45 ℃ after the addition is finished, and carrying out complete reaction on most raw materials after 6-8 hours. Dissolving methanol and thionyl chloride in water, adjusting the pH to 7-8 with sodium bicarbonate, extracting with EA, collecting the EA layer, drying and concentrating to obtain a light yellow solid crude product 909mg with a yield of 84.4%.

2 Synthesis of Compound TB-31-3:

accurately weighing a compound TB-31-2(900mg, 4.62mmol) in a three-necked bottle, using argon for protection, then injecting 10ml of dried dichloromethane into the three-necked bottle, after the addition is finished, moving to-78 ℃, pre-cooling for 0.5H, dropwise adding DIBAL-H (10ml, 9.85mmol) into a reaction solution, continuously stirring for 2H after the addition is finished, and monitoring the reaction solution by TLC thin-layer chromatography to ensure that the raw materials are completely reacted. Adding 1mL of methanol at-78 ℃ to quench the reaction, heating to 0 ℃, adding a proper amount of 10% aqueous solution of citric acid, continuing stirring for 0.5h, performing DCM extraction, collecting a dichloromethane layer, drying and concentrating to obtain a light yellow solid 690mg, wherein the yield is 90.6%.

3 Synthesis of Compound TB-31-4:

accurately weighing a compound TB-31-3(600mg, 3.59mmol), adding the compound TB-31-3 into a reaction bottle, dissolving the compound TB-31-3 with 15ml of dichloromethane, adding Dess-martin (3.35g, 7.9mmol) into the reaction bottle under ice bath, reacting at room temperature after the addition is finished, and completely reacting the raw materials of a reaction solution after 1 hour. The reaction was quenched with aqueous sodium thiosulfate, the pH was adjusted to 7-8 with sodium bicarbonate, a small amount of DCM was added and stirred until distinct layers separated, the DCM was extracted, the DCM layer was collected, dried and concentrated to give a brown liquid 620mg, which was solid after standing in 100% yield.

Synthesis of 4 Compound TB-33-4

Synthesis with reference to the Synthesis of TB-31-4

Synthesis of 5 Compound TB-34-4

Synthesis with reference to the Synthesis of TB-31-4

B. Synthesis of the Compounds of the invention (see in particular the examples below)

Example 1 Synthesis of Compounds TA-1, TA-14, TA-15, TA-16-a, TA-16-b, TA-16-c, TA-17, TB-1, TB-4, TB-6, TB-35, TB-36

Wherein, the structure of TA-16 is as follows:

the structure of TA-16-a is:

the structure of TA-16-b is:

the structure of TA-16-c is:

dissolving amine (1.0 times equivalent) and various substituted aldehydes (1.2-2.0 times equivalent) in Dichloromethane (DCM), adding dihydropyridine (1.4 times equivalent) and an appropriate amount of 4A molecular sieve, dropping trifluoroacetic acid (TFA, 0.5 times equivalent), refluxing at 40 deg.C for 12 hours. See patent CN108689936A for a method of reductive amination. Yield: 20 to 80 percent.

EXAMPLE 2 Synthesis of Compounds TA-4, TA-5, TB-17, TB-26, TB-28, TB-29, TB-30, TB-31, TB-32, TB-33, TB-34, TB-37

Synthesis was performed in reference example 1 with the synthesis yield: 17 to 88 percent.

EXAMPLE 3 Synthesis of Compound TB-14

Synthesis was performed in reference example 1 with the synthesis yield: and 64 percent.

EXAMPLE 4 Synthesis of Compounds TA-6, TA-7, TB-8

Synthesis was performed in reference example 1 with the synthesis yield: 60 to 80 percent.

EXAMPLE 5 Synthesis of Compounds TA-10, TA-11

Synthesis was performed in reference example 1 with the synthesis yield: 30 to 44 percent.

EXAMPLE 6 Synthesis of Compounds TA-12, TA-13, TB-9

Synthesis was performed in reference example 1 with the synthesis yield: 70 to 80 percent.

EXAMPLE 7 Synthesis of Compound TB-16

Reference example 1 was synthesized to give a dark yellow foamy solid, synthesis yield: 87 percent of

EXAMPLE 8 Synthesis of Compound TA-2 of the invention

1 Synthesis of Compound TA-2-2:

firstly, adding raw materials TA-2-1(2g, 14.39mmol) and hexamethylenetetramine (2.014g, 14.39mmol) into a reaction bottle, adding trifluoroacetic acid (18ml) into the reaction bottle, stirring, heating to 115 ℃ after the solid is dissolved, reacting for 5 hours, changing the reaction liquid from colorless transparency to reddish brown in the reaction process, completely reacting after 5 hours, and stopping the reaction. Adding a large amount of water, extracting with ethyl acetate for 3 times, washing with saturated saline solution, drying with anhydrous sodium sulfate, evaporating to dryness, drying, and concentrating to obtain crude product. Then, the product liquid is collected by column chromatography and concentrated to obtain a pure product of 500mg of light yellow solid with the yield of 20.1 percent.

2 Synthesis of Compound TA-2-3:

adding a compound TA-2-2(700mg,4.19mmol) and hydroxylamine hydrochloride (960,13.81mmol) into a reaction bottle, dissolving the compound TA-2-2 and hydroxylamine hydrochloride with 10mL (90% ethanol), slowly adding sodium hydroxide (1510mg,37.75mmol) in batches, generating white fog during the process of slowly adding the sodium hydroxide, releasing heat during the reaction, gradually changing the reaction liquid from brown to reddish brown, stirring the reaction solution at room temperature for 30min after the sodium hydroxide is added, completely reacting the reaction solution, cooling the reaction solution to room temperature, removing the ethanol by rotary evaporation, adding a small amount of water to dissolve the reaction solution, adjusting the pH to be alkalescent by using dilute hydrochloric acid, and then adjusting the pH to be alkalescent by using water and dichloromethane: isopropanol (3:1), drying the organic layer over anhydrous sodium sulfate, and concentrating to give compound TA-2-3 as a yellow-brown solid, 610 mg.

3 Synthesis of Compound TA-2-4:

putting a compound TA-2-3(110mg, 0.26mmol) and triphenylphosphine (100mg, 0.39mmol) into a round-bottom flask, placing the round-bottom flask at 4 ℃, dissolving the compound with tetrahydrofuran, dissolving diethyl azodicarboxylate with tetrahydrofuran under the protection of nitrogen, slowly dropping the mixed solution into a constant-pressure dropping funnel, continuously stirring the mixture to react for 1.5 hours completely, performing rotary evaporation and concentration to obtain a crude product, and performing column chromatography purification (PE: EA is 20: 1) to obtain 21mg of a colorless transparent oily substance with the yield of 57%.

4 Synthesis of Compound TA-2-5:

compound TA-2-5 was prepared as a yellow solid from compound TA-2-4 with reference to the synthetic method of compound a5, yield: 68 percent.

5 Synthesis of Compound TA-2:

synthesis was performed in reference example 1 to give a pale yellow solid, yield: 28 percent.

EXAMPLE 9 Synthesis of Compounds TA-3, TB-12

(1) Synthesis of Compound TB-12

Synthetic route to compound 5:

1 Synthesis of Compound TB-12-2:

adding compound TB-12-1(1g,5.12mmol), N-bromosuccinimide (1.83g,10.26mmol) and benzoyl peroxide (310mg,2.56mmol) into a reaction flask, dissolving with 20mL carbon tetrachloride, refluxing at 80 deg.C for 6h, removing carbon tetrachloride by concentrating under reduced pressure, and purifying by column chromatography to obtain milky white crystal 1.15g with yield of 82%.

2 Synthesis of Compound TB-12-3:

adding compound TB-12-2(200mg,0.73mmol) into a high-pressure reaction kettle, adding 5mL of methanolic ammonia solution, reacting at 80 ℃ for 10h, sucking out the reaction liquid, removing methanol by rotary evaporation, adding a small amount of methanol and dichloromethane, ensuring that a large amount of solid is insoluble, detecting by TLC to obtain a pure product, and filtering to obtain a pure product of 92mg, wherein the yield is 71%.

3 Synthesis of Compound TB-12-4:

compound TB-12-3(200mg, 1.16mmol) was dissolved with ethanol (4mL) and water (4mL), ammonium chloride (31mg, 0.58mmol) and Fe (323mg, 5.78mmol) were added at room temperature, after which stirring was carried out at 80 ℃ for 20min, iron powder was removed by filtration through celite, and after removal of ethanol by rotary evaporation, the mixture was removed with dichloromethane: isopropanol 3:1 and water, drying the organic layer with anhydrous sodium sulfate, concentrating under reduced pressure, and purifying by column chromatography to obtain 135mg of yellow solid, with yield: 79 percent.

Synthesis of Compound 4 TB-12:

compound TB-12-4(100mg,0.67mmol), 2-nitrobenzaldehyde (60mg,0.67mmol) and dihydropyridine ester (113mg,0.67mmol) were added to a reaction flask, dissolved in 3mL of dichloromethane and 3mL of methanol, trifluoroacetic acid (21. mu.L, 0.04mmol) was added dropwise with stirring, refluxed at 45 ℃ for 1.5h, and the reaction mixture was concentrated under reduced pressure and purified by thin layer chromatography to give 76mg of a pale yellow solid in 47% yield.

(2) Synthesis of Compound TA-3

Synthesis of reference Compound TB-12. Yield: 45 percent.

EXAMPLE 10 Synthesis of Compounds of the invention TA-9, TB-15

1 Synthesis of Compound A5-1

Compound A5(70mg,0.19mmol), compound 2a (23mg,0.19mmol) and dihydropyridine ester (67mg,0.26mmol) were dissolved in a mixed solvent of DCM/MeOH, and TFA (2. mu.L, 0.02mmol) was added with stirring. After the addition, the temperature is raised to 45 ℃ for reaction for 4 h. And concentrating the reaction solution under reduced pressure, diluting with EA, and adjusting the pH value to 8-9 by using saturated sodium bicarbonate. Drying, concentrating under reduced pressure, and purifying the crude product by column chromatography to obtain yellow brown solid compound A5-1(50mg,0.1mmol) with yield of 56%.

2 Synthesis of Compound TA-9

Dissolving compound A5-1(50mg,0.1mmol) in methanol, adding palladium carbon, pressurizing to 1MPa with hydrogen in autoclave, stirring at normal temperature for 12h, filtering to remove palladium carbon, concentrating under reduced pressure to remove filtrate, and purifying the crude product by column chromatography to obtain yellow brown semisolid compound TA-9(20mg,0.8mmol) with yield of 80%.

Synthesis of 3 Compound TB-15

Compound TB-15 was prepared in 56% yield from compound A5 starting material according to the synthetic method for compound A5-1.

EXAMPLE 11 Synthesis of the Compound of the invention TA-19

Dissolving TA-19-1(90mg,0.33mmol) and sulfamide (95mg,1mmol) in sulfolane, heating to 170 ℃ to react for 1h, moving the reaction to room temperature, adding water, extracting with dichloromethane, concentrating the organic layer under reduced pressure, and performing column chromatography to obtain a product TA-19(63mg,0.23mmol) with yield: 70 percent.

EXAMPLE 12 Synthesis of the Compounds of the invention TA-20, TA-21

1 Synthesis of Compound 2-2

With 10mL of dioxane and 10mL of H₂O Compound 2-1(1.0g, 8.68mmol) was dissolved and added (Boc) with stirring at room temperature₂O (2.08g, 9.55mmol) and NaOH (1.04g, 26.05mmol) (pH was maintained at about 11), after addition, the reaction was stirred at room temperature for 1h, the pH was adjusted to weak acidity with 1N HCl solution, ethyl acetate was added for extraction, the mixture was washed with saturated sodium chloride, dried over anhydrous magnesium sulfate, concentrated under reduced pressure, and purified by column chromatography to give 1.23g (yield 65.8%) of a pale yellow solid.

2 Synthesis of Compounds 2-3

Compound 2-2(500mg, 2.31mmol) is dissolved in 10mL DCM, left at 0 deg.C and P is addedPh₃(909mg, 3.47mmol), stirring for 5min, slowly adding NBS (618mg, 3.47mmol), after adding, moving to room temperature, stirring for 30min, reacting the raw materials completely, concentrating under reduced pressure, and purifying by column chromatography to obtain 497mg (77.1% yield) of white solid.

3 Synthesis of Compounds 2-4, 3-4

The compound 4-amino-6-bromo-1H-indazole (300mg, 1.42mmol) was dissolved in 6mL DMF and 2-3(473mg, 1.71mmol) and Cs were added with stirring at room temperature₂CO₃(1.39g, 4.26mmol), after the addition, the reaction mixture is transferred to 60 ℃ and stirred for 3h, the raw materials are completely reacted, water is added, ethyl acetate is used for extraction, saturated sodium chloride is used for washing, anhydrous magnesium sulfate is used for drying, reduced pressure concentration is carried out, and column chromatography purification is carried out to obtain light yellow solid compounds 2-4235 mg (yield is 40.5%) and light yellow solid compounds 3-479 mg (yield is 13.6%).

Synthesis of 4 Compound TA-20

Compound 2-4(235mg, 0.58mmol) was dissolved in 6mL DCM and CF was added with stirring at room temperature₃COOH (10% of solvent amount, namely 600 mu L), after the addition is finished, stirring at room temperature for 4h for complete reaction of the raw materials, and using saturated NaHCO₃The solution was adjusted to a slightly alkaline pH, extracted with DCM i.e. isopropanol 3:1, dried over anhydrous magnesium sulfate, concentrated under reduced pressure, and purified by column chromatography to give 108mg (60.9% yield) of a pale yellow solid.

5 Synthesis of Compound TA-21

Compound 3-4(79mg, 0.19mmol) was dissolved in 3mL DCM and CF was added with stirring at room temperature₃COOH (10% of solvent amount, namely 300 mu L), after the addition is finished, stirring at room temperature for 4h for complete reaction of the raw materials, and using saturated NaHCO₃The solution was adjusted to a slightly alkaline pH, extracted with DCM i.e. isopropanol 3:1, dried over anhydrous magnesium sulfate, concentrated under reduced pressure and purified by column chromatography to give a pale yellow solid 29mg (48.7% yield).

EXAMPLE 13 Synthesis of the Compounds of the invention TA-22, TA-23

1 Synthesis of Compound 4-2

Compound 4-1(1.0g, 8.92 mmol) was reacted with 20mL DCM) Dissolving, standing at 0 deg.C, adding PPh₃(3.5g, 13.37mmol), stirring for 5min, slowly adding NBS (2.38mg, 13.37mmol), after adding, moving to room temperature, stirring for 30min, reacting for the raw materials completely, concentrating under reduced pressure, and purifying by column chromatography to obtain 1.10g (yield: 71.0%) of white oily liquid.

2 Synthesis of Compounds 4-3

Compound 4-2(1.1g, 6.32mmol) and m-chloroperoxybenzoic acid (1.64g, 9.48mmol) were dissolved in dry 20mL DCM and reacted at 25 deg.C for 3h with complete reaction of the starting material, concentrated under reduced pressure and purified by column chromatography to a white oily liquid of 400mg (33.3% yield).

3 Synthesis of Compounds 4-4

Compound 4-3(400mg, 2.11mmol) was added to the reaction flask, to which was added 6mL of H₂And O, after the addition is finished, moving to 80 ℃ for reaction for 4h, completely reacting the raw materials, adding water, extracting by ethyl acetate, washing by saturated sodium chloride, drying by anhydrous magnesium sulfate, concentrating under reduced pressure, and purifying by column chromatography to obtain light yellow solid 200mg (the yield is 45.7%).

Synthesis of 4 Compounds TA-22, TA-23

The compound 4-amino-6-bromo-1H-indazole (169mg, 0.80mmol) was dissolved in 4mL DMF and 4-3(200mg, 0.96mmol) and Cs were added with stirring at room temperature₂CO₃(782mg, 2.40mmol) and after the addition, the reaction mixture was transferred to 60 ℃ and stirred for 3h to complete the reaction of the starting materials, water was added, extraction was performed with ethyl acetate, washing was performed with saturated sodium chloride, drying was performed with anhydrous magnesium sulfate, concentration was performed under reduced pressure, and purification was performed by column chromatography to obtain TA-2281 mg (yield 29.8%) as a pale yellow solid compound and TA-2343 mg (yield 15.8%) as a pale yellow solid compound.

EXAMPLE 14 Synthesis of Compound TB-2

Synthesis of 1 Compound TB-2-2

Dissolving compound TB-2-1(150mg,0.62mmol) in 5mL of dichloromethane, adding DMAP (4mg,0.003mmol), dissolving di-tert-butyl dicarbonate in 3mL of dichloromethane, slowly dropping the solution into the reaction solution, reacting at room temperature for 1h for completion of the reaction, concentrating the dichloromethane under reduced pressure to remove the dichloromethane, and purifying by column chromatography to obtain a pale yellow solid of 164mg with a yield of 100%.

Synthesis of 2 Compound TB-2-3

Dissolving compound TB-2-2(164m g,0.62mmol) with ethanol (10mL) and water (5mL), adding ammonium chloride (40mg, 0.31mmol) and Fe (174mg, 3.11mmol) at room temperature, stirring at 80 ℃ for 4h after adding, completely reacting, filtering with diatomite to remove iron powder, extracting with ethyl acetate and water after removing ethanol by rotary evaporation, drying the organic layer with anhydrous sodium sulfate, concentrating by rotary evaporation, purifying by column chromatography to obtain orange powder 100mg, and obtaining the yield: 67%.

Synthesis of 3 Compound TB-2-5

The compounds TB-2-3(100mg,0.43mmol), 2-nitrobenzaldehyde (78mg,0.52mmol) and dihydropyridine ester (108mg,0.43mmol) were added to a reaction flask, dissolved in 3mL of dichloromethane and 3mL of methanol, trifluoroacetic acid (8. mu.L, 0.08mmol) was added dropwise with stirring, the reaction was completed at 45 ℃ under reflux for 1.5h, the reaction mixture was concentrated under reduced pressure and purified by thin layer chromatography to give 65mg of a yellow oil with a yield of 41%.

Synthesis of 4 Compound TB-2

Dissolving the compound 7-5(65mg,0.17mmol) with 2mL dichloromethane, adding 400 μ L trifluoroacetic acid, stirring at room temperature for 2h for complete reaction, diluting with dichloromethane, adding saturated aqueous sodium bicarbonate solution, layering, collecting the organic layer, drying, concentrating, and purifying by column chromatography to obtain 39mg product with 83% yield.

EXAMPLE 15 Synthesis of Compound TB-3 of the invention

1 Synthesis of Compound TB-3-2:

dissolving compound TB-3-1(1g,4.29mmol) in 8mL of ethylene glycol, slowly adding hydrazine hydrate (416 mu L,8.58) dropwise, refluxing at 95 ℃ for 3h, detecting by TLC that the reaction is complete, adding water to separate out a large amount of white solid, and filtering to obtain 1.05g of a product with the yield of 100%.

2 Synthesis of Compound TB-3-3:

compound TB-3-2(500mg,2.32mmol) was dissolved in 5mL of dichloromethane, DMAP (15mg,0.01mmol) was added, di-tert-butyl dicarbonate was dissolved in 5mL of dichloromethane, slowly dropped into the reaction solution, reacted at room temperature for 1 hour, the reaction was completed by TLC, dichloromethane was spun off, and column chromatography purification (PE: EA ═ 20: 1) gave 355mg of a pale yellow solid in 48% yield.

3 Synthesis of Compound TB-3-5:

compound TB-3-3(193mg,0.61mmol) was dissolved in DMF, potassium carbonate (164mg,1.83mmol) and water (35. mu.L, 3.2mmol) were added at room temperature, the reaction was warmed to 5 hours, the reaction solution was cooled to room temperature, 3-nitrobenzyl iodide (170mg,0.61mmol) was added, and the reaction was completed at room temperature. Extracting with water and ethyl acetate, washing with saturated saline, drying, concentrating, and purifying the crude product by column chromatography (PE: EA 60: 1) to obtain a pale yellow oily substance 110mg with a yield of 40%.

4 Synthesis of Compound TB-3:

dissolving compound TB-3-5(100mg,0.22mmol) with 2mL dichloromethane, adding 400 μ L trifluoroacetic acid, stirring at room temperature for 2h, detecting by TLC for complete reaction, diluting with dichloromethane, adding saturated sodium bicarbonate aqueous solution, layering, collecting organic layer, drying, concentrating, and purifying by column chromatography to obtain product 59mg with yield of 78%.

EXAMPLE 16 Synthesis of Compound TB-5 of the invention

4-amino-6-bromo-1H-indazole (50mg,0.185mmol) was dissolved in methylene chloride (3mL), placed in an ice bath, o-nitrobenzoic acid (118.2mg,0.708mmol) was added dropwise, EDCI (62.4mg,0.329mmol), HOBT (48mg,0.329mmol), DIEA (77.5uL,0.470mmol) were added, and the mixture was reacted at 0 ℃ for 5 minutes, and then warmed to room temperature (30 ℃) and stirred overnight. Washing the reaction solution with water (1mL), then washing with saturated sodium bicarbonate (1.5mL), spin-drying the organic phase, and carrying out column chromatography to obtain TB-5. 27.1mg of a yellow solid, yield 40.1%.

EXAMPLE 17 Synthesis of Compound TB-7 of the invention

By taking 4-amino-6-bromo-1H-indazole and o-nitrobenzyl bromide as raw materials and referring to the method for synthesizing 2-4 by using the compound 2-3, TB-7 is obtained with a yield of 63%.

EXAMPLE 18 Synthesis of Compound TB-10 of the invention

4-amino-6-bromo-1H-indazole (152mg,0.71mmol)), 2-nitrobenzoic acid (100mg,0.59) were dissolved in DMF and EDCI (105mg,0.71mmol), HOBt (137mg,0.71mmol), DIEA (197. mu.L, 1.18mmol) were added with stirring at room temperature and reacted completely at room temperature for 6H. Adding water into the reaction solution, extracting for 3 times by ethyl acetate, washing by saturated saline solution, drying and concentrating to obtain a crude product, and purifying by column chromatography to obtain a yellow solid 51mg with the yield of 24%.

EXAMPLE 19 Synthesis of Compound TB-11 of the invention

Synthesis was performed in reference example 1 with the synthesis yield: 65 percent.

EXAMPLE 20 Synthesis of Compound TB-13

Synthesis of 1 Compound TB-13-2

Dissolving compound TB-13-1(1000mg, 5.2mmol) with ethanol (4mL) and water (4mL), adding ammonium chloride (139mg, 2.6mmol) and Fe (1.46g, 26mmol) at room temperature, stirring at 80 ℃ for 20min after addition, completing the reaction, filtering with diatomite to remove iron powder, extracting with ethyl acetate and water after removing ethanol by rotary evaporation, drying the organic layer with anhydrous sodium sulfate, and concentrating by rotary evaporation to obtain 800mg of yellow solid, yield: 94 percent.

2 Synthesis of Compound TB-13

The compound (100mg,0.61mmol) was dissolved in DMF, potassium carbonate (255mg,1.86mmol) was added at room temperature, activation was performed at room temperature for 30min, a solution of 2-nitrobenzyl iodide (170mg,0.61mmol) in DMF was added, and then the reaction was completed at room temperature for 3 h. Extracting with water and ethyl acetate, washing with saturated saline, drying, concentrating, and purifying the crude product with column chromatography dichloromethane to obtain light yellow solid 60mg with yield of 50%.

EXAMPLE 21 Synthesis of Compound TB-38 of the invention

1 Synthesis of Compound TB-38-2

Dissolving TB-38-1(300mg,1.3mmol) in DMF, adding cesium carbonate (1.2g,3.9mmol), metering 2-bromopropane (148 uL, 1.56mmol) at 45 ℃, reacting for 4h at 45 ℃ after the addition is completed, adding a large amount of water, extracting with ethyl acetate, concentrating the organic layer, and carrying out column chromatography to obtain 254mg of a product, wherein the yield is as follows: 72 percent.

2 Synthesis of Compound TB-38-3

The compound TB-38-3 was obtained as a yellow solid with a yield of 60% by using the compound TB-38-2 as a starting material and referring to the synthetic method of the compound A5.

Synthesis of 3 Compound TB-38

Synthesis was performed in reference example 1 with the synthesis yield: and 55 percent.

EXAMPLE 22 Synthesis of Compounds of the invention TB-18, TB-19, TB-20, TB-21, TB-22, TB-23, TB-24, TB-25, TB-27

See for synthesis patent CN 107033087A.

EXAMPLE 23 Synthesis of compound TA-18 of the invention

See patent CN108689936A for synthesis.

The beneficial effects of the present invention are demonstrated by the following experimental examples.

Experimental example 1 confirmation of the Structure of the Compound of the present invention

Nuclear magnetism of the compound prepared in the example of the present invention¹H-NMR) and Mass Spectrometry (MS), the results are shown in table 1. As shown in Table 1, the present inventors have succeeded in preparing the above-mentioned compounds.

TABLE 1 Nuclear magnetic and Mass Spectroscopy results of the Compounds of the invention

Experimental example 2 evaluation of inhibitory Activity of the Compound of the present invention against TDO and IDO1

1. Experimental methods

(1) Enzyme inhibitory Activity test experiment

Human N-terminal IDO1 and TDO were expressed in E.coli and purified by nickel affinity chromatography. The measurement was carried out by UV absorption using recombinant hIDO1, hTDO and L-tryptophan as substrates. To test the inhibitory activity of compounds on TDO and IDO1 enzymes, recombinant hTDO (100nM) and hIDO1(100nM) were mixed with a concentration of compound at room temperature in a mixture containing 400mM tryptophan, 40mM ascorbic acid, 200. mu.g/ml catalase, 20. mu.M methylene blue, Ca²⁺，Mg²⁺And culturing in a potassium phosphate-free buffer solution culture system. For negative control (blank), 5 μ L of assay buffer was added instead of enzyme, and 10% DMSO was added instead of inhibitor. For the positive control, Epacadostat was added instead of the inhibitor. After 1 hour of incubation, 30% trichloroacetic acid was added to each system and incubated at 65 ℃ for 15 minutes to terminate the enzyme reaction and convert N-formyl kynurenine to kynurenine. Then, 100. mu.L of the supernatant from each system was mixed with an equal volume of acetic acid containing DMAB (dimethylaminobenzaldehyde, 3%, w/v) and the optical density was detected at 480nm wavelength using Multiscan spectroscopy Mk3(Thermo Fisher). Kynurenine concentrations were determined from L-kynurenine standard curves. Finally, data was processed using GraphPad Prism 5.0 software.

By the above experimental method, the inhibitory activity of the compounds of the present invention against TDO and IDO1 was tested. The inhibitory activity of specific compounds at a concentration of 10. mu.M is shown in Table 2-1. Wherein A represents an inhibition rate of more than 70%, B represents an inhibition rate of 50-69%, C represents an inhibition rate of 20-49%, and D represents an inhibition rate of less than 20%.

(2) Inhibition assay for IDO1 cells

HeLa cells were seeded in 24-well plates for 24 hours, then INF γ (50ng/mL) was added and treated with inhibitor for 24 hours. DMSO (0.5%) and Epacadostat (25nM) were used as negative and positive controls, respectively. IDO1 activity was determined by measuring the concentration of L-kynurenine in the cell culture medium. 400 μ L of the medium was mixed with 180 μ L of 30% trichloroacetic acid and centrifuged at 13.000rpm for 10 minutes. The supernatant (100. mu.L) was transferred to a new 96-well plate and an equal volume of freshly prepared 2% w/v p-dimethylaminobenzaldehyde in acetic acid was added. Optical density was measured at 480nm using Multiscan spectroscopy Mk3(Thermo Fisher). Kynurenine concentrations were determined from L-kynurenine standard curves. Each assay was performed in triplicate and data are presented as mean ± standard deviation.

(3) Determination of TDO cell inhibition

The TDO-overexpressing cell line a172 was used to test hTDO activity in the cellular environment. After overnight seeding in 96-well plates (2X 104 cells/well), A172 cells were treated with a concentration of compound and L-Trp (20. mu.g/ml) for 24 hours. Then, cell culture medium (300. mu.L/well) was transferred to the culture tube and mixed with 90. mu.LTCA (30%, w/v). Next, the culture tube was transferred to a water bath at 65 ℃ for 30 minutes to convert N-formyl kynurenine into kynurenine. After centrifugation at 13.000rpm for 10 minutes, the supernatant (100. mu.L) was transferred to a new 96-well microplate and an equal volume of freshly prepared 2% w/v solution of p-dimethylaminobenzaldehyde in acetic acid was added. Optical density was measured at 480nm using Multiscan spectroscopy Mk3(Thermo Fisher). Kynurenine concentrations were determined from L-kynurenine standard curves. Finally, data was processed using GraphPad Prism 5.0 software.

2. Results of the experiment

Table 2-1 shows the data on the inhibitory activity of the compounds of the present invention on TDO and IDO 1. It can be seen that the compounds prepared by the present invention are effective in inhibiting TDO, IDO1, and in some cases even both TDO and IDO 1. In particular, the compounds TA-16, TA-15, TB-17, TB-1, TA-1, TA-11, TA-14, TA-18, TB-2, TB-4, TB-6, TB-11, TB-14, TB-20 and TB-23 have obvious inhibiting effect on TDO and/or IDO1 (see Table 2-2).

TABLE 2-1 data on the inhibitory Activity of partial Compounds of the invention on TDO and IDO1

TABLE 2-2 inhibitory Activity of partial Compounds of the invention on TDO, IDO1 data IC50(ug/mL)

EXAMPLE 3 enzyme selectivity test of Compounds of the invention

1. Experimental methods

The inhibition of two other enzymes involved in the metabolism of TYP (kynurenine-3-monooxygenase (KMO), indoleamine 2,3-dioxygenase 2(IDO2)) by the active compounds prepared according to the invention (TA-16, TB-17, TB-1, TA-15, TB-23) was investigated separately in order to evaluate the enzyme selectivity of the groups.

(1) KMO test method

After reaction of recombinant rhKMO with reduced coenzyme II and L-kynurenine as substrates, the UV absorption signal measured at a wavelength of 340nm has a correlation with the amount of reduced coenzyme II remaining in the reaction. Experimental group 100. mu.L of the reaction mixture was added to 384-well plates and reacted in buffers (50mM sodium phosphate, pH7.5, 0.1% Brij-35) containing 200. mu.L of reduced coenzyme II, 400nM L-kynurenine, KMO (2250 ng/well), and various concentrations of inhibitors. The reaction mixture was incubated at room temperature for 90 minutes and the UV absorbance signal was measured. For negative controls (blank), assay buffer was added instead of reducing coenzyme II.

(2) IDO2 test method:

the test compound was dissolved in 20% DMSO, and 10 μ L of this solution was added to 200 μ L of the reaction solution so that the final concentration of DMSO was 1% in all the reaction solutions. All reactions were carried out at room temperature. For the experimental group 200. mu.l of IDO2 assay buffer contained 400nM IDO2, inhibitor, tryptophan and coexisting reaction components. The reaction mixture was incubated at room temperature for 180 minutes. The UV absorbance was measured by reacting recombinant IDO2 with L-tryptophan as a substrate, based on the fact that the UV absorbance at 321nm was positively correlated with the amount of N-formylkynurenine, a reaction product of IDO 2. For the negative control (blank), 10mL of assay buffer was added instead of IDO 2.

2. Results of the experiment

Tables 2-3 show the inhibition rates of KMO and IDO2 at 10. mu.M and 100. mu.M concentrations in each group, and it can be seen that these compounds have only weak inhibitory activities against these two enzymes involved in TYP metabolism. In combination with the data from example 2, it is evident that the compounds prepared according to the invention have selective inhibitory activity on TDO and/or IDO 1.

Tables 2-3 enzyme selectivity testing of some of the compounds of the invention

Experimental example 4 antitumor Activity of the Compound of the present invention against CT26 Colon cancer mice

The purpose of this experiment was to examine the in vivo anti-tumor effect of the compounds of the invention. In the experiment, Balb/c mouse subcutaneous colon cancer cell line CT26 in-vivo tumor model is used for testing the in-vivo anti-tumor activity of compounds TA-16, TB-17 and TA-1.

1. Experimental Material

The 1640 medium was purchased from Gibco BRL, DMSO, PEG400 from SIGMA, doxorubicin from Dalian America Biotechnology, Inc., fetal bovine serum from grassland Green-field Biotechnology, Inc., pancreatin from Biyunnan Biotechnology, Inc., mouse colon cancer cell line CT26 from American ATCC, and Balb/c mice from Dudu Daokou laboratory animals, Inc.

2. Experimental methods

When CT26 cells grew to mid-late logarithmic growth, all cells were harvested after digestion and resuspended to 1 × 107 cells/ml using serum-free media. Subsequently, the cells were inoculated at 100. mu.L/mouse right back. When the tumors grew to 100mm3, animals were randomized into 7 groups (7-8 per group) and dosing was initiated. Tumor volume was measured every three days during the experiment using a vernier caliper and the weight of the mice was weighed. After 12 days of continuous administration, the tumor volume of the control group was too large, the experiment was stopped, a tumor growth curve and a mouse body weight change curve were plotted based on the measured data, and the tumor inhibition rate of each group of compounds to the CT26 model was calculated.

Grouping experiments: (1) drug solvent control (5% DMSO + 20% PEG400+ 75% water); (2) positive control INCB 02436030 mg/kg q.d. oral administration; (3) -TA-16150 mg/kg q.d. oral administration; (4) TA-1-150mg/kg q.d. oral administration; (5) TB-1780 mg/kg q.d. oral administration, (6) TA-16100 mg/kg q.d. oral administration in combination with doxorubicin 5mg/kg/week intravenous administration; (7) adriamycin 5mg/kg/week was administered intravenously.

3. Results of the experiment

As can be seen from figures 1, 2,3 and 4, TA-16, TB-17 and TA-1 all have certain inhibition effect on CT26 tumor of colon cancer in mice in vivo. Among them, the antitumor effects of group (3) (TA-16150 mg/kg) and group (5) (TB-1780 mg/kg) were very significant (P <0.05), and group (6) (167 in combination with doxorubicin) showed better therapeutic effects than group (7) (doxorubicin alone). The phenomena of rash, diarrhea, death and the like of animals are not found in the administration process.

Experiments prove that the compound prepared by the invention has obvious tumor inhibition effect on CT26 colon cancer mice.

Experimental example 5 pharmacokinetic study of the Compound of the present invention

1. Experimental methods

Drug absorption and brain distribution studies were performed on compound TA-16 using Kunming mice. The Kunming mice are orally administrated with 100mg/kg once, 3-5 mice are anesthetized at each time point of 0.5h, 1h, 2h, 4h, 8h, 12h and 24h respectively, and after blood is taken from eyepit, the brain is killed and taken out. After purification by extraction, the drug concentration in blood and brain was measured by HPLC at each time point.

2. Results of the experiment

The results are shown in fig. 14, and it can be seen that compound TA-16 can be detected in mouse brain in mouse pharmacokinetic experiments, and its drug concentration in brain is lower than that in blood.

Experimental example 6 Effect of the Compound of the present invention on learning and memory function of D-galactose-induced AD model mouse

1. Experimental Material

(1) Experimental drugs and reagents

Compound TA-16 prepared according to the invention; xidezhen, lot number: 7K893T, Tianjin Huajin pharmaceutical Co., Ltd; d-galactose, batch No.: 2017060101, Dougenkoron Chemicals, Inc.; sodium carboxymethylcellulose, batch number: 20120727 Chengduo chemical reagent factory; total superoxide dismutase (T-SOD) assay kit, lot No.: AOO1-1, specification: 100T/96 sample, Nanjing, built bioengineering research institute Co., Ltd; malondialdehyde (MDA) assay kit, lot No.: AOO3-1, specification: 100T/96 sample, Nanjing, was built into bioengineering research institute, Inc.

(2) Laboratory apparatus

Electronic balance, model FA1204L, shanghai yuepin scientific instruments ltd; EthoVision XT Morris water maze system, NOLDUS nodata (beijing) information technology, llc; PAT-8 dodging experiment video analysis system, Gengtai alliance software Co.

(3) Laboratory animal

SPF-grade KM male mice, body weight (20 ± 2g), 100 (104 out), purchased from mastered animals laboratories ltd, animal quality certification No.: n0.51203500006569, its license number: SCXK (Chuan) 2015-030. 2. Experimental methods

The experiment was carried out according to the flow chart shown in fig. 15.

2.1 Experimental groups and dosing

When grouping, randomly grabbing mice according to weight layers, a blank group (K), a D-galactose model group (M), a Xidezhen positive group as Y, and a compound TA-16 drug group (167), wherein the total number of the groups is 4, and each group is 10. K. The M groups are intragastrically administered with 5/1000CMC-Na aqueous solution with corresponding volume, the Y and 167 groups are intragastrically administered with corresponding drug (dissolved in 5/1000CMC-Na aqueous solution) with corresponding volume, 1 time of continuous administration is 1 day for 40 days, the administration dose of each group is shown in table 3, and the administration volume is 10 ml/kg. After 1 hour of administration, group K was given a corresponding volume of physiological saline by subcutaneous injection on the back of the neck, and the other groups were given a corresponding volume of D-galactose physiological saline solution by subcutaneous injection on the back of the neck, at an administration dose of 500mg/kg, at an administration volume of 10ml/kg, 1 day 1 time.

TABLE 3 dosage form

2.2 learning and memory function testing

2.2.1 Water maze experiment

And (3) directional navigation test: once daily for 5 days beginning on day 31 of dosing. The mouse is slowly placed from the opposite quadrant of the quadrant where the platform is located facing the barrel wall, and the time from entering water to finding the platform is recorded, so as to avoid the latency. If the mouse does not find the platform within 1min, the mouse needs to be manually guided to the platform and stays for 5s-15s, and the escape latency is recorded as 60 s. If the mouse finds the platform within 1min, the system automatically records the escape latency.

And (3) space exploration testing: and after the directional navigation experiment is finished for 24 hours, removing the platform, slowly putting the mouse into the relative quadrant of the quadrant where the platform is located from the surface of the barrel wall, recording the time for finding the set position of the platform by the system, setting the swimming accumulated time of the quadrant where the platform is located within 60s, setting the frequency of entering the quadrant where the platform is located, and the total swimming distance.

2.2.2 dark avoidance experiments

After the water maze was finished for 24 hours, the dark avoidance experiment was performed in the following way:

the first day: one mouse is put into each light box, the dark box is not electrified, and the mouse can move freely between the light box and the dark box and adapt to 2 minutes. After 2 minutes, the partition was closed, the mouse was placed in the light box, the dark box was powered on, and the partition was immediately removed at the beginning of the experiment. Recording the time of the electric shock when the electric shock is applied to the dark box from the bright box for the first time, namely the escape latency, and recording as 300S if the escape latency is more than 300S; and recording the number of electric shocks of the mice in 300S from the light box to the dark box.

The next day: the mice were placed in the light box, the dark box was powered on, and the partition was immediately removed at the start of the experiment. Recording the time of the electric shock when the electric shock is applied to the dark box from the bright box for the first time, namely the escape latency, and recording as 300S if the escape latency is more than 300S; and recording the number of electric shocks of the mice in 300S from the light box to the dark box.

2.3 kit assay

Taking the brain tissue with the separated hippocampus and the residual cerebral cortex, and according to the brain tissue: homogenizing normal saline at a ratio of 1:9, centrifuging 200 μ l 3500r/min to obtain supernatant, measuring protein content and T-SOD, storing the supernatant at-80 deg.C, and measuring MDA.

2.4 statistical treatment

All experimental data are expressed as mean ± standard deviation (X ± SD), and the comparison between data groups adopts two-factor analysis of variance and one-factor analysis of variance, and the difference is statistically significant when P is less than 0.05.

2.5. Taking materials

On the next day after the dark-avoiding experiment is finished, blood is taken from the eyeball after 1 hour of administration; immediately cutting off the head and taking out the brain, separating the brain on ice, and fixing the left half part in 10% formaldehyde solution for later use; the hippocampus, cortex, and other parts in the right half were isolated, wrapped in tinfoil paper and labeled, respectively, and stored in liquid nitrogen.

3. Results of the experiment

3.1 Water maze experiment

3.1.1 Directional cruise experiment

As shown in table 4, compared with the blank group, after five days of training, the escape latencies of the model group are all longer and have no obvious change, while the escape latencies of the blank group are in a trend of shortening, wherein the statistical difference is obvious on the 5 th day (P <0.001), which indicates that the molding is successful. None of the escape latencies on

days

1, 2, and 3 were statistically different compared to the model group (P > 0.05); compound TA-16 group escape latency at day 4 (P <0.01) was statistically different; day 5 compound TA-16 group had statistical differences in escape latency (P < 0.01).

The compound TA-16 can improve the learning and memory functions of D-galactose induced Alzheimer Disease (AD) model mice comprehensively.

TABLE 4 Directional cruise avoidance latency

Note: in comparison to the set of models,^*P<0.05，^**P<0.01，^***P<0.001

as can be seen from fig. 5, the two-factor anova with 5-day escape latency of directional cruise shows that five-day training results in shorter and shorter escape latency of the K blank group. The escape latency for the M model group did not change significantly. The results of the two-factor anova for the number of training days and the group showed that the number of training days had a statistical difference in the effect on the escape latency compared to the K blank group, and the results showed that F (4,24) ═ 3.601, P ═ 0.0195, and P < 0.05; the group factors also have statistical differences in latency avoidance, and the results show that F (1,6) is 21.49, P is 0.0036, and P is 0.05; however, the two-factor analysis of variance on training days and groups showed no statistical difference in the analysis, and the results showed that F (4,24) was 2.179, P was 0.1019, and P was >0.05, indicating that the model was successful.

Compared with the model group, for the training day factor, the results of the compound TA-16 group showed that F (4,24) ═ 3.813, P ═ 0.0155, and P <0.05, which were statistically different; compound TA-16 group F (1,6) ═ 33.23, P ═ 0.0012, P <0.05 for a group of factors; the two-factor cross-analysis results for training days and groups showed that compound TA-16 group F (4,24) was 1.438, P was 0.2520, and P > 0.05.

In conclusion, the compound TA-16 has certain improvement effect on the learning and memory functions of the D-galactose induced AD model mouse.

3.1.2 space exploration experiments

From table 5 and fig. 6, it can be seen that in the space exploration experiment, compared with the blank group, the escape latency of the model group has no statistical significance, and the percentage of swimming time in the target quadrant (P <0.05), the number of times of entering the quadrant (P <0.01), and the cumulative swimming distance (P <0.05) all have statistical differences, which indicates that the model modeling of the AD model caused by D-galactose is successful. Compared with the model group, the ZT group (P <0.01) has statistical difference in escape latency; compound TA-16 group (P <0.05) had statistical differences in target quadrant swimming time percentage; number of target quadrant entries, compound TA-16 group (P <0.001) had statistical differences; the swimming distance was accumulated and there was no difference between each group and the model group.

In conclusion, the compound TA-16 has the effect of improving the spatial memory capacity of the D-galactose induced AD model mouse.

TABLE 5 results of the space exploration experiment

Note: in comparison to the set of models,^*P<0.05，^**P<0.01，^***P<0.001

3.2 dark avoidance experiments

As can be seen from table 6 and fig. 7, in the dark avoidance experiment, the model group has a longer escape latency, a statistical difference (P <0.05), a larger number of errors, and a statistical difference (P <0.01), compared to the blank group, indicating that the molding is successful. The escape latency of compound TA-16 group was significantly increased compared to the model group with statistical differences (P < 0.01); the number of errors was also significantly reduced in compound TA-16 group, with statistically significant differences (P < 0.001).

Comprehensively, the compound TA-16 can improve the learning and memory ability of the AD model mouse caused by the D-galactose.

TABLE 6 darkening-prevention test results

Note: in comparison to the set of models,^*P<0.05，^**P<0.01，^***P<0.001

3.3T-SOD and MDA kit assay results for residual brain tissue after separation of Hippocampus and cortex

As can be seen from table 7 and fig. 8, compared with the blank group, the SOD activity and MDA content in the model group were lower and statistically different (P <0.01), indicating successful molding. Compared with the model group, the SOD activity of the compound TA-16 group is higher, and the statistical difference is realized (P <0.01), the MDA content is lower, and the statistical difference is realized (P < 0.05).

Comprehensively, the compound TA-16 can improve the antioxidant capacity of the AD model mouse caused by the D-galactose.

TABLE 7 Experimental results for T-SOD and MDA kits

Note: in comparison to the set of models,^*P<0.05，^**P<0.01，^***P<0.001

in conclusion, the results of a water maze experiment and a darkness avoidance experiment show that the compound TA-16 can well improve the learning and memory abilities of the D-galactose AD model mouse, and the T-SOD and MDA kit determination results of the residual brain tissues after the separation of the hippocampus and the cortex show that the compound TA-16 can improve the antioxidant ability of the D-galactose AD model mouse.

Experimental example 7 study on the protective effect of the compound of the present invention on MPTP-induced C57BL/6 Parkinson model mouse

1. Experimental Material

(1) Experimental drugs and reagents

Compound TA-16 prepared according to the invention; madopa, batch No.: SH3580, Shanghai Roche pharmaceuticals, Inc.; MPTP, lot number: M0610A, S0620A, Dalian Meilun Biotechnology Limited; anti-Tyrosine Hydroxylase (TH) antibody (ab137869), lot: GR3219792-1, Abcam China; anti-alpha-syn antibody (ab51253), lot: GR3191908-8, Abcam China.

(2) Laboratory apparatus

ZZ-6 autonomous activity tester, batch number: TM050608, chengtai science and technology limited; OFT-100 rat and rat open-field activity experiment system, Gengtai Union science and technology Limited.

(3) Laboratory animal

SPF grade C57BL/6 male mouse, body weight (20. + -.2 g), 70 (72), purchased from Woods laboratory animals Ltd, animal certification number: 51203500006753, license number: SCXK (Chuan) 2015-030. 2. Experimental methods

The experiment was carried out according to the flow chart shown in fig. 9.

2.1 Experimental groups and dosing

When the mice are divided into groups, the mice are randomly grabbed according to weight layers, and the mice are divided into 8 blank groups (K), 11 MPTP model groups (M), 11 MEDOPA + MPTP groups (Y) and 10 TA-16+ MPTP groups (167), and the total number of the groups is 4 and 40. Feeding in IVC animal room, and adaptively feeding for 7 days. On the 8 th day, the blank group was injected with a corresponding volume of saline, the model group and each administration group were injected with freshly prepared MPTP saline, once a day for 7 consecutive days, at a dose of 30mg/kg and a volume of 10ml/kg, and the doses of each group are shown in Table 8. On day 15, K, M two groups were gavaged with 5/1000CMC-NA aqueous solution of corresponding volume, and Y and compound TA-16 two groups were gavaged with corresponding drug of corresponding volume (5/1000CMC-NA dissolved), once a day for 14 days.

TABLE 8 dosage form

2.2 behavioural testing

2.2.1 Pole climbing experiment

A wooden stick with the length of 50cm and the diameter of 1cm is manufactured, the wooden stick is perpendicular to the ground, a layer of gauze is coated outside the wooden stick to prevent slipping, a soft ball with the diameter of 2.5cm is placed at the top end of the wooden stick, and the lower end of the wooden stick is fixed. During the experiment, the mouse head is placed on the ball upwards, the time (T-turn) from the time when the mouse head is placed on the ball from the top end of the ball upwards to the time when the mouse head downwards climbs down to the rod is recorded, and then the time (T-la) from the time when the mouse climbs up to the rod to the bottom of the climbing rod is recorded, so that the double forelimbs contact the bottom plate of the fixed rod. And if the pause or reverse climbing occurs in the midway, restarting the test.

2.2.2 open field experiment

And (3) adopting an open field experiment system, and automatically recording the static time, the movement time and the total movement distance of the mouse within 5 min. After each mouse is done, the open field experiment is cleaned, the smell and the foreign matters are removed, and the interference on the subsequent mouse behaviours is avoided.

2.2.3 autonomic Activity recording

The autonomic activity of the mice is measured as the number of activities in which the animal is affected by excitatory or inhibitory drug intervention, including the number of movements to cessation, the number of upright probes, the number of back combing of the animal. After each mouse is done, the autonomous activity recorder is cleaned, smell and foreign matters are removed, and interference on subsequent mouse behaviours is avoided.

2.2.4 suspension experiments

A long cotton rope is fixed at a height of about 50cm above the ground, the front claw of the mouse is used for grabbing the rope, the behavior of the mouse is observed, and the time that the double claws of the mouse are hung on the rope and do not fall off is recorded. Scoring 0 if 0s < t <5 s; score 1 if 6s < t <10 s; score 2 if 11s < t <15 s; score 3 if 16s < t <20 s; if t >20s, score 4.

2.3 taking materials

After the behavior is finished, blood is taken from eyeballs, centrifuged, taken supernatant and frozen at-80 ℃. After blood is taken, taking brains immediately, selecting 4 mice randomly in each group, taking and fixing the half brains in 10% formaldehyde solution for pathological examination; the brains of the remaining mice in each group were harvested and separated from the substantia nigra, striata, cortex and other parts on ice, and the brains were weighed, wrapped in tinfoil paper, labeled and stored in liquid nitrogen.

2.4 immunohistochemical staining

Samples stored in 10% formaldehyde solution were sent to Kyowas Biotech Inc. for immunohistochemical staining of mouse substantia nigra TH dopamine positive neurons, from which a pathological examination report was made.

2.5Western blot

The substantia nigra and striatum preserved in liquid nitrogen are used for detecting the content of alpha-synuclein.

2.6 statistical treatment

All experimental data are expressed as means ± standard deviation (X ± SD), comparisons between data groups are analyzed using nonparametric and one-way variances, and T-test, with differences expressed as P <0.05 being statistically significant.

3. Results of the experiment

3.1 Pole climbing experiment

As can be seen from Table 11 and FIG. 12, the model mice showed significantly longer T-turn and T-la in the pole climbing experiments compared to the blank group, and had statistical differences (P <0.05), indicating that the Parkinson model was successful. Compared with the model group, the compound TA-16 group mice have obviously shortened T-turn in the pole climbing experiment and statistical difference (P is less than 0.05), which indicates that the compound TA-16 has an effect of improving the limb coordination ability of the MPTP-induced Parkinson model mice.

TABLE 11 pole climbing test results

Note: in comparison to the set of models,^*P<0.05,^**P<0.01,^***P<0.001。

3.2 open field experiment

As can be seen from table 9 and fig. 10, compared with the blank group, the movement time and movement distance of the model group mice in the open field experiment were significantly shortened, and the statistical difference (P <0.01) was observed, indicating that the parkinson model was successful. Compared with the model group, the compound TA-16 group mice have obviously prolonged movement time and movement distance in the open field experiment and have statistical difference (P <0.05), which indicates that the compound TA-16 has positive influence on the movement capability of the MPTP-induced Parkinson model mice.

TABLE 9 open field test results

Note: in comparison to the set of models,^*P<0.05,^**P<0.01,^***P<0.001。

3.3 autonomic Activity recording

As can be seen from table 10 and fig. 11, the number of times of the autonomous activity in the model group mice was significantly reduced compared to the blank group, and the number of times of the autonomous activity was statistically different (P <0.05), indicating that the parkinson model was successful. Compared with the model group, the times of the independent activity war and the times of the independent activity of the compound TA-16 group mice are obviously increased and have statistical differences (P is less than 0.01), which indicates that the compound TA-16 has a promoting effect on the independent activity of the MPTP-induced Parkinson model mice.

TABLE 10 autonomic Activity test results

Note: in comparison to the set of models,^*P<0.05,^**P<0.01,^***P<0.001。

3.4 suspension experiment

As can be seen from table 12 and fig. 13, the scores in the model group mouse suspension experiment were not significantly different and were not statistically different (P >0.05), but the scores tended to decrease compared to the blank group. Compared with the model group, the YT group mice have no obvious difference in score in suspension experiments, have no statistical difference (P >0.05), and have a rising trend in score.

TABLE 12 results of the suspension experiment

Note: in comparison to the set of models,^*P<0.05,^**P<0.01,^***P<0.001。

the experimental results show that the compound TA-16 has certain improvement on the motor capacity and limb coordination capacity of the MPTP-induced Parkinson model mouse, and the compound TA-16 has certain treatment effect on the MPTP-induced Parkinson model mouse.

In conclusion, the compound shown in the formula I, or the pharmaceutically acceptable salt or the solvate thereof can selectively inhibit TDO and IDO1, and has a remarkable inhibitory effect on TDO and/or IDO 1. In addition, the compound prepared by the invention has obvious anti-tumor effect, has certain treatment effect on Parkinson's disease and Alzheimer's disease, and has good application prospect in the field of medicine preparation.

Claims

1. A compound represented by formula I, or a pharmaceutically acceptable salt, or solvate thereof:

wherein, A ring is selected from

Wherein R is_dSelected from H, C1-4 alkoxy, R_eSelected from C1-4 alkyl;

and when ring A is

R₃、R₄Is H, R₁When it is bromine, R₆Is not:

when ring A is

R₃、R₄Is H, R₁When it is methyl, R₆Is not:

when ring A is

R₁、R₃、R₄When is H, R₆Is not:

2. the compound of claim 1, wherein: in a compound of formula I:

a ring is selected from

R₆is selected from

-NH₂Wherein: x is selected from NH and O; e is selected from C ═ O, 0-2 methylene groups; r₂Selected from substituted or unsubstituted 4-6 membered saturated carbocyclic or heterocyclic ring, substituted or unsubstituted 4-6 membered unsaturated carbocyclic or heterocyclic ring, substitutedOr an unsubstituted fused ring,

Wherein R is_dSelected from H, C1-4 alkoxy, R_eSelected from C1-4 alkyl;

3. The compound of claim 2, wherein: in a compound of formula I:

a ring is selected from

R₆is selected from

-NH₂Wherein X is selected from NH and O; e is selected from C ═ O, 0-2 methylene groups; r₂Selected from substituted or unsubstituted 4-6 membered saturated nitrogen heterocycle, 4-6 membered unsaturated nitrogen heterocycle, substituted or unsubstituted 4-6 membered saturated carbon heterocycle, substituted or unsubstituted benzene ring, indolyl,

The R is₂The substituent on the substituent is selected from hydroxyl and nitroCarboxy, halogen, halogenated or non-halogenated C1-3 alkyl, C1-3 alkoxy,

Wherein R is_dSelected from H, C1-3 alkoxy, R_eSelected from C1-3 alkyl;

4. A compound according to any one of claims 1 to 3, wherein: the structure of the compound is shown as a formula I-1:

wherein E is selected from 0-2 methylene groups;

R₁selected from H, halogen, halogenated C1 alkyl, cyano, amino, -COOR_fWherein R is_fIs selected from methyl; r₄、R₃Each independently selected from H, halogen;

Wherein R is_dSelected from H, C1-3 alkoxy, R_eSelected from C1-3 alkyl.

5. The compound of claim 4, wherein: a compound of formula I-1:

e is selected from 0-1 methylene;

Wherein R is_dSelected from H, methoxy, R_eSelected from methyl.

6. The compound of claim 5, wherein: a compound of formula I-1:

e is selected from 0-1 methylene;

R₃、R₄selected from H, halogen, preferably H, bromine;

7. A compound according to any one of claims 1 to 3, wherein: the structure of the compound is shown as a formula I-2:

8. A compound according to any one of claims 1 to 3, wherein: the structure of the compound is shown as a formula I-3:

R₆selected from amino,

R₃、R₄is selected from H;

R₁selected from halogens, preferably bromine.

9. A compound according to any one of claims 1 to 3, wherein: in a compound of formula I:

a ring is selected from

R₆Is selected from

R2 is selected from substituted or unsubstituted benzene ring, and the substituent is selected from hydroxyl and nitro;

R₁、R₃、R₄each independently selected from H.

10. The compound of any one of claims 1-9, wherein: the structure of the compound is:

11. use of an indazole derivative for the preparation of TDO and/or IDO1 inhibitors, wherein: the indazole derivative is

12. Use according to claim 11, characterized in that: the inhibitor is a medicament for treating neurodegenerative diseases, tumors, infectious diseases associated with cellular immune activation.

13. Use according to claim 12, characterized in that: the neurodegenerative disease is preferably Parkinson disease, Alzheimer disease and depression; the tumor is malignant tumor, preferably colon cancer and brain glioma.