CN106928229B

CN106928229B - Application of tryptanthrin and derivatives thereof in preparation of hIDO2 inhibitor

Info

Publication number: CN106928229B
Application number: CN201511023955.XA
Authority: CN
Inventors: 匡春香; 杨春
Original assignee: Suzhou Ruying Biomedical Co ltd
Current assignee: Suzhou Ruying Biomedical Co., Ltd.
Priority date: 2015-12-30
Filing date: 2015-12-30
Publication date: 2021-03-19
Anticipated expiration: 2035-12-30
Also published as: WO2017114260A1; CN106928229A

Abstract

The invention provides an indoleamine2,3-dioxygenase2 inhibitor and application thereof. Specifically, the invention provides a compound of formula I or a pharmaceutically acceptable salt thereof, which has excellent indoleamine2,3-dioxygenase2 inhibiting and antitumor effects. The invention also provides a pharmaceutical composition containing the compound and application of the compound in inhibiting indoleamine2, 3-dioxygenase.

Description

Application of tryptanthrin and derivatives thereof in preparation of hIDO2 inhibitor

Technical Field

The present invention relates to the field of pharmaceutical chemistry, and more particularly to compounds of formula I and their use in inhibiting indoleamine2,3-dioxygenase 2.

Background

Indoleamine2,3-dioxygenase1 (indoamine 2,3-dioxygenase1, IDO1) is the first rate-limiting enzyme in mammals that catalyzes the metabolism of tryptophan along the kynurenine pathway (kynurenine pathway). Research shows that IDO1 has high expression in various tumor tissues, can inhibit the proliferation of lymphocytes by reducing the tryptophan concentration in the tumor microenvironment and plays an important role in tumor immune escape, so that the high expression of IDO1 is considered to be one of important factors causing the body to generate immune tolerance to tumors.

Ball et al, in 2007, discovered an enzyme that is very similar to IDO1 in terms of coding gene sequence, molecular structure and biological activity, and was named indoamine 1 (3-dioxygenase-like protein), i.e., the present indoleamine2,3-dioxygenase2 (indoamine 2,3-dioxygenase2, IDO 2). IDO2 is located downstream of IDO1 gene, and structurally the sequence of IDO2 is highly similar to that of IDO1, located on chromosome eight of human and mouse, and highly expressed in kidney, reproductive system and liver of mouse. It has been found that IDO2 is expressed in cancer tissues of stomach, colon, pancreas and kidney cancers. Several studies have shown that IDO2, although less active than IDO1 and not dominant, also catalyzes the degradation of tryptophan. Research shows that IDO2 is closely related to IDO1, and the two probably act together to participate in major diseases or physiological activities such as tumorigenesis, tumor immune tolerance, inflammatory reaction and the like, so that IDO2 is expected to be an effective drug target for treating the major diseases of human beings after IDO 1.

The important role of IDO1 in tumor immune tolerance is acknowledged, and the gene expression, signal transduction, and application in the treatment of diseases (including cancer, aids, alzheimer's disease, depression, cataract and other serious diseases) with pathological features of IDO1 mediated tryptophan metabolic pathway are hot research points. The research on the biological characteristics, functions and immune tolerance of IDO2 is relatively less, and the development of a high-efficiency inhibitor for selectively inhibiting IDO2 is urgently needed in the field, so that a new drug target and a new idea are provided for treating human major diseases mediated by IDO2 alone or by both IDO1 and IDO 2.

Disclosure of Invention

The invention aims to provide a compound for inhibiting activity of indoleamine2,3-dioxygenase2(IDO2) and application thereof.

In a first aspect of the invention, there is provided the use of a compound of formula I, or a pharmaceutically acceptable salt thereof, for the manufacture of a pharmaceutical composition or formulation for inhibiting the activity of indoleamine2,3-dioxygenase2(IDO2),

wherein the content of the first and second substances,

r1, R2, R3 and R4 are each independently selected from the group consisting of: H. CH (CH)₃Halomethyl, F, Cl, Br, and-NO₂。

In another preferred embodiment, the R1, R2, R3 and R4 are the same or different.

In another preferred embodiment, at least two of R1, R2, R3 and R4 are H.

In another preferred embodiment, R1, R2 and R4 are all H.

In another preferred embodiment, R1, R2 and R3 are all H.

In another preferred embodiment, both R2 and R4 are H.

In another preferred embodiment, at least one of R2 and R3 is halogen, methyl, or-NO₂。

In another preferred embodiment, one of R2 and R3 is halogen, methyl, or-NO₂And the other is H.

In another preferred embodiment, one of R2 and R3 is halogen or-NO₂And the other is H.

In another preferred embodiment, one of R2 and R3 is-NO₂And the other is H.

In another preferred embodiment, the compound of formula I is selected from the group consisting of:

in another preferred embodiment, the compound of formula I is selected from the group consisting of: compound 1, compound 4.

In another preferred embodiment, the compound of formula I is selected from the group consisting of: compound 2, compound 3, and compound 8.

In another preferred embodiment, the indoleamine2,3-dioxygenase2 is human indoleamine2,3-dioxygenase 2.

In another preferred embodiment, the pharmaceutical composition or formulation is also used to inhibit the activity of indoleamine2,3-dioxygenase 1(IDO 1).

In another preferred embodiment, said pharmaceutical composition or formulation is also for use in the prevention or treatment of diseases associated with IDO 2.

In another preferred embodiment, the "disease related to IDO 2" includes diseases mediated by IDO2 alone or diseases mediated by IDO1 and IDO2 together.

In another preferred embodiment, the disease comprises a disorder of tryptophan metabolism.

In another preferred embodiment, the disease is selected from the group consisting of: tumorigenesis, tumor immune tolerance, cancer, AIDS, Alzheimer's disease, depression, and cataracts.

In another preferred embodiment, the cancer is selected from the group consisting of: liver cancer, lung cancer, cervical cancer, breast cancer, melanoma, pancreatic cancer, colon cancer, kidney cancer, prostate cancer, and the like.

In another preferred embodiment, the pharmaceutical composition comprises 0.001-99 wt%, preferably 0.1-90 wt%, more preferably 1-80 wt% of the compound of formula I or its pharmaceutically acceptable salt, based on the total weight of the composition.

In another preferred embodiment, the dosage form of the pharmaceutical composition is an oral dosage form or an injection dosage form.

In another preferred example, the oral dosage form comprises tablets, capsules, films, granules and the like, and also comprises sustained-release or non-sustained-release dosage forms.

In another preferred embodiment, the pharmaceutical composition may further comprise other pharmaceutically active ingredients.

In another preferred embodiment, the other pharmaceutically active ingredient comprises a compound, an antibody, a nucleic acid molecule, an anti-tumor immune cell, or a combination thereof.

In another preferred embodiment, the compound comprises chemotherapeutic drugs for treating tumors, such as cisplatin and paclitaxel.

In another preferred embodiment, the antibody comprises an anti-tumor antibody, such as an antibody against Her2, an antibody against VEGF2, or a combination thereof.

In another preferred embodiment, the anti-tumor immune cells comprise CAR-T cells.

In a second aspect of the invention, there is provided an in vitro non-therapeutic method of inhibiting indoleamine2,3-dioxygenase2 activity comprising the steps of:

(a) indoleamine2,3-dioxygenase2 is contacted with a compound of formula I according to the first aspect of the invention, or a pharmaceutically acceptable salt thereof, such that the activity of indoleamine2,3-dioxygenase2 is inhibited.

In another preferred embodiment, the indoleamine2,3-dioxygenase2 is in a free state or is expressed in a cell.

In another preferred embodiment, in step (a), the compound of formula I, or a pharmaceutically acceptable salt thereof, is added to a cell culture system such that it is contacted with indoleamine2,3-dioxygenase 2.

In another preferred embodiment, the cell is a normal cell or a tumor cell.

In another preferred embodiment, the cell is a mammalian cell.

In another preferred embodiment, the cell is a human cell.

In a third aspect of the invention, there is provided a method of inhibiting indoleamine2,3-dioxygenase2 or treating a disease associated with IDO2, said method comprising: (i) administering to a subject in need thereof a compound of formula I as described in the first aspect of the invention, or a pharmaceutically acceptable salt thereof.

In another preferred embodiment, the subject includes human and non-human mammals.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Drawings

FIG. 1 shows the inhibition of IDO2 activity by the compounds of the present invention.

Detailed Description

The inventor of the invention has studied extensively and intensively, and found a compound with a structure shown as formula I for the first time unexpectedly that the activity of IDO2 can be obviously inhibited. Experiments show that the compound shown in the formula I has a better inhibiting effect on IDO2, the inhibiting effect is better than that of an IDO2 inhibitor 1-MT (1-methyl tryptophan) which is universal in vitro and in vivo experiments, and the compound shown in the formula I is a reversible inhibitor. The compound of formula I can be used for treating or preventing human major diseases including cancer, which are mediated by IDO2 alone or IDO1 and IDO2 jointly. On the basis of this, the present invention has been completed.

Term(s) for

As used herein, "a compound of the present invention", "tryptanthrin and its derivatives of the present invention", or "a compound of formula I" are used interchangeably to refer to a compound of formula I, or a racemate, a enantiomer, or a pharmaceutically acceptable salt thereof. It is to be understood that the term also includes mixtures of the above components.

In the formula, each group is as defined above.

The compound of the invention not only has an inhibiting effect on IDO2, but also has a certain inhibiting effect on IDO 1.

In the present invention, pharmaceutically acceptable salts of the compounds of formula I are also included. The term "pharmaceutically acceptable salt" refers to a salt of a compound of the present invention with an acid or base that is suitable for use as a pharmaceutical. Pharmaceutically acceptable salts include inorganic and organic salts. One preferred class of salts is that formed by reacting a compound of the present invention with an acid. Suitable acids for forming the salts include, but are not limited to: inorganic acids such as hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid, phosphoric acid, etc., organic acids such as formic acid, acetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, picric acid, methanesulfonic acid, phenylmethanesulfonic acid, benzenesulfonic acid, etc.; and acidic amino acids such as aspartic acid and glutamic acid.

Tryptanthrin is a known compound, and the compound of formula I of the present invention can be prepared by methods well known to those skilled in the art, and there is no particular limitation on the reaction parameters of each step.

Indoleamine2,3-dioxygenase

Indoleamine2,3-dioxygenase1 (indoamine 2,3-dioxygenase1, IDO1) is the first rate-limiting enzyme in mammals that catalyzes the metabolism of tryptophan along the kynurenine pathway (kynurenine pathway). Research shows that IDO1 has high expression in various tumor tissues, can inhibit the proliferation of lymphocytes by reducing the tryptophan concentration in the tumor microenvironment, and plays an important role in tumor immune escape.

As used herein, "indoleamine 2,3-dioxygenase 2", indoamine 2,3-dioxygenase2(IDO2), is an enzyme found in 2007 that is similar to IDO1 in coding gene sequence, molecular structure and biological activity. IDO2 is located downstream of IDO1 gene, and at the amino acid level, human IDO1 and IDO2 are approximately 43% identical. Furthermore, IDO2 also has expression characteristics different from IDO1, IDO2 is expressed mainly in renal tubules, liver and sperm, but IDO1 and IDO2 are both expressed by antigen presenting dendritic cells. Studies have shown that IDO1 and IDO2 have different expression patterns and different kinetic properties, and therefore IDO2 may have a different function than IDO 1. It has been found that IDO2 is expressed in cancer tissues of stomach cancer, colon cancer, pancreatic cancer and renal cancer.

Sorensen study and Munn et al, cultured in a mixture of IDO1 positive dendritic cells and T lymphocytes, and treated with IDO1 and IDO2 specific inhibitors D-1-methyltrypophan (D-1 MT) and L-1-methyltrypophan (L-1 MT) to show that T cells all have the expression of proliferation, indicating that inhibition of IDO1 or IDO2 activity promotes the proliferation of lymphocytes. Hou et al found that the specific inhibitor D-1-MT of IDO2 and cyclophosphamide were combined to have a significant tumor-reducing effect in melanoma studies, which was more significant than the specific inhibitor L-1-MT of IDO 1. But also the survival rate is obviously prolonged in the D-1-MT group. The research result shows that the inhibition of IDO2 has more effects on the aspects of delaying the growth of the tumor and reducing the tumor than the inhibition of IDO1, and the inhibition is supposed to play an important role in immune tolerance.

The research of Merlo et al finds that compared with the wild type rheumatoid arthritis mouse, the IDO2 gene knockout mouse has reduced pathogenic autoimmune antibody and antibody secretion cells in vivo and reduced joint inflammation, while the IDO1 gene knockout mouse does not have the phenomenon, and indicates that IDO2 is an important mediating molecule for generating autoantibody and generating inflammation.

Type of inhibition

The inventors have found that the type of inhibition of IDO2 and/or IDO1 is different for different compounds. In the present invention, the inhibition types mainly include: competitive, anti-competitive, non-competitive, and mixed-type competitive.

Compositions and methods of administration

The present invention provides a composition for inhibiting indoleamine2,3-dioxygenase 2. The composition includes (but is not limited to): pharmaceutical compositions, food compositions, dietary supplements, beverage compositions, and the like.

In the present invention, the pharmaceutical composition can be directly used for disease treatment, for example, for antitumor treatment. When the medicinal preparation is used, other therapeutic agents such as antitumor medicaments and the like can be used simultaneously.

The invention also provides a pharmaceutical composition comprising a safe and effective amount of a compound of the invention and a pharmaceutically acceptable carrier or excipient. Such vectors include (but are not limited to): saline, buffer, dextrose, water, glycerol, ethanol, powders, and combinations thereof. The pharmaceutical preparation should be compatible with the mode of administration.

In the case of pharmaceutical compositions, the compositions of the present invention may be prepared in the form of injections, for example, by conventional methods using physiological saline or aqueous solutions containing glucose and other adjuvants. Pharmaceutical compositions, such as tablets and capsules, can be prepared by conventional methods. Pharmaceutical compositions such as injections, solutions, tablets and capsules are preferably manufactured under sterile conditions. The pharmaceutical combination of the present invention may also be formulated as a powder for inhalation by nebulization. The amount of active ingredient administered is a therapeutically effective amount, for example from about 1 microgram per kilogram of body weight to about 5 milligrams per kilogram of body weight per day. In addition, the indoleamine2,3-dioxygenase2 inhibitors of the present invention may also be used with other therapeutic agents.

For the pharmaceutical compositions of the present invention, administration to a subject in need thereof (e.g., human and non-human mammals) can be by conventional means. Representative modes of administration include (but are not limited to): oral administration, injection, aerosol inhalation, etc.

In the case of pharmaceutical compositions, a safe and effective amount of the drug is administered to the mammal, wherein the safe and effective amount is generally at least about 10 micrograms/kg body weight, and in most cases no more than about 8 mg/kg body weight, preferably the dose is from about 10 micrograms/kg body weight to about 1 mg/kg body weight. Of course, the particular dosage will depend upon such factors as the route of administration, the health of the patient, and the like, and is within the skill of the skilled practitioner.

The main advantages of the invention include:

(a) the compound of formula I has a better inhibiting effect on IDO 2.

(b) The compounds of formula I of the present invention also have an inhibitory effect on IDO 1.

(c) The compounds of formula I of the present invention have better therapeutic potential for IDO1 or IDO2 mediated tumor immune escape.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out under conventional conditions or conditions recommended by the manufacturers. Unless otherwise indicated, percentages and parts are by weight.

Universal material

The nucleotide sequences of IDO1 and IDO2 are disclosed, and the active IDO1 and IDO2 in the examples are human-derived IDO1 and IDO2, which can be prepared by conventional molecular cloning means.

In the examples, the inhibitors to be tested include IDO inhibitors L-1-MT and D-1-MT (L-1-methyltryptophan, D-1-methyltryptophan, both commercially available) which are commonly used in vitro and in vivo experiments at present, and the compounds of the present invention selected from the group consisting of:

example 1

Preliminary screening for enzyme level IDO2 inhibitors

In a 500. mu.L standard assay system, 50mmol/L potassium phosphate buffer (pH 7.5), 200. mu.g/mL catalase, 40mmol/L ascorbic acid, 20. mu. mol/L methylene blue, a substrate L-tryptophan (0-40mM) at an appropriate concentration, and an IDO2 inhibitor to be tested (including the compound of the present invention, L-1-MT, and D-1-MT) at a final concentration of 10. mu.M are mixed, the mixture is subjected to a 37 ℃ water bath for 5min, then IDO2 is added to the mixture, the mixture is subjected to a reaction at 37 ℃ for 30min, and after the completion of the enzymatic reaction, 200. mu.L 30% (w/v) trichloroacetic acid is added to terminate the reaction, and then the mixture is heated in a 65 ℃ water bath for 15min to complete the conversion of the reaction product from N-formylkynurenine to kynurenine. Centrifuging at 138000 Xg for 10min, sucking 100 μ L supernatant, mixing with 2 ‰ (w/v) acetic acid solution of dimethylaminobenzaldehyde, reacting kynurenine with the solution to yellow, and detecting absorbance at 492nm with enzyme-labeling instrument.

The initial screening results are shown in figure 1, and the inhibition rate of 9 compounds of the invention on IDO2 is remarkably higher than that of L-1-MT and D-1-MT (the inhibition rate is less than 8 percent). Among them, compound 8 has the best inhibitory effect on IDO2, with an inhibitory rate of 63%; the inhibition rate of the rest compounds on IDO2 is 18-47%. The preliminary screening result of the compound of this example was the subsequent IC₅₀Value, Ki value determination, and inhibition type determination provide the basis for data validation.

Example 2

Inhibition type and Ki value determination of the Compounds of the invention

In 500. mu.L of the detection system described in example 1, substrate L-tryptophan was added at different concentrations (20, 30, 40mM or 20, 25, 35mM) and at each substrate concentration, a gradient of the inhibitor to be detected (including the compound of the present invention, L-1-MT and D-1-MT) was added at different concentrations, and no inhibitor was added to the control group, the mixture was subjected to a 37 ℃ water bath for 5min, 10. mu.L of IDO2 (about 1. mu.M) was added, the reaction was carried out at 37 ℃ for 30min, 200. mu.L of 30% (w/v) trichloroacetic acid was added after the completion of the enzymatic reaction, and then the reaction product was heated in a 65 ℃ water bath for 15min to complete the conversion from N-formylkynurenine to kynurenine. Centrifuging at 138000 Xg for 10min, sucking 100 μ L supernatant, mixing with 2 ‰ (w/v) acetic acid solution of dimethylaminobenzaldehyde, reacting kynurenine with the solution to yellow, and detecting absorbance at 492nm with enzyme-labeling instrument. Determining the type of the inhibitor by a Dixon mapping method (1/v- [ i ]); ki values of the inhibitors can be obtained by plotting [ S ]/v to [ i ].

The similar method determines the inhibition type and Ki value of the inhibitor to be tested on IDO 1.

The results are shown in the following table: the tryptanthrin and the derivatives thereof are reversible inhibitors of IDO2, wherein the compound 8 is a counter-competitive inhibitor, the Ki value for inhibiting IDO2 is 0.97 mu M and reaches a nanomolar level (0.97 mu M, namely 970nM) which is only 0.23 percent of L-1-MT, and in addition, the compound 8 has strong inhibition effect on IDO1, and the Ki value is 0.054 and is only 0.16 percent of L-1-MT. Ki values of compound 2 and compound 3 are 3.51 and 5.62 mu M respectively, and are both less than 10 mu M; even compound 4, which has a relatively poor inhibitory effect, has a Ki value of 1/4 for L-1-MT. Therefore, the compound of the present invention has an excellent inhibitory effect on IDO 2. In addition, the compound of the invention can inhibit both IDO1 and IDO2, thereby having better therapeutic potential for IDO1 or IDO2 mediated tumor immune escape.

Note: ND: no measurement was made; and (2) preparing: not measured.

Example 3

Half-effective inhibitory concentration IC of the compounds of the invention₅₀Determination of value

Determination of IC's for inhibition of IDO1 and IDO2 by Compounds of the invention at in vitro and cellular levels, respectively₅₀The value is obtained.

In vitro level (enzyme level): in 500. mu.L of the assay system described in example 1, 30mM of the substrate L-tryptophan and the inhibitors (including the compound of the present invention, L-1-MT and D-1-MT) at different concentration gradients were added, and the reaction was carried out in the same manner as in example 1 except that the control group was not added with the inhibitors and the other treatments were carried outAfter completion, the absorbance was measured at 492nm using a microplate reader. The inhibition rate is plotted against the inhibitor concentration, and the IC is calculated by using the modified Kouyan method₅₀The value is obtained.

Cellular level: u87MG cell line (ATCC No.: HTB-14) was cultured in DMEM high-sugar medium containing 10% fetal bovine serum at 37 ℃ in 5% CO₂Culturing in an incubator. And (3) blowing and beating the cells, uniformly passaging the cells in a 6-well plate, and performing transfection when the cells grow to 80-90% of fusion. The procedure was performed according to the Lipofectamine 2000 instructions with slight modifications: serum-containing media was aspirated and the cells were washed 2 times with PBS, 1500. mu.L of serum-free media was added to each well. The plasmid and liposome were added to an EP tube pre-filled with 125. mu.L of Opti-MEM medium at a ratio of 1:2 (2.5 ng plasmid and 5. mu.L liposome per well) and gently mixed, the two were mixed after 5min, incubated at room temperature for 20min and then added dropwise to the medium of the cells to be transfected, at 37 ℃ with 5% CO₂Culturing in culture box for 6 hr, changing into DMEM medium containing 10% serum, culturing for 18 hr, and culturing at 2.5 × 10⁴The cells/well density was seeded in 96-well plates at 37 ℃ with 95% humidity and 5% CO₂Culturing for 6h in an incubator to allow cells to adhere to the wall, adding compounds to be detected (including the compound of the invention, L-1-MT and D-1-MT) with different concentration gradients, supplementing the total volume of each well with a cell culture medium containing L-tryptophan (with a final concentration of 200 mu M, filtering and sterilizing) to 200 mu L, incubating for 24h, taking 140 mu L of supernatant to another 96-well plate, adding 10 mu L of 30% (w/v) trichloroacetic acid, heating at 65 ℃ for 15min, and centrifuging at 13800 Xg for 5 min. 100 mu L of supernatant is mixed with glacial acetic acid solution of 2 thousandth (w/v) of p-diaminobenzaldehyde with the same volume, and after the mixture is fully mixed, the absorbance value is detected at 492nm by using a microplate reader. The experiments were grouped into pcDNA3.1(+) -IDO2 transfected (experimental), pcDNA3.1(+) transfected (empty plasmid control) and untransfected (blank control) groups, each with three replicates. The inhibition rate is plotted against the inhibitor concentration, and the IC is calculated by using the modified Kouyan method₅₀Value of

The results are shown in the table below, and the tryptanthrin and the derivatives thereof of the invention are both effective inhibitors of IDO2, and have good inhibitory effect on IDO 1. Among them, Compound 8 had excellent inhibitory effects on both IDO1 and IDO2, of Compound 8IC at enzyme level and cellular level₅₀The values are both significantly lower than that of the general inhibitors L-1-MT and D-1-MT, and the IDO2 inhibitory activity is 185 times and 334 times that of the L-1-MT and D-1-MT respectively (in terms of cell level IC)₅₀Value as an example). IC of enzyme level of Compound 8, Compound 2, Compound 3₅₀The values were 1.87, 7.68 and 8.26. mu.M (each less than 10. mu.M), respectively, and the enzyme level IC of L-1-MT was determined₅₀ Values 1/30, 1/8, 1/7 indicate that the above three compounds have about 30-fold, 8-fold and 7-fold inhibitory activity against hIDO2 at the enzyme level, respectively, as compared to L-1-MT. In conclusion, the compounds of the present invention have excellent IDO2 inhibitory effects at the enzyme level and at the cell level.

Note: ND: no measurement was made; and (2) preparing: not measured.

All data in this invention are mean values from 3 independent experiments (3 replicates of the same compound were set for each experiment). The relative standard deviation RSD (relative deviation/average value) of each group of experimental data calculated by a formula is less than 1.5 percent, which shows that the repeatability and reproducibility of the data are better.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Claims

1. The use of a compound of formula I or a pharmaceutically acceptable salt thereof for the preparation of a pharmaceutical composition or formulation for inhibiting the activity of indoleamine2,3-dioxygenase2,

wherein the content of the first and second substances,

r1, R2, R3 and R4 are each independently selected from the group consisting of: H. CH (CH)₃Halomethyl, F, Cl, Br, and-NO₂

And, the pharmaceutical composition or formulation is also useful for preventing or treating diseases associated with IDO 2;

the 'disease related to IDO 2' is a disease mediated by IDO2 alone or a disease mediated by IDO1 and IDO2 together.

2. The use according to claim 1, wherein at least two of R1, R2, R3 and R4 are H.

3. The use according to claim 1, wherein R1, R2 and R4 are all H.

4. The use according to claim 1, wherein R1, R2 and R3 are all H.

5. The use according to claim 1, wherein at least one of R2 and R3 is halogen, methyl, or-NO₂。

6. The use of claim 1, wherein one of R2 and R3 is halogen, methyl, or-NO₂And the other is H.

7. The use according to claim 1, wherein one of R2 and R3 is-NO₂And the other is H.

8. The use according to claim 1, wherein the compound of formula I is selected from the group consisting of:

9. the use according to claim 8, wherein the compound of formula I is selected from the group consisting of: compound 1, compound 4.

10. The use according to claim 8, wherein the compound of formula I is selected from the group consisting of: compound 2, compound 3, and compound 8.

11. The use according to claim 1, wherein the indoleamine2,3-dioxygenase2 is human indoleamine2,3-dioxygenase 2.

12. The use according to claim 1, wherein the pharmaceutical composition or formulation is further for inhibiting the activity of indoleamine2,3-dioxygenase 1.

13. The use according to claim 1, wherein the disease is a disorder of tryptophan metabolism.

14. The use according to claim 1, wherein the disease is selected from the group consisting of: tumorigenesis, tumor immune tolerance, cancer, AIDS, Alzheimer's disease, depression, and cataracts.

15. The use of claim 1, wherein the pharmaceutical composition comprises 0.001 to 99 wt% of the compound of formula I or a pharmaceutically acceptable salt thereof, based on the total weight of the composition.

16. The use according to claim 15, wherein the pharmaceutical composition comprises 0.1 to 90 wt% of the compound of formula I or a pharmaceutically acceptable salt thereof, based on the total weight of the composition

17. The use according to claim 16, wherein the pharmaceutical composition comprises 1 to 80 wt% of the compound of formula I, or a pharmaceutically acceptable salt thereof, based on the total weight of the composition.

18. The use according to claim 1, wherein the pharmaceutical composition further comprises other pharmaceutically active ingredients, wherein the other pharmaceutically active ingredients are selected from the group consisting of: a compound, an antibody, a nucleic acid molecule, an anti-tumor immune cell, or a combination thereof.

19. The use of claim 18, wherein the compound is a chemotherapeutic agent for treating a tumor;

the antibody is an anti-tumor antibody; and/or

The anti-tumor immune cell is a CAR-T cell.

20. An in vitro non-therapeutic method of inhibiting indoleamine2,3-dioxygenase2 activity comprising the steps of:

(a) contacting indoleamine2,3-dioxygenase2 with a compound of formula I, or a pharmaceutically acceptable salt thereof, as claimed in claim 1, thereby inhibiting the activity of indoleamine2,3-dioxygenase 2.

21. The method of claim 20, wherein indoleamine2,3-dioxygenase2 is in a free state or is expressed in a cell.

22. The method of claim 20, wherein in step (a), the compound of formula I, or a pharmaceutically acceptable salt thereof, is added to a cell culture system such that it is contacted with indoleamine2,3-dioxygenase 2.