CN106928230B - Application of N-aryl, benzyl tryptanthrin and derivatives thereof in preparation of hIDO2 inhibitor - Google Patents

Application of N-aryl, benzyl tryptanthrin and derivatives thereof in preparation of hIDO2 inhibitor Download PDF

Info

Publication number
CN106928230B
CN106928230B CN201511024731.0A CN201511024731A CN106928230B CN 106928230 B CN106928230 B CN 106928230B CN 201511024731 A CN201511024731 A CN 201511024731A CN 106928230 B CN106928230 B CN 106928230B
Authority
CN
China
Prior art keywords
compound
ido2
formula
group
use according
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN201511024731.0A
Other languages
Chinese (zh)
Other versions
CN106928230A (en
Inventor
匡春香
杨春
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Suzhou Ruying Biomedical Co Ltd
Original Assignee
Suzhou Ruying Biomedical Co ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Suzhou Ruying Biomedical Co ltd filed Critical Suzhou Ruying Biomedical Co ltd
Priority to CN201511024731.0A priority Critical patent/CN106928230B/en
Priority to PCT/CN2016/111319 priority patent/WO2017114261A1/en
Publication of CN106928230A publication Critical patent/CN106928230A/en
Application granted granted Critical
Publication of CN106928230B publication Critical patent/CN106928230B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
    • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
    • C07D487/04—Ortho-condensed systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
    • A61K31/5375—1,4-Oxazines, e.g. morpholine
    • A61K31/5377—1,4-Oxazines, e.g. morpholine not condensed and containing further heterocyclic rings, e.g. timolol
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Landscapes

  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Animal Behavior & Ethology (AREA)
  • Epidemiology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Pharmacology & Pharmacy (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)

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 N-aryl, benzyl 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),
Figure BDA0000896425190000021
in the formula,
R1is hydrogen or fluorine;
R2is-R5-NR3R4；
Said R5Is substituted or unsubstituted C1-C3 alkylene, or none;
said R3、R4Each independently selected from the group consisting of: H. substituted or unsubstituted C1-C4 alkyl, substituted or unsubstituted C2-C4 alkenyl, substituted or unsubstituted C2-C4 alkynyl, substituted or unsubstituted C3-C6 cycloalkyl;
or R3、R4Together with the adjacent nitrogen atom, form a substituted or unsubstituted 5-7 membered heterocyclic ring, wherein said 5-6 membered saturated ring has 1-2 nitrogen atoms, and 0-2 heteroatoms selected from the group consisting of: o, S, respectively;
the substitution means that one or more hydrogen atoms on the group are substituted with a substituent selected from the group consisting of: C1-C4 alkyl, C1-C4 haloalkyl, an amine protecting group (preferably t-butyloxycarbonyl), and halogen.
In another preferred embodiment, R is5Is methylene.
In another preferred embodiment, R1Is F.
In another preferred embodiment, when said R is3、R4When taken together with an adjacent nitrogen atom to form a substituted or unsubstituted 5-6 membered saturated ring, the nitrogen atom on the ring may optionally have an amine protecting group thereon.
In another preferred embodiment, the 5-7 membered heterocyclic ring is not a heteroaromatic ring.
In another preferred embodiment, the 5-7 membered heterocyclic ring is a saturated heterocyclic ring, preferably a 5-6 membered saturated heterocyclic ring.
In another preferred embodiment, the 5-7 membered heterocyclic ring contains only one or two heteroatoms.
In another preferred embodiment, all heteroatoms in said 5-to 7-membered heterocyclic ring are N.
In another preferred embodiment, R3、R4Each independently selected from the group consisting of: C1-C4 alkyl; or R3、R4Together with the adjacent nitrogen atom, form a substituted or unsubstituted 5-6 membered saturated ring, wherein said 5-6 membered saturated ring has 1 or 2 nitrogen atoms, and optionally 1 is selected fromHeteroatoms of the following group: and O.
In another preferred embodiment, R3、R4Not H at the same time.
In another preferred embodiment, the-NR is3R4Is a cyclic imine.
In another preferred embodiment, the-NR is3R4Is a substituted or unsubstituted group selected from:
Figure BDA0000896425190000031
wherein,
Figure BDA0000896425190000033
represents a linking site;
the substitution means that one or more hydrogen atoms on the group are substituted with a substituent selected from the group consisting of: C1-C4 alkyl, halogen.
In another preferred embodiment, R1Is F.
In another preferred embodiment, the compound of formula I is selected from the group consisting of:
Figure BDA0000896425190000032
Figure BDA0000896425190000041
in another preferred embodiment, the compound of formula I is selected from the group consisting of: compound 2, and compound 5.
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 0.001-99wt%, 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 present 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.
Figure BDA0000896425190000061
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.
The compounds of formula I according to the invention can be prepared by methods well known to the person skilled in the art, without particular limitation to the reaction parameters of the individual steps.
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:
Figure BDA0000896425190000091
Figure BDA0000896425190000101
example 1
Preliminary screening for enzyme level IDO2 inhibitors
In a 500-microliter standard detection system, 50mmol/L potassium phosphate buffer (pH 7.5), 200-g/mL catalase, 40mmol/L ascorbic acid, 20-micromol/L methylene blue, substrate L-tryptophan with appropriate concentration and IDO2 inhibitor to be detected (including the compound of the invention, L-1-MT and D-1-MT) with final concentration of 10-microliter are mixed, the mixed solution is put into a water bath at 37 ℃ for 5 minutes, IDO2 is added into the mixed solution, the mixture is reacted at 37 ℃ for 30 minutes, 200-microliter 30% (w/v) trichloroacetic acid is added after the enzymatic reaction is finished to terminate the reaction, and then the mixture is heated in a water bath kettle at 65 ℃ for 15 minutes 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.
As shown in FIG. 1, the compound of the present invention has significantly higher IDO2 inhibition rate than L-1-MT and D-1-MT under the condition of pH 7.5. The results of the preliminary screening of the compounds of this example provide a basis for data validation for subsequent determination of IC50 values, Ki values, and inhibition type.
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 compounds of the invention are all reversible inhibitors of IDO2, wherein, compound 2 is a non-competitive inhibitor, the Ki value for inhibiting IDO2 is 1.96 mu M and is only 0.46 percent of L-1-MT, and in addition, compound 2 also has stronger inhibiting effect on IDO1, and the Ki value is 4.12 and is slightly higher than the Ki value for inhibiting IDO 2. The compound of the invention can inhibit both IDO1 and IDO2, thereby having better therapeutic potential for IDO1 or IDO2 mediated tumor immune escape.
Figure BDA0000896425190000111
Note: NI: there was no inhibition; 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): 30mM of the substrate L-tryptophan and inhibitors (including the compound of the present invention, L-1-MT and D-1-MT) at different concentration gradients were added to 500. mu.L of the detection system described in example 1, the control group was treated in the same manner as in example 1 except that no inhibitor was added, and the absorbance at 492nm was measured using a microplate reader after the reaction was completed. 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. Blowing and beating the cells to be evenly passagedTransfection was performed in 6-well plates until 80% -90% of the cells had grown confluent. 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 compounds of the invention are effective inhibitors of IDO2, and have good inhibitory effect on IDO 1. Among them, Compound 2 had excellent inhibitory effects on both IDO1 and IDO2, IC of Compound 2 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 inhibition rates are 23.1 times and 41.6 times of the inhibition rates of the general inhibitors L-1-MT and D-1-MT respectively (in terms of cell level IC)50Value as an example). In conclusion, the compounds of the present invention have excellent IDO2 inhibitory effects at the enzyme level and at the cell level.
Figure BDA0000896425190000121
Figure BDA0000896425190000131
Note: NI: there was no inhibition; 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 (15)

1. Use of a compound of formula I, or a pharmaceutically acceptable salt thereof, for the preparation of a pharmaceutical composition or formulation for inhibiting indoleamine2,3-dioxygenase2 activity, for the prevention or treatment of disorders associated with IDO 2; and the disease related to IDO2 is a disease mediated by IDO2 alone or IDO1 and IDO2 are jointly involved in the mediated disease;
Figure 927299DEST_PATH_IMAGE001
(I)
in the formula,
R1is hydrogen or fluorine;
R2is-R5-NR3R4；
Said R5Is a substituted or unsubstituted C1-C3 alkylene group, or a single bond;
said-NR3R4Is a substituted or unsubstituted group selected from:
Figure 175878DEST_PATH_IMAGE002
Figure 915295DEST_PATH_IMAGE003
；
wherein "
Figure 198509DEST_PATH_IMAGE004
"denotes a linking site;
the substitution means that one or more hydrogen atoms on the group are substituted with a substituent selected from the group consisting of: C1-C4 alkyl, halogen.
2. The use according to claim 1, wherein R is5Is methylene.
3. The use according to claim 1, wherein R is1Is F.
4. The use according to claim 1, wherein the compound of formula I is selected from the group consisting of:
Figure 817709DEST_PATH_IMAGE005
Figure 428950DEST_PATH_IMAGE006
Figure 96692DEST_PATH_IMAGE007
Figure 359046DEST_PATH_IMAGE008
Figure 821251DEST_PATH_IMAGE009
Figure DEST_PATH_IMAGE010
。
5. the use according to claim 4, wherein the compound of formula I is selected from the group consisting of:
Figure 388630DEST_PATH_IMAGE011
。
6. the use according to claim 1, wherein the indoleamine2,3-dioxygenase2 is human indoleamine2,3-dioxygenase 2.
7. The use according to claim 1, wherein the pharmaceutical composition or formulation is further for inhibiting the activity of indoleamine2,3-dioxygenase 1.
8. The use according to claim 1, wherein the disease is a disorder of tryptophan metabolism.
9. 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.
10. The use of claim 1, wherein the pharmaceutical composition comprises 0.001 to 99wt% of the compound of formula I or a pharmaceutically acceptable salt thereof, based on the total weight of the composition.
11. The use of claim 1, wherein the pharmaceutical composition further comprises an additional pharmaceutically active ingredient selected from the group consisting of: a compound, an antibody, a nucleic acid molecule, an anti-tumor immune cell, or a combination thereof.
12. The use of claim 11, wherein the compound is a chemotherapeutic agent for treating a tumor;
the antibody is an anti-tumor antibody;
the anti-tumor immune cell is a CAR-T cell.
13. 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.
14. The method of claim 13, wherein the indoleamine2,3-dioxygenase2 is in a free state or is expressed in a cell.
15. The method of claim 13, 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.
CN201511024731.0A 2015-12-30 2015-12-30 Application of N-aryl, benzyl tryptanthrin and derivatives thereof in preparation of hIDO2 inhibitor Active CN106928230B (en)

Priority Applications (2)

Application Number Priority Date Filing Date Title
CN201511024731.0A CN106928230B (en) 2015-12-30 2015-12-30 Application of N-aryl, benzyl tryptanthrin and derivatives thereof in preparation of hIDO2 inhibitor
PCT/CN2016/111319 WO2017114261A1 (en) 2015-12-30 2016-12-21 Uses of n-aryl, benzyltryptanthrin and derivative thereof in preparing hldo2 inhibitor

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201511024731.0A CN106928230B (en) 2015-12-30 2015-12-30 Application of N-aryl, benzyl tryptanthrin and derivatives thereof in preparation of hIDO2 inhibitor

Publications (2)

Publication Number Publication Date
CN106928230A CN106928230A (en) 2017-07-07
CN106928230B true CN106928230B (en) 2021-03-19

Family

ID=59224534

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201511024731.0A Active CN106928230B (en) 2015-12-30 2015-12-30 Application of N-aryl, benzyl tryptanthrin and derivatives thereof in preparation of hIDO2 inhibitor

Country Status (2)

Country Link
CN (1) CN106928230B (en)
WO (1) WO2017114261A1 (en)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111135178A (en) * 2018-11-02 2020-05-12 苏州如鹰生物医药有限公司 Use of tryptanthrin derivatives for the treatment of neurological disorders
CN110183454B (en) * 2019-06-20 2022-01-07 同济大学 Tryptanthrin containing 1,2, 3-triazole and preparation method and application thereof

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103570726A (en) * 2013-07-15 2014-02-12 上海天慈生物谷生物工程有限公司 N-alkyl tryptanthrin derivative, as well as preparation method and application thereof
CN103570727A (en) * 2013-11-12 2014-02-12 复旦大学 N-benzyl tryptanthrin derivative, as well as preparation method and use thereof

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103570726A (en) * 2013-07-15 2014-02-12 上海天慈生物谷生物工程有限公司 N-alkyl tryptanthrin derivative, as well as preparation method and application thereof
CN103570727A (en) * 2013-11-12 2014-02-12 复旦大学 N-benzyl tryptanthrin derivative, as well as preparation method and use thereof

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
吲哚胺-2,3双加氧酶与乳腺癌免疫耐受关系的研究进展;张天，等;《细胞与分子免疫学杂志》;20131231;第29卷(第8期);第893-895页 *

Also Published As

Publication number Publication date
WO2017114261A1 (en) 2017-07-06
CN106928230A (en) 2017-07-07

Similar Documents

Publication Publication Date Title
RU2560683C2 (en) Anticancer agent involving combinations of kinase inhibitory compounds
RU2423980C2 (en) Combinations containing epothilones and protein kinase inhibitors and pharmaceutical application thereof
KR101475167B1 (en) Combination anti-cancer therapy
AU2012246490B2 (en) Therapeutic agent for tumor
JP6864990B2 (en) A cell death inducer for cells having a BRAF gene mutation, a growth inhibitor of the cells, and a pharmaceutical composition for treating a disease caused by abnormal growth of the cells.
JP6090836B2 (en) Anti-tumor activity enhancer of chemotherapeutic agent
JP2023504046A (en) Combination therapy with diaryl macrocycles
SG187828A1 (en) Novel combination therapy for the treatment of cancer
CN106928229B (en) Application of tryptanthrin and derivatives thereof in preparation of hIDO2 inhibitor
TW202200581A (en) Sik-3 inhibitors and uses thereof
CN106928230B (en) Application of N-aryl, benzyl tryptanthrin and derivatives thereof in preparation of hIDO2 inhibitor
CN112386593A (en) Antineoplastic medicine composition containing cideramide and application thereof
JP7016883B2 (en) A pharmaceutical composition for the prevention and treatment of cancer containing an malate-aspartate shuttle inhibitor and an anticancer agent as active ingredients.
AU2021250199B2 (en) Deuterated oxophenylarsine compound and use thereof
CN115403583B (en) Compound for targeted degradation of FAK protein and application thereof
AU2019341976A1 (en) Cancer combination therapy using quinoline carboxamide derivative
JP7381115B2 (en) Compositions and their application in the preparation of medicines for cancer treatment
WO2013059548A1 (en) Compositions and methods for treating cancer using jak2 inhibitor
US20210252000A1 (en) THERAPEUTIC AGENT CONTAINING PYRAZOLO[3,4-d]PYRIMIDINE COMPOUND AS ACTIVE INGREDIENT
Sementino et al. AKT and the Hallmarks of Cancer
Greco Synthesis and biological evaluation of pyrazolo [3, 4-d] pyrimidine derivatives active as SGK1, Fyn and Src kinases inhibitors
US20160367555A1 (en) Use of a Receptor-Type Kinase Modulator for Treating Polycystic Kidney Disease
CN113262223A (en) Application of nilotinib and pharmaceutically acceptable salt thereof in preparation of medicines for treating multiple myeloma
CN115835888A (en) Stable heavy isotopes in amide functions and uses thereof
JP2015163592A (en) Method for treating cancer by combined use of anti-cancer agents

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
TA01 Transfer of patent application right

Effective date of registration: 20190910

Address after: Unit 207-3, R&D Building 168 Yuanfeng Road, Yushan Town, Kunshan City, Suzhou City, Jiangsu Province

Applicant after: Suzhou Ruying Biomedical Co., Ltd.

Address before: 215000 No. 198 Jin Shan Road, Suzhou hi tech Development Zone, Jiangsu, China

Applicant before: SUZHOU KANGZHENG BIOLOGICAL MEDICINE CO., LTD.

TA01 Transfer of patent application right
GR01 Patent grant
GR01 Patent grant