CN116459259A

CN116459259A - Application of hydroxypyrimidine compound in preparation of IDO1 inhibitor

Info

Publication number: CN116459259A
Application number: CN202310705617.2A
Authority: CN
Inventors: 山广志; 刘伊彤; 朱志玲; 左利民; 李怡然; 赵学佳; 赵婷; 姜艺菲; 连晓芳; 周霞; 赵圣楠
Original assignee: Institute of Medicinal Biotechnology of CAMS
Current assignee: Institute of Medicinal Biotechnology of CAMS
Priority date: 2023-06-15
Filing date: 2023-06-15
Publication date: 2023-07-21
Anticipated expiration: 2043-06-15
Also published as: CN116459259B

Abstract

The invention provides an application of a hydroxypyrimidine compound in preparation of an IDO1 inhibitor, and belongs to the technical field of pharmaceutical chemistry. The invention provides an application of a hydroxypyrimidine compound with a structure shown in a formula I and/or pharmaceutically acceptable salts thereof in preparation of IDO1 inhibitors or tumor immunotherapy medicaments. The hydroxypyrimidine compounds have obvious enzyme inhibition activity on IDO1 protein at cellular level and biochemical level, and can be used for preparing small-molecule inhibitors targeting anti-tumor target IDO1 and tumor immunotherapy medicaments.Formula I.

Description

Application of hydroxypyrimidine compound in preparation of IDO1 inhibitor

Technical Field

The invention relates to the technical field of pharmaceutical chemistry, in particular to application of a hydroxypyrimidine compound in preparation of an IDO1 inhibitor.

Background

Indoleamine 2,3-dioxygenase (IDO 1) is a heme-containing monomeric enzyme that catalyzes the conversion of L-tryptophan (L-Trp) to kynurenine (Kyn) and its derivatives along the kynurenine pathway (Meireson et al, "IDO Expression in Cancer: different Compartment, different Functionality.

Overexpression of IDO1 in the tumor microenvironment results in local tryptophan level depletion and accumulation of the series kynurenine derivatives. Low levels of L-Trp cause activation of stress responsive kinase (GCN 2) and inactivation of mammalian rapamycin (serine/threonine protein kinase) complex (mTORC 1), interfere with T cell signaling pathways, arrest the G1 phase cell cycle, cause T cell differentiation or apoptosis, and thus disrupt immune regulation mechanisms, forming immunosuppressive microenvironments, promoting tumor local tolerance (Amobi et al, "-Tryptophan Catabolism and Cancer Immunotherapy Targeting IDO Mediated Immune Suppression", adv Exp Med Biol, 1036:129-144, 2017;Brincks et al., "Indoximod opposes the immunosuppressive effects mediated by IDO and TDO via modulation of AhR function and activation of mTORC1", oncotarget, 11 (25): 2438-2461, 2020); the downstream metabolite kynurenine of the kynurenine pathway activates the aromatic hydrocarbon receptor (AhR), promotes the differentiation of human fork head box protein P3 (FOXP 3) regulatory T cells (Tregs), inhibits anti-tumor immune responses, ultimately leading to the failure of effector T cells and activation of Tregs immunosuppressive functions, assisting in tumor evasion immune surveillance (Platten et al, "Cancer Immunotherapy by Targeting IDO1/TDO and Their Downstream Effectors", front Immunol, 5:673, 2014; moon et al, "Targeting the indoleamine 2,3-dioxygenase pathway in cancer", J Immunother Cancer, 3:51, 2015). Therefore, IDO1 is an important target for anti-tumor immune research and anti-tumor drug development.

IDO1 inhibitors are a recent development focus, and various structural types of IDO1 inhibitors have been currently introduced into the clinical setting, indoximod is a derivative inhibitor of a natural substrate analog (Kuffel et al, "Kynurenine: a biomarker for in vitro and in vivo modulation of indoleamine, 3-dioxygenase by 1-Methyl- [ D ] -gyrophan (NSC 721782)", cancer Research, 65 (9_support): 982-982, 2005); epacadenostat (INCB 024360) is a heme-binding IDO1 inhibitor of the hydroxyamidine class and exhibits substrate competition (Liu et al, "Selective inhibition of IDO1 effectively regulates mediators of antitumor immunity", blood, 115 (17): 3520-3530, 2010); navoximod is a class of heme binding inhibitors containing 4-phenylimidazole substituted aromatic hydrocarbons (Zhou et al, "Discovery of novel indoleamine 2,3-dioxygenase 1 (IDO 1) inhibitors by virtual screening", comput Biol Chem, 78:306-316, 2019); linrodostat (BMS-986025) is a heme targeting iron-free to replace Apo-IDO1 (Cherney et al, "Conformative-Analysis-Guided Discovery of 2,3-Disubstituted Pyridine IDO1 Inhibitors", ACS Med Chem Lett, 12 (7): 1143-1150, 2021); PF-06840003 is a tryptophan non-competitive, non-heme binding IDO1 inhibitor (Sun, "Advances in the discovery and development of selective heme-displacing IDO1 inhibitors", expert Opin Drug Discov (10): 1223-1232, 2020). However, to date, only a few drugs have entered the clinical trial stage and have not been approved by the FDA. Further searching for IDO1 inhibitors with novel structure and high activity is a scientific problem to be solved in the field of tumor immunotherapy.

Disclosure of Invention

The invention aims to provide an application of a hydroxypyrimidine compound in preparing an IDO1 inhibitor, wherein the hydroxypyrimidine compound has obvious inhibitory activity on IDO1 protein at a cellular level and a biochemical level.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides an application of a hydroxypyrimidine compound and/or pharmaceutically acceptable salt thereof in preparing an IDO1 inhibitor, wherein the hydroxypyrimidine compound has a structure shown in a formula I:

formula I.

The invention provides an application of a hydroxypyrimidine compound and/or pharmaceutically acceptable salt thereof in preparing tumor immunotherapy medicaments, wherein the hydroxypyrimidine compound has a structure shown in a formula I:

formula I.

Preferably, the medicament comprises an active ingredient and a pharmaceutically acceptable carrier, wherein the active ingredient is a hydroxypyrimidine compound and/or pharmaceutically acceptable salt thereof, and the content of the active ingredient in the medicament is 0.1-99.9wt%.

Preferably, the tumor comprises cervical cancer, breast cancer, brain cancer, colon cancer or non-small cell lung cancer.

Preferably, the pharmaceutically acceptable carrier comprises one or more of water for injection, propylene glycol, mannitol, glycerol, stearic acid, sodium chloride, dextrin, glucose, starch, sucrose, lactose, sodium carboxymethyl starch, polyethylene glycol, alginic acid and polysorbate 80.

Preferably, the dosage form of the medicament comprises tablets, capsules, pills, granules, syrups, emulsions, suspensions, aerosols, injections or suppositories.

Preferably, the mode of administration of the drug includes intravenous, oral, inhalation or rectal administration.

The invention provides an application of a hydroxypyrimidine compound with a structure shown in a formula I and/or pharmaceutically acceptable salts thereof in preparation of an IDO1 inhibitor. The hydroxypyrimidine compounds have obvious enzyme inhibition activity on IDO1 protein at cellular level and biochemical level, and can be used for preparing small-molecule inhibitors targeting anti-tumor target IDO1 and tumor immunotherapy medicaments.

Drawings

FIG. 1 is a graph showing the results of the pharmacophore model 9 constructed in example 1;

FIG. 2 is a graph showing the IDO1 protein inhibition dose-response curve (EC) for cellular level compound 29 in example 2 ₅₀ ) IDO1 protein inhibition dose-response curve (IC) for biochemical level compound 29 ₅₀ ）；

FIG. 3 is EC of Compound 1 ₅₀ A curve;

FIG. 4 is EC of Compound 6 ₅₀ A curve;

FIG. 5 is EC of Compound 9 ₅₀ A curve;

FIG. 6 is EC of Compound 17 ₅₀ A curve;

FIG. 7 is EC of Compound 25 ₅₀ A curve;

FIG. 8 is EC of Compound 28 ₅₀ A curve;

FIG. 9 is EC of Compound 29 ₅₀ A curve;

FIG. 10 is EC of Compound 31 ₅₀ A curve;

FIG. 11 is EC of Compound 32 ₅₀ A curve;

FIG. 12 is a schematic illustration of a combinationEC of object 33 ₅₀ A curve;

FIG. 13 is EC of Compound 40 ₅₀ A curve;

FIG. 14 is a graph showing the binding of compound 29 to IDO1 protein in example 3;

FIG. 15 is a three-dimensional diagram of the interaction of IDO1 protein with Compound 29 in example 4;

FIG. 16 is a two-dimensional graph showing the interaction of IDO1 protein with Compound 29 in example 4.

Detailed Description

formula I.

According to the invention, IDO1 protein is used as a target, a comprehensive virtual screening strategy is adopted, a pharmacophore modeling (HipHop) and a hierarchical molecular docking means are utilized to carry out series screening on Spics and Chembridge databases, and 40 hit compounds are selected and purchased through careful visual screening; through the evaluation of the IDO1 protein inhibitory activity at the cellular level and the biochemical level, the obvious inhibitory activity of the hydroxypyrimidine compound (marked as compound 29, compound ID 7399426) on the IDO1 protein at the cellular level and the biochemical level is finally found. SPR further corroborated studies indicate that compound 29 has direct inhibitory binding to IDO1 protein, and the molecular docking model further illustrates the possible mechanism of action between the two at the molecular level, supporting the conclusion that compound 29 can act as an IDO1 inhibitor with potential research and development prospects.

formula I.

In the invention, the medicament comprises an active ingredient and a pharmaceutically acceptable carrier, wherein the active ingredient is a hydroxypyrimidine compound and/or pharmaceutically acceptable salt thereof, and the content of the active ingredient in the medicament is preferably 0.1-99.9wt%, more preferably 1-99wt%, further preferably 10-90wt%, and even more preferably 30-85wt%. In the present invention, the tumor preferably includes cervical cancer, breast cancer, brain cancer, colon cancer or non-small cell lung cancer. In the present invention, the pharmaceutically acceptable carrier preferably includes one or more of water for injection, propylene glycol, mannitol, glycerin, stearic acid, sodium chloride, dextrin, dextrose, starch, sucrose, lactose, sodium carboxymethyl starch, polyethylene glycol, alginic acid and polysorbate 80. In the present invention, the dosage form of the drug preferably includes tablets, capsules, pills, granules, syrups, emulsions, suspensions, aerosols, injections or suppositories. The preparation method of the medicament in the form of the medicament is not particularly limited, and the method well known to the person skilled in the art can be adopted. In the present invention, the administration mode of the drug preferably includes intravenous injection, oral administration, inhalation or rectal administration.

The technical solutions of the present invention will be clearly and completely described in the following in connection with the embodiments of the present invention. It will be apparent that the described embodiments are only some, but not all, embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

The human cervical cancer cell line Hela used in the examples below was purchased from ATCC; the positive inhibitor epacoadostat was purchased from MCE company; catalase was purchased from Sigma-Aldrich company; interferon-gamma (IFN-gamma) was purchased from Jin Ruisi biotechnology limited; 5-aminolevulinic acid (ALA) is available from Accela; isopropyl- β -D-thiogalactoside (IPTG), ascorbic acid, L-Trp, methylene blue, p-dimethylaminobenzaldehyde (p-DMAB) and imidazole were all purchased from beijing belvedere technologies inc; enspire microplate reader system was purchased from Perkinelmer Inc. of USA.

HeThe la cells were cultured in MEM medium containing 10% fetal bovine serum and 100 U.mL ^-1 Penicillin and 100 U.mL ^-1 Streptomycin; hela cells at 37℃with 5% CO ₂ And culturing and passaging in a constant temperature incubator.

Example 1 database virtual screening

The structural formula of the 6 known IDO1 positive inhibitors used in this example are shown below:

。

using Discovery Studio 2019 software, constructing a pharmacophore model 9 by using Common Feature Pharmacophore Generation program and based on the 6 known IDO1 positive inhibitor structures, preprocessing IDO1 crystal structure (PDB ID: 6O 3I) based on a preparation Protein module, preprocessing and screening a Chembridge and Specs database containing about 176 thousands of compounds based on a Prepare or Filter Ligands module, and forming a 3D class drug database;

screening and reserving the 3D drug-like database by taking a pharmacophore model 9 as a 3D pharmacophore query type, and butting to a butting site of IDO1 through LibDock and CDOCKER module levels; screening the compounds 10% before docking scoring by PAINS and ADMET characteristics to obtain drug-like compounds, clustering by using FCFP_6 fingerprint, and selecting 186 compounds with ideal binding conformation for further visual inspection; finally, 40 commercially available compounds obtained by virtual screening were used as candidates for subsequent bioassays.

Fig. 1 is a graph showing the results of the pharmacophore model 9 constructed in this example, and fig. 1 shows that blue is a hydrophobic feature, orange is an aromatic ring feature, purple is a hydrogen bond donor feature, and green is a hydrogen bond acceptor feature.

The structural formula of the 40 candidates is shown below:

；/>；/>；

；/>；/>；；/>；/>；

；/>；/>；

。

EXAMPLE 2 evaluation of IDO1 protein inhibitory Activity

Cell level: heLa cells were grown at 2X 10 ⁴ Density of wells (200. Mu.L) inoculated in 96 well plates, placed at 37℃in 5% CO ₂ Culturing overnight in a constant temperature incubator. The next day, the original medium was discarded, and 200. Mu.L of cell culture medium containing 100ng/mL of human interferon-gamma, 50. Mu. M L-Trp and different concentrations of test compound was replaced with 5% CO at 37 ℃ ₂ Culturing in an incubator for 48h, mixing 140 mu L of supernatant with 10 mu L of 40% (w/v) trichloroacetic acid solution, and incubating at 65 ℃ for 15min; the mixture was then centrifuged at 2500rpm for 10min to remove the precipitate. Mixing 100 μL of supernatant with 2% (w/v) p-DMAB-glacial acetic acid solution, incubating at room temperature for 10min, reading absorbance at 490nm wavelength, calculating IDO1 inhibition rate, and calculating EC using GraphPad Prism 9 ₅₀ Values.

Biochemical level: in a 100. Mu.L reaction system, the reagent consists of 50mM potassium phosphate buffer (pH=6.5), 10mM ascorbic acid, 10. Mu.M methylene blue, 100. Mu.g/mL catalase, 100. Mu. M L-Trp, 100nM IDO1 and compounds to be tested in different concentrations. The reaction was stopped by adding 40. Mu.L of 40% (w/v) trichloroacetic acid solution at 37℃for 30 min. The sample was incubated at 65℃for 15min to promote Kyn production, and finally 60. Mu.L of a 3.75% (w/v) solution of p-DMAB-glacial acetic acid was added for color development and absorbance measurement at 490nm was performed. Nonlinear regression analysis using GraphPad Prism 9 to determine IC ₅₀ Values.

The inhibition of IDO1 protein activity by compounds at the cellular level was initially evaluated by measuring the change in absorbance at 490nm of the Kyn and p-DMAB plus reaction products in the cell culture supernatant. Initial screening inhibition assay was performed on 40 candidates with epacoadostat as positive control, wherein 11 compounds showed inhibition rate of more than 50%, and the results of the 11 compounds cell level and biochemical level IDO1 protein level inhibition activity are shown in fig. 2 to 13 and table 1, wherein fig. 2 is IDO1 protein inhibition dose-response curve (EC ₅₀ ) IDO1 protein inhibition dose-response curve (IC) for biochemical level compound 29 ₅₀ ) FIG. 3 is EC of Compound 1 ₅₀ Curve, FIG. 4 is EC for Compound 6 ₅₀ Curve, FIG. 5 is EC for Compound 9 ₅₀ Curve, FIG. 6 is EC for Compound 17 ₅₀ FIG. 7 is a graph of EC for Compound 25 ₅₀ FIG. 8 is a graph of EC of Compound 28 ₅₀ FIG. 9 is a graph of EC for compound 29 ₅₀ FIG. 10 is a graph of EC for Compound 31 ₅₀ FIG. 11 is a graph of EC of Compound 32 ₅₀ Curve, FIG. 12 is EC for Compound 33 ₅₀ FIG. 13 is a graph of EC for compound 40 ₅₀ A curve. The results show that the compound 29 is an active candidate containing a hydroxypyrimidine skeleton, has obvious IDO1 protein inhibition effect, and can inhibit the EC of IDO1 protein at the cellular level ₅₀ IC with a value of 53.93+ -1.22 μm and biochemical level ₅₀ The values were 179.+ -. 8.12. Mu.M.

TABLE 1 determination of IDO1 protein inhibitory Activity of 11 Compounds of example 2

Note that: "/" indicates inactive; n=3, and the experimental results are expressed as mean ± standard deviation.

EXAMPLE 3 surface plasmon resonance SPR analysis

Specific binding between compound 29 and IDO1 protein was evaluated based on the Biacore T200 biomolecular interaction analysis system. The CM5 chip was activated by EDC/NHS, then the high purity IDO1 protein was immobilized by amino coupling and the remaining unbound carboxyl sites were blocked with ethanolamine. The running buffer was 5% DMSO PBS-P solution (20mM PBS,2.7mM KCl,137mM NaCl,0.05% tween 20, ph=7.4). Specifically, a series of compound solutions with different concentration gradients (the concentration of the compound 29 is 3.90 mu M, 7.81 mu M, 15.63 mu M, 31.25 mu M, 62.50 mu M, 250 mu M and 500 mu M) are prepared, and the response signals under the concentrations are measured by respectively flowing through the IDO1 protein surface; the binding time and dissociation time were set to 100s and the running flow rate was 30. Mu.L/min. The binding response values at each concentration were recorded in real time and the equilibrium dissociation constant (K) of the test compound was calculated using Biacore T200 Evaluation Software _D ) Value, K _D The value reflects the magnitude of the affinity of the compound for the target, with smaller values having stronger affinities.

The presence of direct interactions between compound 29 and IDO1 protein was tested and the specific results are shown in fig. 14. FIG. 14 is a graph showing the binding between compound 29 and IDO1 protein in example 3. As can be seen from FIG. 14, K between compound 29 and IDO1 protein _D About 65.92. Mu.M, indicating that it has inhibitory binding activity with the purified IDO1 protein.

EXAMPLE 4 molecular docking model Studies

In order to explore the interaction mechanism of the inhibitory activity of compound 29 in depth, molecular docking studies were performed in this example. IDO1 crystal structure (PDB ID: 6O 3I) was pre-treated with DS software, and docking sites were defined within a 12A radius sphere centered on the natural ligand LKP502, with sphere centers x, y, z coordinates 10.721, 27.366, 164.210, respectively. The CDOCKER module is used for precise molecular docking to accurately predict the binding mode between the receptor and the ligand.

FIGS. 15 and 16 are models of predicted binding of IDO1 protein to compound 29 in example 4, and FIG. 15 is a three-dimensional plot of IDO1 protein interactions with compound 29. As can be seen from FIG. 15, instead of pyrimidine occupying a hydrophobic pocket above heme, the oxygen atom of hydroxypyrimidine of compound 29 forms a coordination interaction with heme iron atom; the pyrimidine ring of compound 29 also forms three pi-pi interactions with phenylalanine 163 (PHE 163) and the pyrrolyl group of heme, and at the same time forms an Amide-pi hydrophobic interaction with alanine 264 (ALA 264), and in addition, the oxygen atom of hydroxypyrimidine forms a hydrogen bond interaction with ALA 264. FIG. 16 is a two-dimensional graph showing interaction of IDO1 protein with compound 29. From FIG. 16, it is clear that two hydrogen bonds are formed between the oxygen atom and the hydrogen atom of the amide bond of compound 29 and serine at side chain 235 (SER 235) and glycine at 262 (GLY 262), respectively, which provides a reasonable explanation for the interaction and combination of the two, and supports the conclusion that compound 29 is used as a precursor of novel IDO1 inhibitor.

The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Claims

1. The application of a hydroxypyrimidine compound and/or pharmaceutically acceptable salt thereof in preparing an IDO1 inhibitor, wherein the hydroxypyrimidine compound has a structure shown in a formula I:

formula I.

2. The application of a hydroxypyrimidine compound and/or pharmaceutically acceptable salts thereof in preparing tumor immunotherapy medicaments is disclosed, wherein the hydroxypyrimidine compound has a structure shown in a formula I:

formula I.

3. The use according to claim 2, wherein the medicament comprises an active ingredient and a pharmaceutically acceptable carrier, the active ingredient is a hydroxypyrimidine compound and/or a pharmaceutically acceptable salt thereof, and the content of the active ingredient in the medicament is 0.1-99.9wt%.

4. The use according to claim 2 or 3, wherein the tumour comprises cervical cancer, breast cancer, brain cancer, colon cancer or non-small cell lung cancer.

5. The use according to claim 3, wherein the pharmaceutically acceptable carrier comprises one or more of water for injection, propylene glycol, mannitol, glycerol, stearic acid, sodium chloride, dextrin, glucose, starch, sucrose, lactose, sodium hydroxymethyl starch, polyethylene glycol, alginic acid and polysorbate 80.

6. The use according to claim 3 or 5, wherein the pharmaceutical dosage form comprises a tablet, capsule, pill, granule, syrup, emulsion, suspension, aerosol, injection or suppository.

7. The use according to claim 6, wherein the mode of administration of the medicament comprises intravenous, oral, inhalation or rectal administration.