CN112574178A

CN112574178A - Deuterated benzimidazole compound and application thereof as EP300/CBP inhibitor

Info

Publication number: CN112574178A
Application number: CN202011005085.4A
Authority: CN
Inventors: 樊磊; 王飞; 罗童川; 李兴海; 陈元伟
Original assignee: Haichuang Pharmaceutical Co Ltd
Current assignee: Haichuang Pharmaceutical Co Ltd; Hinova Pharmaceuticals Inc
Priority date: 2019-09-27
Filing date: 2020-09-22
Publication date: 2021-03-30

Abstract

The invention discloses a deuterated benzimidazole compound and application thereof as an EP300/CBP inhibitor. Specifically provided is a deuterated benzimidazole compound shown in formula I, or a salt thereof, or a conformational isomer thereof, or a crystal form thereof, or a solvate thereof. The compound has high selectivity to EP300/CBP, and can effectively inhibit the activity of EP 300/CBP; meanwhile, compared with a non-deuterated compound, the compound provided by the invention has the advantages that the metabolic stability and the pharmacokinetic performance are obviously improved. In addition, the compound has excellent inhibition effect on various tumor cells including prostate cancer cells, leukemia cells, breast cancer cells and multiple myeloma cells.Therefore, the compound has good application prospect in preparing EP300/CBP inhibitors and medicines for preventing and/or treating tumors and regulating regulatory T cells.

Description

Deuterated benzimidazole compound and application thereof as EP300/CBP inhibitor

Technical Field

The invention belongs to the field of pharmaceutical synthesis industry, and particularly relates to a deuterated benzimidazole compound with a novel structure and application thereof as an EP300/CBP inhibitor.

Background

Histone Acetyltransferases (HATs) and Histone Deacetylases (HDACs) can affect histone acetylation, and recruitment and normal function of HATs and HDACs are key regulatory steps in gene expression and cell cycle, and functional defects of these enzymes may lead to a variety of diseases including tumors.

The EP300/CBP family, consisting of highly homologous adenovirus E1A-associated 300kDa protein (EP300) and the binding protein (CBP) of cyclic adenosine monophosphate response element binding protein (CREB), is one of the major members of the Histone Acetyltransferase (HAT) family. EP300/CBP is involved in cell cycle progression and cell growth, differentiation and development, is a very important class of co-activators, and can regulate the functions of a variety of key transcriptional regulators. Researches show that the EP300/CBP is highly expressed and activated in various tumors, and the EP300/CBP is closely related to various tumor diseases and is a tumor treatment target with a great application prospect. Thus, EP300/CBP inhibitors are receiving increasing attention from researchers.

Researchers at the Structural genomics society of oxford university (SGC) first designed and synthesized the benzimidazole compound 9a, but 9a had poor selectivity for CBP and BRD4 (CBP: IC50 ═ 4 μmol · L)^-1，BRD4：IC50＝6.3μmol·L^-1). Then, compound 10a (CBP: IC50 ═ 0.12 μmol · L) was obtained by further optimization^-1，BRD4：IC50＝2.4μmol·L^-1) And 11a (SGC-CBP30, CBP: IC50 ═ 69nmol^-1、Kd＝21nmol.L^-1，p300：Kd＝32nmol.L^-1)。

However, the EP300/CBP inhibitors reported at present still cannot meet the clinical requirements, and therefore, it is very important to research more EP300/CBP inhibitors with novel structure, higher selectivity and better inhibitory activity.

Deuterated drugs refer to replacement of a portion of the hydrogen atoms in a drug molecule with deuterium. Because deuterium is close to hydrogen in shape and volume in a drug molecule, deuterated drugs generally retain the biological activity and selectivity of the original drug. Since the C-D bond is more stable than the C-H bond, the C-D bond is less likely to be broken during the chemical reaction of the deuterated drug, and the half-life period of the deuterated drug may be prolonged.

However, due to the complex metabolic processes of biological systems, the pharmacokinetic properties of drugs in the body are influenced by various factors and show corresponding complexity. The change in pharmacokinetic properties of deuterated drugs represents a great chance and unpredictability compared to corresponding non-deuterated drugs. Deuteration at some sites, not only does not prolong half-life, but may shorten it (Scott l. harbeson, Roger d. tung. deuterium in Drug Discovery and Development, P405-406.), deteriorating its pharmacokinetic properties; on the other hand, hydrogen at some positions on a drug molecule is also not easily deuterated due to steric hindrance and the like, so that the deuteration of the drug is not random and a site capable of deuteration is unpredictable.

Therefore, the development of the EP300/CBP inhibitor which has excellent pharmacokinetic properties and high activity simultaneously has a very good application prospect.

Disclosure of Invention

The invention aims to provide a high-activity EP300/CBP inhibitor with better metabolic stability and pharmacokinetic performance.

The invention provides a compound shown as a formula I, or a salt, a conformational isomer, a crystal form or a solvate thereof:

wherein R is₀～R₂₅Each independently selected from H, deuterium, C₁-C₄Alkyl radical, C₃-C₆Cycloalkyl, C substituted by more than 1 deuterium₁-C₄Alkyl, C substituted by more than 1 deuterium₃-C₆A cycloalkyl group; and R is₀～R₂₅At least one group contains deuterium.

Further, R₀～R₂₅Each independently selected from H, deuterium, methyl, CD₃(ii) a And R is₀～R₂₅In which at least one group is deuterium or CD₃。

Further, R₀、R₁、R₁₀Each independently selected from methyl or CD₃，R₂～R₉、R₁₁～R₂₅Each independently selected from hydrogen or deuterium; preferably, R₁₀Is a CD₃。

Further, the structure of the compound is shown as a formula II-1 or a formula II-2:

wherein R is₀～R₂₅As described above.

Further, the compound is one of the following compounds:

the invention also provides application of the compound or a deutero-compound thereof, or a salt thereof, or a conformational isomer thereof, or a crystal form thereof, or a solvate thereof in preparation of an EP300 inhibitor, a CBP inhibitor or an EP300/CBP inhibitor.

Further, the EP300/CBP inhibitor is a medicament for preventing and/or treating tumors and malignant diseases of hematopoietic stem/progenitor cells of myeloid lines and regulating regulatory T cells;

preferably, the tumor is selected from hematological malignancy, gastric cancer, intestinal cancer, cervical cancer, bladder cancer, laryngeal cancer, liver cancer, lung cancer, breast cancer, ovarian cancer, prostate cancer, lymphoma or multiple myeloma, and the myeloid hematopoietic stem/progenitor cell malignancy is leukemia;

more preferably, the lymphoma is non-hodgkin's lymphoma, diffuse large B cell lymphoma and the leukemia is acute myeloid leukemia.

The invention also provides a medicament for treating diseases, which is a preparation prepared by taking the compound, or a deuteron, or a salt, or a conformational isomer, or a crystal form, or a solvate thereof as an active ingredient and adding pharmaceutically acceptable auxiliary materials.

The invention also provides a combined drug which contains the compound, or a deuterogen thereof, or a salt thereof, or a conformational isomer thereof, or a crystal form thereof, or a solvate thereof, which are prepared from unit preparations with the same or different specifications and used for simultaneous or separate administration, other drugs with anti-tumor effects, and a pharmaceutically acceptable carrier.

Further, the other drugs with the anti-tumor effect are chemotherapeutic drugs or radiotherapy drugs, preferably, the chemotherapeutic drugs are targeted drugs; or, the other medicine with the anti-tumor effect is selected from one or more than two of CDK4/6 inhibitor, Parp inhibitor, androgen receptor inhibitor and immune checkpoint inhibitor.

Definitions of terms used in connection with the present invention: the initial definitions provided herein for a group or term apply to that group or term throughout the specification unless otherwise indicated; for terms not specifically defined herein, the meanings that would be given to them by a person skilled in the art are to be given in light of the disclosure and the context.

"EP 300/CBP inhibitor" refers to an inhibitor that is capable of inhibiting both EP300 and CBP activity.

"EP 300" is "P300" and "EP 300/CBP inhibitor" is "P300/CBP inhibitor".

The minimum and maximum values of the carbon atom content in the hydrocarbon group are indicated by a prefix, e.g. prefix C_a-C_bAlkyl represents any alkyl group containing from "a" to "b" carbon atoms. E.g. C₁-C₄The alkyl group is a straight-chain or branched alkyl group having 1 to 4 carbon atoms.

Cycloalkyl refers to a saturated or unsaturated cyclic hydrocarbon substituent; the cyclic hydrocarbon may be monocyclic or polycyclic. For example, "C₃-C₆The cycloalkyl refers to a saturated or unsaturated cycloalkyl with 3-6 carbon atoms in the ring.

By "pharmaceutically acceptable" is meant that the carrier, diluent, excipient, and/or salt formed is generally chemically or physically compatible with the other ingredients comprising a pharmaceutical dosage form and physiologically compatible with the recipient.

"salts" are acid and/or base salts of a compound or a stereoisomer thereof with inorganic and/or organic acids and/or bases, and also include zwitterionic (inner) salts, as well as quaternary ammonium salts, such as alkylammonium salts. These salts can be obtained directly in the final isolation and purification of the compounds. The compound, or a stereoisomer thereof, may be obtained by appropriately (e.g., equivalentlymixing) a certain amount of an acid or a base. These salts may form precipitates in the solution which are collected by filtration, or they may be recovered after evaporation of the solvent, or they may be prepared by reaction in an aqueous medium followed by lyophilization.

The salts described in the present invention include pharmaceutically acceptable salts. The salt in the invention can be hydrochloride, sulfate, citrate, benzene sulfonate, hydrobromide, hydrofluoride, phosphate, acetate, propionate, succinate, oxalate, malate, succinate, fumarate, maleate, tartrate or trifluoroacetate of the compound.

The experimental result shows that the compound has high selectivity to EP300/CBP and can effectively inhibit the activity of EP 300/CBP; meanwhile, compared with a non-deuterated compound, the compound provided by the invention has the advantages that the metabolic stability and the pharmacokinetic performance are obviously improved. In addition, the compound has excellent inhibition effect on various tumor cells including prostate cancer cells, leukemia cells, breast cancer cells and multiple myeloma cells. Therefore, the compound has good application prospect in preparing EP300/CBP inhibitors and medicines for preventing and/or treating tumors and regulating regulatory T cells.

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

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

Detailed Description

The raw materials and equipment used in the invention are known products and are obtained by purchasing commercial products.

Example 1 Synthesis of Compound 100 of the invention

(1) Synthesis of Compound 4-bromo-N- ((1r,4r) -4-methoxycyclohexyl) -2-nitroaniline (Int 1)

SM1(22 g; 0.1mol) was dissolved in acetonitrile (110mL), potassium carbonate (16.6 g; 0.12mol) and trans-4-methoxycyclohexylamine (12.9 g; 0.1mol) were added, and the mixture was heated under reflux for 3 h. The reaction was poured into water (500mL), filtered and washed with water to give Int 1(30 g; 0.09mol) as an orange solid in 91.2% yield.

(2) Compound 4-bromo-N¹Synthesis of (- (1r,4r) -4-methoxycyclohexyl) benzene-1, 2-diamine (Int 2)

Int 1(30 g; 0.09mol) was dissolved in tetrahydrofuran (300mL), water (100mL), aqueous ammonia (28%, 60mL) and finally sodium dithionite (62 g; 0.36mol) were added and stirred at room temperature for 16 hours. The layers were separated by settling, the aqueous phase was extracted with ethyl acetate (50 mL. times.2), the organic phases were combined, washed with saturated brine (200mL), dried over anhydrous sodium sulfate and spun dry to give Int 2(24 g; 0.08mol) in 88.2% yield.

(3) Synthesis of Compound (S) -N- (5-bromo-2- (((1r,4S) -4-methoxycyclohexyl) amino) phenyl) -6-oxopiperidine-2-carboxamide (Int 3)

Int 2(24 g; 0.08mol), (S) -6-oxopiperidine-2-carboxylic acid (12 g; 0.084mol) and DIPEA (12 g; 0.096mol) were added to dichloromethane (240mL), propylphosphoric anhydride (50% in EA; 61 g; 0.096mol) was added dropwise with stirring at room temperature, and after the addition was complete, stirring was continued for 3 hours at room temperature. The Int 3(27 g; 0.064mol) was obtained by column chromatography in 79% yield.

(4) Synthesis of compound (S) -6- (5-bromo-1- ((1r,4S) -4-methoxycyclohexyl) -1H-benzo [ d ] imidazol-2-yl) piperidin-2-one (Int 4)

To acetic acid (270mL) was added Int 3(27 g; 0.064mol) and stirred at 50 ℃ for 5 days. Removing acetic acid by spinning, adjusting the pH value to 7-8 by using saturated sodium bicarbonate aqueous solution, extracting by using dichloromethane (100mL multiplied by 3), and washing an organic phase by using saturated saline water (100 mL). After drying over anhydrous sodium sulfate, spin-drying gave Int4(20.7 g; 0.051mol) with a yield of 80%.

(5) Synthesis of compound (S) -6- (5-bromo-1- ((1r,4S) -4-methoxycyclohexyl) -1H-benzo [ d ] imidazol-2-yl) -1- (3, 4-difluorophenyl) piperidin-2-one (Int 5)

Pyridine (41 mL; 0.52mol), copper acetate monohydrate (12.4 g; 0.066mol) and 3, 4-difluorophenylboronic acid (29 g; 0.2mol) were added in this order to methylene chloride (200mL) in which Int4(20.7 g; 0.051mol) was dissolved, and the mixture was stirred at room temperature for 5 hours. Washing with water and washing with saturated salt water. The Int 5(20 g; 0.04mol) was obtained by column chromatography with a yield of 75.7%.

(6) Synthesis of compound (S) -1- (3, 4-difluorophenyl) -6- (5- (3, 5-dimethylisoxazol-4-yl) -1- ((1r,4S) -4-methoxycyclohexyl) -1H-benzo [ d ] imidazol-2-yl) piperidin-2-one (Int 6)

Int 5(20 g; 0.04mol) was added to a mixed solvent of 1, 4-dioxane (180mL) and water (30mL), followed by addition of Pd (dppf) Cl₂(2.9 g; 0.004mol) and cesium carbonate (26 g; 0.08mol), under nitrogen, and stirred at 100 ℃ for 16 hours. After column chromatography, Int 6(19 g; 0.036mol) was obtained in 92.2% yield.

(7) Synthesis of compound (S) -1- (3, 4-difluorophenyl) -6- (5- (3, 5-dimethylisoxazol-4-yl) -1- ((1r,4S) -4-hydroxycyclohexyl) -1H-benzo [ d ] imidazol-2-yl) piperidin-2-one (Int 7)

Int 6(54 mg; 0.1mmol) was dissolved in dichloromethane (5mL), cooled to 0 ℃ in an ice bath and a solution of boron tribromide (75 mg; 0.3mmol) in dichloromethane (1mL) was added dropwise with stirring. After the addition was complete, the temperature was maintained and stirring was continued for 2 hours. The reaction was dropped into ice water. The organic phases were combined, washed with saturated brine (15mL) and dried over anhydrous sodium sulfate. Spin-dry to obtain Int 7(31 mg; 0.06mmol), 58.9% yield.

(8) Synthesis of compound (S) -1- (3, 4-difluorophenyl) -6- (5- (3, 5-dimethylisoxazol-4-yl) -1- ((1r,4S) -4-deuterated methylcyclohexyl) -1H-benzo [ d ] imidazol-2-yl) piperidin-2-one (100)

Int 7(31 mg; 0.06mmol) was dissolved in tetrahydrofuran (1mL), sodium hydride (60% dispersion in minor oil; 3.6 mg; 0.09mmol) was added with stirring for 2 minutes under cooling in an ice-water bath, and deuterated iodomethane (9 mg; 0.06mmol) was added. The reaction was allowed to stand at this temperature for 30 minutes, poured into ice water (5mL), extracted with methylene chloride (5 mL. times.3), washed with saturated brine (15mL), and dried over anhydrous sodium sulfate. Column chromatography gave compound 100(26 mg; 0.048mmol) in 81.2% yield. Mass (M + H) 538.3. H NMR (400MHz, DMSO) δ 7.79(d, J ═ 8.6Hz,1H),7.70(s,1H), 7.41-7.30 (m,2H),7.15(d, J ═ 8.5Hz,1H),7.06(s,1H),5.77(s,1H),4.38(s,1H),3.40(s,2H),2.56(s,2H),2.41(s,3H),2.27(d, J ═ 22.7Hz,4H),2.18(d, J ═ 13.8Hz,2H),2.01(d, J ═ 14.7Hz,3H),1.80(s,2H), 1.42-1.34 (m,2H),1.25(d, J ═ 9.2Hz,1H)

Example 2 Synthesis of Compound 101 of the invention

The synthetic route for the compound (S) -1- (3, 4-difluorophenyl) -6- (5- (3, 5-dimethylisoxazol-4-yl) -1- ((1r,4S) -4-deuterated methylcyclohexyl) -1H-benzo [ d ] imidazol-2-yl) piperidin-2-one-3, 3-didedeuterium (101) is as follows:

100(54 mg; 0.1mmol) was dissolved in CD₃OD/D₂Adding NaOD (41 mg; 1mmol) to O (3mL/0.5mL) at 10 deg.C, stirring for 12 hr, and performing rotary column chromatography to obtain compound 101(36) with yield of 70%. Mass (M + H) 540.2. H NMR (400MHz, DMSO) δ 7.79(d, J ═ 8.6Hz,1H),7.70(s,1H), 7.41-7.30 (m,2H),7.15(d, J ═ 8.5Hz,1H),7.06(s,1H),5.77(s,1H),4.38(s,1H),3.40(s,2H),2.56(s,2H),2.41(s,3H),2.01-2.27(m,7H),1.80(s,2H), 1.42-1.34 (m,2H),1.25(d, J ═ 9.2Hz,1H)

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

Experimental example 1 stability of hepatocyte microsome metabolism of Compound of the present invention

(1) Experimental Material

The microsomes were stored in a-80 ℃ freezer and the information is given in Table 1. Before use, the mixture is melted in a water bath at 37 ℃ and placed on ice for standby after being melted. Other reagents were purchased through local suppliers.

TABLE 1 liver microsome information

Species of species	Goods number	Batch number	Line of	Sex	Supply ofBusiness support
						Human being	452117	38295	N/A	Mixing	Corning
Mouse	M1000	1910002	ICR/CD-1	Male part	Xenotech

(2) Experimental procedure

Step 1: the reaction system was configured as shown in Table 2

Table 2: reaction system configuration method

Step 2: the reaction was preincubated in a water bath at 37 ℃ for 10 minutes. To the reaction system, 40. mu.L of a10 mM NADPH solution was added, and the final concentration of NADPH was 1mM each. As a negative control, 40. mu.L of ultrapure water was used in place of the NADPH solution.

And step 3: the reaction was initiated by adding 4 μ L of 200 μ M test compound or non-deuterated control compound to the reaction, with a final concentration of 2 μ M drug.

And 4, step 4: mu.L of each reaction was removed at 0, 15, 30, 45 and 60 minutes and quenched with 4-fold cold acetonitrile containing internal standards (200nM alprazolam, 200nM labetalol, 2. mu.M ketoprofen, 200nM caffeine). The samples were centrifuged at 3,220g for 45 minutes. After the centrifugation is finished, 90 mu L of supernatant and 90 mu L of ultrapure water are uniformly mixed for LC-MS/MS analysis and detection.

(3) Data analysis

All data calculations were performed by Microsoft Excel software. Peak areas were detected by extracting ion spectra. The in vitro half-life (t) of the parent drug was determined by linear fitting of the percentage disappearance of the parent drug to time_1/2). Half life in vitro (t)_1/2) By slope calculation:

invitrot_1/2＝-(0.693/k)

the in vitro half-life (t) is then determined using the following formula_1/2) Conversion to in vitro clearance (in. mu.L/min/mg):

(4) results of the experiment

Table 3: results of stability of hepatocyte microsome metabolism by Compounds of the invention

The results show that the in vitro half-life of the compounds of the invention in mouse and human hepatocyte microsomes is significantly prolonged and the in vitro clearance rate is significantly reduced compared to non-deuterated control compounds. Indicating that the compounds of the invention have significantly improved metabolic stability over the non-deuterated control compounds.

Experimental example 2 mouse pharmacokinetics of the Compound of the present invention

(1) Experimental methods

Healthy adult ICR mice 18 (compound 9 animals per mode of administration, 3 animals per time point) were fasted overnight (free drinking water), administered by tail vein injection and gavage, respectively, blood was collected at 0.1ml and anticoagulated with EDTA-K2, plasma was separated by centrifugation at 4 ℃ for 5min, and stored at-80 ℃ for testing.

The plasma concentration-time curve is drawn and the main pharmacokinetic parameters are calculated using WinNonlin 6.3 software.

Time to peak T_maxAnd peak concentration C_maxAll adopt measured values;

area under the time curve AUC_allThe value: calculating by adopting a trapezoidal method; AUC_inf＝AUC_all+C_t/k_eCt is the blood concentration at the last measurable time point, k_eTo eliminate the rate constant;

elimination of half-life t_1/2＝0.693/k_e；

Mean residence time MRT ═ AUMC/AUC;

clearance rate CL ═ D/AUC_inf(D is the administered dose);

volume V of steady state distribution_ss＝CL x MRT。

Absolute bioavailability F ═ AUC (AUC)_i.g.x D_i.v.)/(AUC_i.v.x D_i.g.)×100％

(2) Results of the experiment

Table 4: mouse pharmacokinetic results for Compounds of the invention

Remarking: "IV (1 mpk)" in the table means administration by tail vein injection at a dose of 1mg per kg; PO (10mpk) indicates intragastric administration at a dose of 10 mg per kg.

As can be seen from the results in Table 4, the compounds of the present invention have a higher AUC in mice than the non-deuterated control compounds_allValues, longer elimination half-life, lower clearance, higher absolute bioavailability. Illustrative of the inventionThe substance has significantly improved pharmacokinetic properties over the non-deuterated control compound.

Experimental example 3 inhibitory Activity of the Compound of the present invention on CBP/EP300

A. Experimental methods

CBP and EP300 AlphaScreen assay:

1. preparing 1-fold concentration detection buffer

2. Preparation of the Compounds

Compounds were first diluted 1000-fold to final concentration using Precision autosampler (3-fold gradient dilution):

1) 50 μ L of compound stored at a concentration of 10mM was transferred to A2 wells of a 96-well plate using an Echo autosampler.

2) Transfer 30 μ L of DMSO to a1 well, A3 to a12 well with Precision.

3) Precision was used to transfer 15. mu.L from A2 to A3, in turn from A10 to A11. Completing 1: and 3, gradient dilution.

4) The compound plate was centrifuged at 1000 rpm for 1 minute.

3. Preparing a test board

Transfer 20nL to assay plate from each concentration of compound plate with Echo autosampler: from plate-configured a1 to plate-configured a1 and a2, and from plate-configured a2 to plate-configured A3 and a 4.

4. Bromodomain (BRD) binding detection assay

1) Preparing 2-fold protein and polypeptide solution

Proteins and polypeptides were dissolved in 1-fold concentration of assay buffer.

2) 10 μ L of protein and polypeptide solution was transferred to each well of 3 to 24 columns in the assay plate, and 10 μ L of 1-fold assay buffer was transferred to wells of 1 to 2 columns in the assay plate as a negative control.

3) The plates were centrifuged at 1000 rpm for 1 minute.

4) Incubate at room temperature for 15 minutes.

5) Acceptor and donor beads were formulated with 1-fold detection buffer to 2-fold acceptor and donor solutions.

6) Transfer 2-fold of the receptor and donor solutions to the assay plate.

Add 15. mu.L of acceptor and donor solutions and avoid light.

7) The assay plate was centrifuged at 1000 rpm for 1 minute and incubated at room temperature for 60 minutes.

5. The endpoint was read using ensspire and Alpha modes.

6. Fitting of curves

Inputting experimental data into an Excel document and obtaining the inhibition rate Inh% by using equation (1):

equation (1): inh% (Max-Signal)/(Max-Min) × 100

Wherein, Max: control with DMSO, Max: a low control with the addition of DMSO was used,

inputting the obtained data into XL-Fit software and obtaining IC by using equation (2)₅₀The value of (c):

equation (2) Y ═ Bottom + (Top-Bottom)/(1+10^ ((Logicc))₅₀-X)×Hill Slope))

B. Results of the experiment

Table 5: IC of each compound on CBP BRD and EP300 BRD₅₀

The results show that the compound of the invention has obvious inhibitory activity on both CBP BRD and EP300 BRD, and the inhibitory activity on CBP BRD and EP300 BRD is obviously higher than that of the non-deuterated control compound.

Experimental example 4 inhibitory Effect of the Compound of the present invention on proliferation of prostate cancer CWR22RV1 cells

1. The experimental steps are as follows:

subculturing the prostate cancer CWR22RV1 cells with a cell culture solution, inoculating the cells with good growth state into a 96-well plate, wherein each well has 80 mu L of cells, the number of the cells in each well is 1500, and the temperature is 37 ℃ and the CO content is 5 percent₂The cells were incubated overnight in a cell incubator.

② the drug is prepared into 30mM stock solution by dimethyl sulfoxide (DMSO). Immediately before use, the mixture was diluted 3 times with DMSO and then 3 times in a gradient to obtain 9 concentration gradients, and the compound was diluted 200 times with the culture medium (to ensure that the DMSO concentration in the culture system was 0.1%), and the concentration was repeated in 2 wells. mu.L of the diluted compound was added to the cell culture well (final concentration 10. mu.M, 3.3. mu.M, 1.1. mu.M …) and mixed by gentle shaking. Additional 3 negative control wells containing cells only and 3 blank control wells containing culture medium only (6 wells each containing 20. mu.L of culture medium diluted 200-fold in DMSO) were also provided.

2. The detection method comprises the following steps:

(1) after 6 days of incubation, 10. mu.L of CCK-8 was added to each well and incubation was continued for 2.5 hours at 37 ℃ in a 5% CO2 cell incubator.

(2) Absorbance (OD value) was measured at 450nm using a multifunctional microplate reader.

(3) The data were analyzed using the Dose-response-inhibition equation in the software GraphPad Prism6 to yield IC₅₀The value is obtained.

3. Results of the experiment

IC for inhibiting activity of compound of the invention on CWR22RV1 cells₅₀The (nM) results are shown in Table 6.

A represents IC₅₀Less than or equal to 500 nM; b represents IC₅₀Greater than 500nM and less than or equal to 2000 nM; c represents IC₅₀Greater than 2000 nM.

Table 6: IC of each compound on CWR22RV1 cells₅₀

Compound (I)	IC₅₀
		Compound 100	A
Compound 101	A

As can be seen, the compound has obvious inhibitory effect, IC, on prostate cancer CWR22RV1 cells₅₀As low as 500nM or less.

Experimental example 5 inhibitory Effect of the Compound of the present invention on proliferation of other tumor cells 1 Experimental method

Using the same method as in Experimental example 4, CWR22RV1 cells were replaced with tumor cells shown in Table 7, and IC for inhibition of activity of the compounds of the present invention on these tumor cells was tested and calculated₅₀(nM) and the results are shown in Table 7.

2. Results of the experiment

In Table 7, A represents IC₅₀Less than or equal to 500 nM; b represents IC₅₀Greater than 500nM and less than or equal to 2000 nM.

Table 7: IC of Compounds on other tumor cells₅₀

The compound prepared by the invention has obvious inhibition effect on the proliferation of other prostate cancer cells, leukemia cells, breast cancer cells and multiple myeloma cells. The compound of the invention has good inhibition effect on various tumors.

Experimental example 6 pharmacodynamic evaluation of the compound of the present invention in human acute myeloid leukemia cell strain MOLM-16 subcutaneous xenografted NOD

1. Cell culture

Human acute myeloid leukemia MOLM-16 cells were cultured in RPMI1640 medium containing 20% fetal bovine serum. The exponential growth phase of MOLM-16 cells were collected and PBS resuspended to appropriate concentrations for subcutaneous tumor inoculation in mice.

2. Animal model

Experimental mice were inoculated subcutaneously on the right back with 1X 10⁷MOLM-16 cells, which are resuspended in 1:1 PBS and matrigel (0.1 ml/piece) to periodically observe the growth of tumor, until the tumor grows to the average volume of 150 (the average tumor volume is 100-³The administration was randomized and divided into groups according to the tumor size and the mouse body weight. The vehicle was given the same dose as the control group. The day of tumor cell inoculation was defined as day 0.

After the start of the administration, the body weight and tumor size of the mice were measured twice a week. Tumor volume calculation formula: tumor volume (mm)³)＝1/2×(a×b²) (wherein a represents a long diameter and b represents a short diameter).

Student director (tm) (version number 3.1.399.19, supplier student system, Inc.) software was used in the experiments to collect data, including measurements of the long and short diameter of tumors and weighing of animal body weights.

3. Evaluation index

Relative tumor proliferation rate, T/C (%), i.e. the percentage value of the relative tumor volume or tumor weight of the treated and control groups at a certain time point. The calculation formula is as follows:

T/C％＝TRTV/CRTV×100％

wherein, TRTV: mean Relative Tumor Volume (RTV) in treatment groups; CRTV: mean Relative Tumor Volume (RTV) of control group; RTV-Vt/V0, V0 is the tumor volume of the animal at the time of the grouping, and Vt is the tumor volume of the animal after treatment.

Or T/C% ═ TTW/CTW × 100%

Wherein, TTW: mean tumor weights at the end of treatment group experiments; CTW: mean tumor weights at the end of the control experiment.

Relative tumor inhibition, TGI (%), calculated by the formula:

TGI％＝(1-T/C)×100％

where T and C are the Relative Tumor Volume (RTV) or Tumor Weight (TW) at a particular time point in the treated and control groups, respectively.

4. Results of the experiment

Table 8: efficacy of each Compound in MOLM-16 subcutaneous xenograft mouse model

The experimental results show that the compound has good treatment effect on human acute myeloid leukemia model animals, and the treatment effect of the compound is obviously improved compared with that of a non-deuterated compound.

In conclusion, the invention provides a deuterated benzimidazole compound with a novel structure, the compound has high selectivity on EP300/CBP, and can effectively inhibit the activity of EP300/CBP, and simultaneously, compared with a non-deuterated compound, the deuterated compound provided by the invention has remarkably improved metabolic stability and pharmacokinetic performance. In addition, the compound has excellent inhibition effect on various tumor cells including prostate cancer cells, leukemia cells, breast cancer cells and multiple myeloma cells. Therefore, the compound has good application prospect in preparing EP300/CBP inhibitors and medicines for preventing and/or treating tumors and regulating regulatory T cells.

Claims

1. A compound represented by formula I, or a salt thereof, or a conformational isomer thereof, or a crystalline form thereof, or a solvate thereof:

2. The compound according to claim 1, or a salt thereof, or a conformational isomer thereof, or a crystalline form thereof, or a solvate thereof, characterized in thatThe method comprises the following steps: r₀～R₂₅Each independently selected from H, deuterium, methyl, CD₃(ii) a And R is₀～R₂₅In which at least one group is deuterium or CD₃。

3. The compound according to claim 2, or a salt thereof, or a conformational isomer thereof, or a crystalline form thereof, or a solvate thereof, characterized in that: r₀、R₁、R₁₀Each independently selected from methyl or CD₃，R₂～R₉、R₁₁～R₂₅Each independently selected from hydrogen or deuterium; preferably, R₁₀Is a CD₃。

4. A compound according to any one of claims 1-3, or a salt thereof, or a conformational isomer thereof, or a crystalline form thereof, or a solvate thereof, characterized in that: the structure of the compound is shown as a formula II-1 or a formula II-2:

wherein R is₀～R₂₅The method according to any one of claims 1 to 3.

5. The compound of claim 4, or a salt thereof, or a conformational isomer thereof, or a crystalline form thereof, or a solvate thereof, wherein: the compound is one of the following compounds:

6. use of a compound of any one of claims 1-5, or a deuterode thereof, or a salt thereof, or a conformational isomer thereof, or a crystalline form thereof, or a solvate thereof, in the preparation of an EP300 inhibitor, a CBP inhibitor, or an EP300/CBP inhibitor.

7. Use according to claim 6, characterized in that: the EP300/CBP inhibitor is a medicament for preventing and/or treating tumors and malignant diseases of hematopoietic stem/progenitor cells of myeloid lines and regulating and controlling regulatory T cells;

8. A medicament for treating disease characterized by: the medicine is a preparation prepared by taking the compound, or a deuteron thereof, or a salt thereof, or a conformational isomer thereof, or a crystal form thereof, or a solvate thereof, as an active ingredient and adding pharmaceutically acceptable auxiliary materials.

9. A combination comprising: a compound of any one of claims 1-5, or a deutero-derivative thereof, or a salt thereof, or a conformational isomer thereof, or a crystal form thereof, or a solvate thereof, and other drugs having an anti-tumor effect, in unit preparations of the same or different specifications for simultaneous or separate administration, and a pharmaceutically acceptable carrier.

10. The combination of claim 9, wherein: the other medicines with the anti-tumor effect are chemotherapeutic medicines or radiotherapy medicines, preferably, the chemotherapeutic medicines are targeted medicines; or, the other medicine with the anti-tumor effect is selected from one or more than two of CDK4/6 inhibitor, Parp inhibitor, androgen receptor inhibitor and immune checkpoint inhibitor.