A deuterated benzimidazole compound and use thereof as an ep300 / cbp inhibitor

Abstract

The application discloses a kind of deuterated benzimidazole compounds and its purposes as EP300 / CBP inhibitor.The specific purpose is to provide a kind of deuterated benzimidazole compound shown in formula I, or its salt, or its conformational isomer, or its crystal form, or its solvate.The compound of the present application has high selectivity to EP300 / CBP, and can effectively inhibit the activity of EP300 / CBP;At the same time, compared with non-deuterated compound, the metabolic stability and pharmacokinetic performance of the compound of the present application are significantly improved.In addition, the compound of the present application has excellent inhibitory effect on various tumor cells, including prostate cancer cells, leukemia cells, breast cancer cells, multiple myeloma cells.Therefore, the compound of the present application has very good application prospect in the preparation of EP300 / CBP inhibitor, and the prevention and / or treatment of tumor, the drug for regulating regulatory T cells.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of drug synthesis, and particularly relates to a novel deuterated benzimidazole compound and use thereof as an EP300 / CBP inhibitor. BACKGROUND

[0002] Histone acetyltransferase (HAT) and histone deacetylase (HDAC) can affect the acetylation of histone, and the recruitment and normal function of HAT and HDAC are key regulatory steps of gene expression and cell cycle, and functional defects of these enzymes can lead to various diseases including tumors.

[0003] The EP300 / CBP family consisting of highly homologous adenovirus E1A-associated 300kDa protein (EP300) and cyclic AMP response element binding protein (CREB)-binding protein (CBP) is one of the main members of the histone acetyltransferase (HAT) family. EP300 / CBP is involved in cell cycle progression and cell growth, differentiation and development, and is a very important coactivator that can regulate the function of various key transcriptional regulators. Studies have shown that EP300 / CBP is highly expressed and activated in a variety of different tumors, and EP300 / CBP is closely related to various tumor diseases, and is a promising tumor treatment target. Therefore, EP300 / CBP inhibitors have attracted more and more attention from researchers.

[0004] Researchers of the Structural Genomics Consortium (SGC) of the University of Oxford first designed and synthesized benzimidazole compound 9a, but 9a has low selectivity for CBP and BRD4 (CBP: IC50= 4 μmol·L -1 , BRD4: IC50= 6.3 μmol·L -1 ). Then, through further optimization, compounds 10a (CBP: IC50= 0.12 μmol·L -1 , BRD4: IC50= 2.4 μmol·L -1 ) and 11a (SGC-CBP30, CBP: IC50= 69 nmol·L -1 , Kd= 21 nmol·L -1 , p300: Kd= 32 nmol·L -1 ) were obtained.

[0005]

[0006] However, the EP300 / CBP inhibitors reported so far still cannot meet the clinical needs, therefore, it is very important to develop more EP300 / CBP inhibitors with novel structure, higher selectivity and better inhibitory activity.

[0007] Deuterated drugs refer to replacing part of hydrogen atoms in drug molecules with deuterium. Since deuterium is close to hydrogen in shape and volume in drug molecules, deuterated drugs generally retain the biological activity and selectivity of the original drugs. Since the C-D bond is more stable than the C-H bond, the C-D bond is less likely to break during chemical reactions, and the half-life may be extended.

[0008] However, due to the complexity of the metabolic process of the biological system, the pharmacokinetic properties of the drug in the body are influenced by many factors and also show corresponding complexity. Compared with the corresponding non-deuterated drugs, the changes in the pharmacokinetic properties of deuterated drugs show great randomness and unpredictability. Deuterium at certain sites not only cannot extend the half-life, but also may shorten it (Scott L. Harbeson, Roger D. Tung. Deuterium in Drug Discovery and Development, P405-406.), and degrade its pharmacokinetic properties; on the other hand, hydrogen at certain positions of the drug molecule is not easy to be deuterated due to steric hindrance and other reasons, therefore, the deuterium of the drug is not at will, and the deuterated sites are unpredictable.

[0009] Therefore, developing an EP300 / CBP inhibitor with excellent pharmacokinetic properties and high activity at the same time will have very good application prospects. SUMMARY

[0010] The purpose of the present application is to provide a high-activity EP300 / CBP inhibitor with better metabolic stability and pharmacokinetic properties.

[0011] The present application provides a compound shown in formula I, or a salt thereof, or a conformational isomer thereof, or a crystal form thereof, or a solvate thereof:

[0012]

[0013] wherein, R0-R 25 are each independently selected from H, deuterium, C1-C4 alkyl, C3-C6 cycloalkyl, C1-C4 alkyl substituted with one or more deuterium, C3-C6 cycloalkyl substituted with one or more deuterium; and at least one of R0-R 25 contains deuterium.

[0014] Further, R0-R 25 are each independently selected from H, deuterium, methyl, CD3; and at least one of R0-R25 at least one of R0, R1, R2, R3, R4, R5, R6, R7, R8, and R9is deuterium or CD3.

[0015] Further, R0, R1, R2, R3, R4, R5, R6, R7, R8, and R9are independently selected from hydrogen or deuterium. 10 11 25 10

[0016] Further, the structure of the compound is shown in formula II-1 or formula II-2:

[0017]

[0018] 25

[0019] Further, the compound is one of the following compounds:

[0020]

[0021] The present application also provides a use of the above-mentioned compound, or a deuterated compound thereof, or a salt thereof, or a conformational isomer thereof, or a crystal form thereof, or a solvate thereof in the preparation of an EP300 inhibitor, a CBP inhibitor, or an EP300 / CBP inhibitor.

[0022] Further, the EP300 / CBP inhibitor is a drug for preventing and / or treating a tumor, a myeloid hematopoietic stem / progenitor cell malignant disease, or a regulatory T cell.

[0023] Preferably, the tumor is selected from a 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 malignant disease is leukemia.

[0024] More preferably, the lymphoma is non-Hodgkin's lymphoma, diffuse large B-cell lymphoma, and the leukemia is acute myeloid leukemia.

[0025] The present application also provides a drug for treating a disease, which is a preparation prepared by taking the above-mentioned compound, or a deuterated compound 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 a pharmaceutically acceptable adjuvant.

[0026] The present application also provides a combination drug containing the above-mentioned compound, or a deuterated compound thereof, or a salt thereof, or a conformational isomer thereof, or a crystal form thereof, or a solvate thereof, and another drug having an anti-tumor effect, which are the same or different in the unit preparation, and are administered simultaneously or separately, and a pharmaceutically acceptable carrier. ​​​​​​

[0027] Further, the other drug having an anti-tumor effect is a chemotherapeutic drug or a radiotherapy drug, preferably, the chemotherapeutic drug is a targeted drug; or, the other drug having an anti-tumor effect is selected from one or more than two of CDK4 / 6 inhibitor, Parp inhibitor, androgen receptor inhibitor, and immune checkpoint inhibitor.

[0028] Definitions of terms used in connection with the present application: Unless otherwise indicated, the initial definition of a group or term provided herein applies throughout the specification. For terms not specifically defined herein, those terms are intended to have the meanings that one of skill in the art would attribute to them.

[0029] “EP300 / CBP inhibitor” refers to an inhibitor capable of inhibiting both EP300 and CBP activity.

[0030] “EP300” is also referred to as “P300”, and “EP300 / CBP inhibitor” is also referred to as “P300 / CBP inhibitor”.

[0031] The minimum and maximum number of carbon atoms in a hydrocarbon group are indicated by a prefix, e.g., the prefix C a -C b Alkyl refers to any alkyl group of “a” to “b” carbon atoms. For example, C1-C4 alkyl refers to a straight or branched chain alkyl group containing from 1 to 4 carbon atoms.

[0032] Cycloalkyl refers to a saturated or unsaturated cyclic hydrocarbon substituent; the cyclic hydrocarbon can be monocyclic or polycyclic. For example, “C3-C6 cycloalkyl” refers to a saturated or unsaturated cyclic alkyl group having from 3 to 6 ring carbon atoms.

[0033] “Pharmaceutically acceptable” refers to a carrier, vehicle, diluent, adjuvant, and / or salt generally compatible with pharmaceutical dosage forms and physiologically compatible with the subject.

[0034] “Salt” refers to an acid and / or base addition salt of a compound or stereoisomer thereof, and also includes zwitterions, and also includes quaternary ammonium salts, e.g., alkylammonium salts. These salts can be formed directly at the final isolation and purification of the compound. They can also be formed by admixing a compound, or stereoisomer thereof, with a quantity of an acid or base, as appropriate (e.g., equivalent amounts). These salts can be collected by filtration as a precipitate from solution, or recovered after evaporation of the solvent, or by lyophilization after reaction in aqueous media.

[0035] The salt in the present application includes a pharmaceutically acceptable salt. The salt in the present application can be a hydrochloride, a sulfate, a citrate, a besylate, a hydrobromide, a hydrofluoride, a phosphate, an acetate, a propionate, a succinate, an oxalate, a malate, a succinate, a fumarate, a maleate, a tartrate or a trifluoroacetate of the compound.

[0036] The experimental results show that the compound of the present application has high selectivity for EP300 / CBP and can effectively inhibit the activity of EP300 / CBP; at the same time, compared with the non-deuterated compound, the metabolic stability and pharmacokinetic performance of the compound of the present application are significantly improved. In addition, the compound of the present application has excellent inhibitory effect on various tumor cells including prostate cancer cells, leukemia cells, breast cancer cells and multiple myeloma cells. Therefore, the compound of the present application has very good application prospect in the preparation of EP300 / CBP inhibitors, and the prevention and / or treatment of tumors, and the regulation of regulatory T cells.

[0037] Obviously, according to the above content of the present application, according to the ordinary technical knowledge and common means in the art, other various forms of modification, replacement or change can be made without departing from the above basic technical idea of the present application.

[0038] The above content of the present application will be further described in detail through the specific embodiments in the form of examples. However, it should not be understood that the scope of the above subject matter of the present application is limited to the following examples. Any technology realized based on the above content of the present application belongs to the scope of the present application. DETAILED DESCRIPTION

[0039] The raw materials and equipment used in the present application are known products, which are obtained by purchasing commercially available products.

[0040] Example 1, synthesis of compound 100 of the present application

[0041]

[0042] (1) Synthesis of compound 4-bromo-N-((1r,4r)-4-methoxycyclohexyl)-2-nitroaniline (Int 1)

[0043]

[0044] Dissolve SM1 (22 g; 0.1 mol) in acetonitrile (110 mL), add potassium carbonate (16.6 g; 0.12 mol) and trans-4-methoxycyclohexylamine (12.9 g; 0.1 mol), heat to reflux for 3 h. Pour the reaction liquid into water (500 mL), filter, wash with water, and obtain orange solid Int 1 (30 g; 0.09 mol), with a yield of 91.2%.

[0045] (2) Synthesis of compound 4-bromo-N 1 Synthesis of ((1 r,4r)-4-methoxycyclohexyl)benzene-1,2-diamine (Int 2)

[0046]

[0047] Int 1 (30 g; 0.09 mol) was dissolved in tetrahydrofuran (300 mL), water (100 mL), aqueous ammonia (28%, 60 mL) was added, and finally sodium hydrosulfite (62 g; 0.36 mol) was added. The mixture was stirred at room temperature for 16 hours. The mixture was allowed to stand and separate into layers. The aqueous phase was extracted with ethyl acetate (50 mL x 2), and the combined organic phases were washed with saturated brine (200 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain Int 2 (24 g; 0.08 mol) with a yield of 88.2%.

[0048] (3) Synthesis of compound (S)-N-(5-bromo-2-(((1 r,4S)-4-methoxycyclohexyl)amino)phenyl)-6-oxopiperidine-2-carboxamide (Int 3)

[0049]

[0050] Int 2 (24 g; 0.08 mol), (S)-6-oxopiperidine-2-carboxylic acid (12 g; 0.084 mol), and DIPEA (12 g; 0.096 mol) were added to dichloromethane (240 mL), and propylphosphonic anhydride (50% in EA; 61 g; 0.096 mol) was added dropwise with stirring at room temperature. After the dropwise addition was completed, the mixture was stirred at room temperature for 3 hours. Column chromatography gave Int 3 (27 g; 0.064 mol) with a yield of 79%.

[0051] (4) Synthesis of compound (S)-6-(5-bromo-1-((1 r,4S)-4-methoxycyclohexyl)-1 H-benzo[d]imidazol-2-yl)piperidin-2-one (Int 4)

[0052]

[0053] Int 3 (27 g; 0.064 mol) was added to acetic acid (270 mL), and the mixture was stirred at 50°C for 5 days. The acetic acid was removed by evaporation, and the pH was adjusted to 7-8 using saturated aqueous sodium bicarbonate solution. The mixture was extracted with dichloromethane (100 mL x 3), and the organic phase was washed with saturated brine. After drying over anhydrous sodium sulfate, the mixture was concentrated under reduced pressure to obtain Int 4 (20.7 g; 0.051 mol) with a yield of 80%.

[0054] (5) Synthesis of compound (S)-6-(5-bromo-l-((lr,4S)-4-methoxycyclohexyl)-lH- benzo[d]imidazol-2-yl)-l-(3,4-difluorophenyl)piperidin-2-one (Int 5)

[0055]

[0056] To a solution of Int 4 (20.7 g; 0.051 mol) in dichloromethane (200 mL), pyridine (41 mL; 0.52 mol), copper acetate monohydrate (12.4 g; 0.066 mol) and 3,4-difluorophenylboronic acid (29 g; 0.2 mol) were added successively and stirred at room temperature for 5 hours. Washed with water and saturated brine. Column chromatography gave Int 5 (20 g; 0.04 mol) with a yield of 75.7%.

[0057] (6) Synthesis of compound (S)-l-(3,4-difluorophenyl)-6-(5-(3,5-dimethylisoxazol-4-yl)-l- ((lr,4S)-4-methoxycyclohexyl)-lH-benzo[d]imidazol-2-yl)piperidin-2-one (Int 6)

[0058]

[0059] Int 5 (20 g; 0.04 mol) was added to a mixture of 1,4-dioxane (180 mL) and water (30 mL), followed by Pd(dppf)Cl2(2.9 g; 0.004 mol) and cesium carbonate (26 g; 0.08 mol), and stirred at 100 °C for 16 hours under nitrogen protection. Column chromatography gave Int 6 (19 g; 0.036 mol) with a yield of 92.2%.

[0060] (7) Synthesis of compound (S)-l-(3,4-difluorophenyl)-6-(5-(3,5-dimethylisoxazol-4-yl)-l- ((lr,4S)-4-hydroxycyclohexyl)-lH-benzo[d]imidazol-2-yl)piperidin-2-one (Int 7)

[0061]

[0062] Int 6 (54 mg; 0.1 mmol) was dissolved in dichloromethane (5 mL) and cooled to 0 °C in an ice bath. Boron tribromide (75 mg; 0.3 mmol) in dichloromethane (1 mL) was added dropwise with stirring. After the addition was complete, the temperature was maintained for 2 h. The reaction was added dropwise to ice water. The solution was partitioned, extracted with dichloromethane (5 mL x 2), the organic layers were combined, washed with saturated brine (15 mL), and dried over anhydrous sodium sulfate. The solution was concentrated to give Int 7 (31 mg; 0.06 mmol) in 58.9% yield.

[0063] (8) Synthesis of compound (S)-1-(3,4-difluorophenyl)-6-(5-(3,5-dimethylisoxazol-4-yl)-1-((1r,4S)-4-deutero-methylcyclohexyl)-1H-benzo[d]imidazol-2-yl)piperidin-2-one (100)

[0064]

[0065] Int 7 (31 mg; 0.06 mmol) was dissolved in tetrahydrofuran (1 mL) and cooled in an ice water bath. Sodium hydride (60% dispersion in mineral oil; 3.6 mg; 0.09 mmol) was added with stirring for 2 min, and deuterated iodomethane (9 mg; 0.06 mmol) was added. The reaction was maintained at this temperature for 30 min, poured into ice water (5 mL), extracted with dichloromethane (5 mL x 3), washed with saturated brine (15 mL), and dried over anhydrous sodium sulfate. Column chromatography gave compound 100 (26 mg; 0.048 mmol) in 81.2% yield. Mass (M+H) = 538.3. H NMR (400 MHz, DMSO) δ 7.79 (d, J = 8.6 Hz, 1H), 7.70 (s, 1H), 7.41 - 7.30 (m, 2H), 7.15 (d, J = 8.5 Hz, 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 = 22.7 Hz, 4H), 2.18 (d, J = 13.8 Hz, 2H), 2.01 (d, J = 14.7 Hz, 3H), 1.80 (s, 2H), 1.42 - 1.34 (m, 2H), 1.25 (d, J = 9.2 Hz, 1H)

[0066] Example 2, Synthesis of compound 101 of the present application

[0067] The synthesis route of compound (S)-1-(3,4-difluorophenyl)-6-(5-(3,5-dimethylisoxazol-4-yl)-1-((1r,4S)-4-deutero-methylcyclohexyl)-1H-benzo[d]imidazol-2-yl)piperidin-2-one-3,3-dideuterium (101) is as follows:

[0068]

[0069] Compound 101 (36) was obtained by dissolving 100 (54 mg; 0.1 mmol) in CD3OD / D2O (3 mL / 0.5 mL), adding NaOD (41 mg; 1 mmol) at 10 degrees, stirring for 12 hours, and column chromatography after spin-drying, with a yield of 70%. Mass spectrum (M+H) = 540.2. H NMR (400 MHz, DMSO) δ 7.79 (d, J = 8.6 Hz, 1H), 7.70 (s, 1H), 7.41-7.30 (m, 2H), 7.15 (d, J = 8.5 Hz, 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 = 9.2 Hz, 1H)

[0070] The beneficial effects of the present application are demonstrated by the following experimental examples.

[0071] Experimental Example 1, Hepatocyte microsomal metabolic stability experiment of the compound of the present application

[0072] (1) Experimental materials

[0073] The microsomes were stored in a -80°C refrigerator, and the specific information is shown in Table 1. Before use, they were first thawed in a 37°C water bath, and after melting, they were placed on ice for use. Other reagents were purchased from local suppliers.

[0074] Table 1. Liver microsomal information

[0075] Species Catalog No. Lot No. Strain Sex Supplier Human 452117 38295 N / A Mixed Corning Mouse M1000 1910002 ICR / CD-1 Male Xenotech

[0076] (2) Experimental steps

[0077] Step 1: Prepare the reaction system according to Table 2

[0078] Table 2: Reaction system preparation method

[0079]

[0080]

[0081] Step 2: The reaction system was pre-incubated at 37°C water bath for 10 minutes. 40 μL of 10 mM NADPH solution was added into the reaction system, and the final concentration of NADPH was 1 mM, respectively. 40 μL of ultrapure water was used as negative control instead of NADPH solution.

[0082] Step 3: 4 μL of 200 μM of the test compound or non-deuterated control compound was added into the reaction, and the reaction was started, and the final concentration of the drug was 2 μM.

[0083] Step 4: 50 μL of the reaction sample was taken out at 0, 15, 30, 45 and 60 minutes, respectively, and quenched with 4 times of cold acetonitrile containing internal standard (200 nM alprazolam, 200 nM labetalol, 2 μM ketoprofen, 200 nM caffeine). The sample was centrifuged at 3,220 g for 45 minutes. After centrifugation, 90 μL of supernatant and 90 μL of ultrapure water were mixed and used for LC-MS / MS analysis.

[0084] (3) Data analysis

[0085] All data calculations were performed by Microsoft Excel software. The peak area was detected by extracting the ion spectrum. The in vitro half-life (t 1 / 2 ) of the parent drug was detected by linear fitting of the percentage of the disappearance of the parent drug and time. 1 / 2 The in vitro half-life (t 1 / 2 ) was calculated by the slope:

[0086] invitro 1 / 2 = -(0.693 / k)

[0087] Then the in vitro half-life (t 1 / 2 ) was converted into in vitro clearance (unit: μL / min / mg) by the following formula:

[0088]

[0089] (4) Experimental results

[0090] Table 3: Hepatocyte microsomal metabolic stability results of the compounds of the present application

[0091]

[0092]

[0093] The results show that the in vitro half-life of the compounds of the present application in mouse hepatocyte microsomes and human hepatocyte microsomes is significantly prolonged, and the in vitro clearance is significantly reduced compared with the non-deuterated control compounds. It is proved that the compounds of the present application have significantly improved metabolic stability compared with the non-deuterated control compounds.

[0094] Experimental Example 2, Mouse Pharmacokinetics of the Compound of the Invention

[0095] (1) Experimental Method

[0096] Eighteen healthy adult ICR mice (9 animals for each administration method of the compound, and 3 animals for each time point) were administered by tail vein injection and gavage, respectively, after overnight fasting (free water). Blood 0.1 ml was collected, anticoagulated with EDTA-K2, centrifuged at 4°C for 5 min to separate plasma, and stored at -80°C for testing.

[0097] The blood concentration-time curve was plotted, and the main pharmacokinetic parameters were calculated using WinNonlin 6.3 software.

[0098] The peak time T max and the peak concentration C max were measured values;

[0099] The area under the concentration-time curve AUC all value was calculated using the trapezoidal method; AUC inf = AUC all + C t / k e , Ct is the blood concentration at the last measurable time point, and k e is the elimination rate constant;

[0100] The elimination half-life t 1 / 2 = 0.693 / k e ;

[0101] The mean residence time MRT = AUMC / AUC;

[0102] The clearance CL = D / AUC inf (D is the dose administered);

[0103] The steady-state distribution volume V ss = CL x MRT.

[0104] The absolute bioavailability F = (AUC i.g. x D i.v. ) / (AUC i.v. x D i.g. ) x 100%

[0105] (2) Experimental Results

[0106] Table 4: Mouse Pharmacokinetics Results of the Compound of the Invention

[0107]

[0108] Note: "IV (1 mpk)" in the table means tail vein injection, and the dose is 1 mg per kg; PO (10 mpk) means oral administration, and the dose is 10 mg per kg.

[0109] As can be seen from the results in Table 4, the compound of the present application has a higher AUC value, a longer elimination half-life, a lower clearance rate, and a higher absolute bioavailability in mice compared with the non-deuterated control compound. all This shows that the compound of the present application has significantly improved pharmacokinetic properties compared with the non-deuterated control compound.

[0110] Experimental Example 3, Inhibitory activity of the compound of the present application on CBP / EP300

[0111] A, Experimental method

[0112] CBP and EP300 AlphaScreen assay experiment:

[0113] 1. Prepare 1x assay buffer

[0114] 2. Prepare the compound

[0115] First, dilute the compound to a final concentration of 1000 times (3-fold gradient dilution) using the Precision automatic pipette:

[0116] 1) Use the Echo automatic pipette to transfer 50 μL of the compound stored at a concentration of 10 mM to well A2 of the 96-well plate.

[0117] 2) Use the Precision to transfer 30 μL of DMSO to wells A1, A3 to A12.

[0118] 3) Use the Precision to transfer 15 μL from A2 to A3, and so on until A10 to A11. Complete the 1:3 gradient dilution.

[0119] 4) Centrifuge the compound plate at 1000 rpm for 1 minute.

[0120] 3. Prepare the assay plate

[0121] Use the Echo automatic pipette to transfer 20 nL from each concentration of the compound plate to the assay plate: from well A1 of the configuration plate to wells A1 and A2 of the assay plate, and from well A2 of the configuration plate to wells A3 and A4 of the assay plate.

[0122] 4. Bromodomain (BRD) binding assay experiment

[0123] 1) Prepare 2x protein and polypeptide solutions

[0124] Dissolve the proteins and polypeptides in 1x assay buffer.

[0125] 2) Transfer 10 μL of protein and peptide solution to each well of columns 3 to 24 of the assay plate and 10 μL of 1x assay buffer to columns 1 and 2 as negative controls.

[0126] 3) Centrifuge the assay plate at 1000 rpm for 1 minute.

[0127] 4) Incubate for 15 minutes at room temperature.

[0128] 5) Receptor beads and donor beads are prepared as 2x receptor and donor solutions in 1x assay buffer.

[0129] 6) Transfer 2x receptor and donor solutions to the assay plate.

[0130] Add 15 μL of receptor and donor solution and avoid light.

[0131] 7) Centrifuge the assay plate at 1000 rpm for 1 minute and incubate for 60 minutes at room temperature.

[0132] 5. Read end point with EnSpire and Alpha mode.

[0133] 6. Curve fitting

[0134] Enter experimental data into Excel document and use equation (1) to obtain % inhibition Inh:

[0135] Equation (1): Inh% = (Max - Signal) / (Max - Min) x 100

[0136] Where Max: control with DMSO, Max: low control with DMSO,

[0137] Enter resulting data into XL-Fit software and use equation (2) to obtain IC 50 values:

[0138] Equation (2): Y = Bottom + (Top - Bottom) / (1 + 10^((LogIC 50 - X) x Hill Slope))

[0139] B. Experimental results

[0140] Table 5: IC50values of various compounds against CBP BRD, EP300 BRD 50

[0141]

[0142]

[0143] The results show that the compound of the present application has obvious inhibitory activity on CBP BRD and EP300 BRD at the same time, and the inhibitory activity on CBP BRD and EP300 BRD is obviously higher than that of the non-deuterated control compound.

[0144] Experimental Example 4, Inhibition of the compound of the present application on the proliferation of prostate cancer CWR22RV1 cells

[0145] 1. Experimental procedure:

[0146] ① The prostate cancer CWR22RV1 cells were subcultured with cell culture solution, and the well-grown cells were inoculated into a 96-well plate at 80 μL per well, and the cell number was 1500 per well. Incubate in a 37℃, 5% CO2 cell incubator overnight.

[0147] ② The drug was prepared into a 30 mM stock solution with dimethyl sulfoxide (DMSO). Before use, dilute 3 times with DMSO, and then dilute 3 times to get 9 concentration gradients. Dilute each concentration of compound 200 times with culture solution (to ensure that the DMSO concentration in the culture system is 0.1%), and repeat 2 wells for each concentration. Take 20 μL of the diluted compound and add it to the cell culture well (final concentration 10 μM, 3.3 μM, 1.1 μM…), gently shake to mix. Set up 3 negative control wells with only cells and 3 blank control wells with only culture solution (6 wells each add 20 μL of DMSO diluted 200 times with culture solution).

[0148] 2. Detection method:

[0149] (1) After 6 days of culture, add 10 μL CCK-8 to each well, and continue to incubate at 37℃, 5% CO2 cell incubator for 2.5 hours.

[0150] (2) Measure the absorbance (OD value) at 450 nm with a multifunctional enzyme marker.

[0151] (3) The data is analyzed by Dose-response-inhibition equation in software GraphPad Prism6, and the IC 50 value is obtained.

[0152] 3. Experimental results

[0153] The IC 50 (nM) of the compound of the present application on the activity inhibition of CWR22RV1 cells is shown in Table 6.

[0154] A indicates IC 50 less than or equal to 500 nM; B indicates IC 50greater than 500 nM and less than or equal to 2000 nM; C represents IC 50 greater than 2000 nM.

[0155] Table 6: IC of each compound on CWR22RV1 cells 50

[0156] Compound IC 50 ]]> Compound 100 A Compound 101 A

[0157] It can be seen that the compound of the present application has obvious inhibitory effect on the proliferation of prostate cancer CWR22RV1 cells, IC 50 as low as 500 nM or less.

[0158] Experimental Example 5, Inhibitory effect of the compound of the present application on the proliferation of other tumor cells 1. Experimental method

[0159] Using the same method as in Experimental Example 4, CWR22RV1 cells were replaced with tumor cells in Table 7, and the IC of the compound of the present application on the inhibition of the activity of these tumor cells was tested and calculated 50 (nM), and the results are shown in Table 7.

[0160] 2. Experimental results

[0161] In Table 7, A represents IC 50 less than or equal to 500 nM; B represents IC 50 greater than 500 nM and less than or equal to 2000 nM.

[0162] Table 7: IC of each compound on other tumor cells 50

[0163]

[0164] It can be seen that the compound prepared in the present application has obvious inhibitory effect on the proliferation of other prostate cancer cells, leukemia cells, breast cancer cells, and multiple myeloma cells. It shows that the compound of the present application has good inhibitory effect on multiple tumors.

[0165] Experimental Example 6, Pharmacodynamic evaluation of the compound of the present application in a NOD.SCID female mouse model of human acute myeloid leukemia cell line MOLM-16 subcutaneous xenograft

[0166] 1. Cell culture

[0167] Human acute myeloid leukemia MOLM-16 cells were cultured in RPMI1640 culture medium containing 20% fetal bovine serum. Exponentially growing MOLM-16 cells were collected, resuspended with PBS to a suitable concentration for subcutaneous tumor inoculation in mice.

[0168] 2. Animal modeling

[0169] The experimental mice were inoculated subcutaneously with 1 x 10 7 MOLM-16 cells were resuspended in 1:1 PBS and Matrigel (0.1 ml / mouse) and observed regularly for tumor growth. When the tumors reached an average volume of ~150 (average tumor volume 100-200) mm 3 The mice were randomly divided into groups according to tumor size and body weight and dosed. The same dose of vehicle was given to the control group. The day of tumor cell inoculation was defined as day 0.

[0170] After the start of dosing, the body weight and tumor size of the mice were measured twice a week. The tumor volume was calculated using the formula: Tumor volume (mm 3 ) = 1 / 2 x (a x b 2 ) (where a represents the long diameter and b represents the short diameter).

[0171] The data, including the measurement of the long and short diameters of the tumors and the weighing of the body weight of the animals, were collected using StudyDirector™ (version number 3.1.399.19, supplier Studylog System, Inc.) software during the experiment.

[0172] 3. Evaluation index

[0173] The relative tumor proliferation rate, T / C (%), was the percentage value of the relative tumor volume or tumor weight of the treatment group and the control group at a certain time point. The calculation formula was:

[0174] T / C % = TRTV / CRTV x 100%

[0175] Where TRTV: the average relative tumor volume (RTV) of the treatment group; CRTV: the average relative tumor volume (RTV) of the control group; RTV = Vt / V0, V0 is the tumor volume of the animal at the time of grouping, and Vt is the tumor volume of the animal after treatment.

[0176] Or T / C % = TTW / CTW x 100%

[0177] Where TTW: the average tumor weight of the treatment group at the end of the experiment; CTW: the average tumor weight of the control group at the end of the experiment.

[0178] The relative tumor inhibition rate, TGI (%), was calculated using the formula:

[0179] TGI % = (1-T / C) x 100%

[0180] Where T and C are the relative tumor volume (RTV) or tumor weight (TW) of the treatment group and the control group, respectively, at a certain time point.

[0181] 4. Experimental results

[0182] Table 8: Pharmacodynamics of each compound in MOLM-16 subcutaneous xenograft mouse model

[0183]

[0184] From the experimental results, it can be seen that the compound of the present application has good therapeutic effect on the animal model of human acute myeloid leukemia, and compared with the non-deuterated compound, the therapeutic effect of the compound of the present application is obviously improved.

[0185] In summary, the present application provides a novel deuterated benzimidazole compound, which has high selectivity to EP300 / CBP and can effectively inhibit the activity of EP300 / CBP. Meanwhile, compared with the non-deuterated compound, the metabolic stability and pharmacokinetic performance of the deuterated compound of the present application are significantly improved. In addition, the compound of the present application has excellent inhibitory effect on various tumor cells including prostate cancer cells, leukemia cells, breast cancer cells and multiple myeloma cells. Therefore, the compound of the present application has very good application prospect in the preparation of EP300 / CBP inhibitor, and the prevention and / or treatment of tumors, and the regulation of regulatory T cells.

Claims

1. Use of compound 100, or a pharmaceutically acceptable salt thereof, or a conformational isomer thereof, as the sole active ingredient in the preparation of an EP300 / CBP inhibitor; wherein the EP300 / CBP inhibitor is a drug for the prevention and / or treatment of acute myeloid leukemia; wherein compound 100 is:

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