Arginine methyltransferase 6 inhibitors and methods of making, pharmaceutical compositions, and uses thereof

Abstract

The application provides an arginine methyltransferase 6 inhibitor and a preparation method, a pharmaceutical composition and a use thereof, relates to the technical field of biological medicine. The arginine methyltransferase 6 inhibitor provided by the application is a new compound obtained on the basis of a large number of design, synthesis and screening. The arginine methyltransferase 6 inhibitor is different from existing PRMTs inhibitors. The arginine methyltransferase 6 inhibitor can selectively degrade arginine methyltransferase 6, thereby regulating the protein level of arginine methyltransferase 6, has antitumor cell activity, can be used for preventing or treating diseases related to abnormal expression of PRMT6, and provides a new choice for development and application of antitumor drugs. The preparation method of the arginine methyltransferase 6 inhibitor provided by the application adopts raw materials which are easy to obtain, has the advantages of simple synthesis method, safety, easy realization and high yield, and can be used for industrial production.

Description

Technical Field

[0001] This application relates to the field of biomedical technology, and in particular to an arginine methyltransferase 6 inhibitor, its preparation method, pharmaceutical composition and use. Background Technology

[0002] Epigenetics involves DNA methylation, histone modifications, chromatin remodeling, and non-coding RNA. Epigenetic dysregulation is a common feature of most cancers, which often occur directly by altering epigenetic mechanisms. Arginine methylation is a common post-translational modification that acts as an epigenetic regulator of transcription, playing a crucial role in DNA damage signaling, precursor messenger RNA splicing, cell signaling, and messenger RNA translation.

[0003] Arginine methyltransferases (PRMTs) are key enzymes involved in the arginine methylation process of proteins, and nine PRMT members have been identified in mammalian cells. By regulating different forms of arginine methylation, PRMTs can be classified into three types: type I, type II, and type III. PRMT6, a member of the type I PRMT family, is the most important protein in the Arg2 (H3R2) methylation of histone H3, participating in the veil-like regulation of gene expression, DNA repair, alternative splicing, cell proliferation and senescence, and DNA methylation. Furthermore, PRMT6 can methylate non-histone substrates such as CRAF, PTEN, BAG5, p21, and GPS2. Aberrant expression of PRMT6 is associated with the development and progression of various diseases. Therefore, the development of novel PRMT6 inhibitor molecules is of great significance. Summary of the Invention

[0004] The purpose of this application is to provide an arginine methyltransferase 6 inhibitor, its preparation method, pharmaceutical composition, and uses, in order to solve the above-mentioned problems.

[0005] To achieve the above objectives, this application adopts the following technical solution:

[0006] This application first provides an arginine methyltransferase 6 inhibitor having the structure shown in Formula I:

[0007]

[0008] Where Z represents pyridine, and the connection method of pyridine is selected from...

[0009] R1 and R2 are independently either H or CH3;

[0010] X is -CH2- or carbonyl, and Y is -CO-NH- or -NH-CO-.

[0011] Linkers are aliphatic chains or polyethylene glycol chains with a length of 1-11 carbons;

[0012] n is 0 or 1.

[0013] Preferably, the arginine methyltransferase 6 inhibitor is selected from one or more compounds shown in the following structural formulas:

[0014]

[0015]

[0016] This application also provides a method for preparing an arginine methyltransferase 6 inhibitor, comprising:

[0017] The first and second raw materials are subjected to a first condensation reaction under alkaline conditions to obtain a first intermediate product. The structural formula of the first raw material is as follows: Y1 is an amino or carboxyl group, and the structural formula of the second raw material is: X1 is an amino or carboxyl group, and n1 is any positive integer from 1 to 11;

[0018] The first intermediate product reacts under first acidic conditions to give a second intermediate product;

[0019] The second intermediate product and the third raw material undergo a second condensation reaction under alkaline conditions to obtain the third intermediate product, wherein the structural formula of the third raw material is as follows: n is 0 or 1;

[0020] The fourth and fifth raw materials were subjected to a reductive amination reaction under second acidic conditions to obtain a fourth intermediate product. The fourth raw material was 5-bromopyridine-3-carboxaldehyde, and the fifth raw material had the following structural formula: R1 and R2 are each independently H or CH3;

[0021] The third intermediate and the fourth intermediate were subjected to a palladium-mediated coupling reaction under alkaline conditions to obtain the fifth intermediate;

[0022] The fifth intermediate was reacted under third acidic conditions to obtain the arginine methyltransferase 6 inhibitor.

[0023] Preferably, the condensing agents used in the first and second condensation reactions are 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 1-hydroxy-7-azobenzotriazole; the base used is N-methylmorpholine; the molar ratio of the first raw material, the second raw material, the base, the 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and the 1-hydroxy-7-azobenzotriazole is 1:(1-3):(3-10):(1-3):(1-3); the molar ratio of the second intermediate product, the third raw material, the base, the 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and the 1-hydroxy-7-azobenzotriazole is 1:(1-3):(3-10):(1-3):(1-3).

[0024] Preferably, the acids used in the first acidic condition and the third acidic condition are each trifluoroacetic acid or hydrochloric acid independently; the molar ratio of the first intermediate product to the acid is 1:(3-10), and the molar ratio of the fifth intermediate product to the acid is 1:(3-10).

[0025] Preferably, the solvent for the reductive amination reaction is dichloromethane or dichloroethane; the acid used in the second acidic condition is glacial acetic acid; and the reductive amination agent used is sodium triacetoxyborohydride.

[0026] Preferably, the palladium is any one of [1,1'-bis(diphenylphosphine)ferrocene]palladium dichloride dichloromethane complex, palladium acetate, or tetra(triphenylphosphine)palladium; the base used in the coupling reaction is any one of sodium carbonate, potassium carbonate, or cesium carbonate; the solvent used in the coupling reaction is a mixture of dioxane and water in a volume ratio of (5:1) to (1:1); and the molar ratio of the third intermediate, the fourth intermediate, the palladium, and the base is 1:(1-2):(0.05-0.15):(1-5).

[0027] Preferably, the reductive amination reaction is carried out at a temperature of 0°C, the coupling reaction is carried out at a temperature of 90–110°C, and the reaction temperatures for the remaining reaction conditions are 5–25°C.

[0028] This application also provides a pharmaceutical composition comprising the above-described arginine methyltransferase 6 inhibitor and its pharmaceutically acceptable excipient.

[0029] This application also provides the use of the above-mentioned arginine methyltransferase 6 inhibitor and the above-mentioned pharmaceutical composition in the preparation of a medicament for the prevention and / or treatment of diseases related to abnormal PRMT6 expression;

[0030] Preferably, the disease is a tumor, including brain cancer, glioblastoma, leukemia, lymphoma, Bannayan-Zonana syndrome, Cowden's disease, Lhermitte-Duclos disease, breast cancer, inflammatory breast cancer, Wilms' tumor, Ewing sarcoma, rhabdomyosarcoma, ependymoma, medulloblastoma, colon cancer, gastric cancer, bladder cancer, head and neck cancer, kidney cancer, lung cancer, liver cancer, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, sarcoma, osteosarcoma, giant cell tumor of bone, or thyroid cancer.

[0031] Compared with the prior art, the beneficial effects of this application include:

[0032] The arginine methyltransferase 6 inhibitor provided in this application is a novel compound obtained based on extensive design, synthesis, and screening. Unlike existing PRMTs inhibitors, this arginine methyltransferase 6 inhibitor can selectively degrade arginine methyltransferase 6, thereby regulating the protein level of arginine methyltransferase 6 and exhibiting anti-tumor cell activity. It can be used to prevent or treat diseases related to abnormal PRMT6 expression, providing a new option for the development and application of anti-tumor drugs.

[0033] The preparation method of the arginine methyltransferase 6 inhibitor provided in this application uses readily available raw materials, and the synthesis method is simple, safe, easy to implement, and has a high yield, making it suitable for industrial production. Attached Figure Description

[0034] Figure 1 The results show the inhibitory effect of compound 8C on MDA-MB-435 and HCC827 cells.

[0035] Figure 2 The results show the effect of compound 8C on the colony-forming ability of MDA-MB-435 and HCC827 cells.

[0036] Figure 3 The results show the degradation of PRMT6 protein in MDA-MB-435 and HCC827 cells by compound 8C;

[0037] Figure 4 The results of PRMT6 protein degradation in MDA-MB-435 and HCC827 cells by compound 8C;

[0038] Figure 5 MDA-MB-435 cells were co-treated with the proteasome inhibitor MG132, the lysosome inhibitor chloroquine or EPZ020411, and the results were obtained by immunoblotting with compound 8C.

[0039] Figure 6MDA-MB-435 cells and HCC827 cells were treated with 0, 10, and 20 μM of compound 8C for 72 hours, respectively, and the apoptosis was detected by flow cytometry. Detailed Implementation

[0040] Example 1: Preparation of (4-oxo-4-((4-(4,4,5,5-tetramethyl-1,3,2-dioxaborhecyclopentan-2-yl)phenyl)amino)tert-butyl)carbamate (compound 2C)

[0041] 4-Aminophenylboronic acid pinacol ester (500 mg, 1.45 mmol), tert-butylmethyl (2-(methylamino)ethyl)carbamate (354 mg, 1.74 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) (501 mg, 2.61 mmol), 1-hydroxy-7-azobenzotriazole (HOAT) (355.7 mg, 2.61 mmol), and N-methylmorpholine (0.74 mL, 7.26 mmol) were added to 20 mL of dimethyl sulfoxide (DMSO) and reacted overnight at room temperature. After the reaction was complete as detected by TLC, the reaction solution was poured into ice water and extracted with dichloromethane. The organic phase was collected, concentrated, and the target product was obtained by column chromatography. 620 mg yellow solid, yield: 75.8%.

[0042] The NMR data are as follows: 1 H NMR (400MHz, DMSO-d6) δ8.94(d,J=1.8Hz,1H),8.84(d,J=2.3Hz,1H),8.74(s,1H),8.39(t,J= 2.1Hz, 1H), 6.92 (t, J = 5.8Hz, 1H), 3.29 (d, J = 6.4Hz, 2H), 3.11 (q, J = 6.1Hz, 2H), 1.37 (s, 9H).

[0043] The structural formula is as follows:

[0044] Example 2 Preparation of 2-amino-N-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborhecyclopentan-2-yl)phenyl)acetamide (compound 2A)

[0045] Using 4-aminophenylboronic acid pinacol ester and BOC-glycine, a reaction similar to that used to synthesize compound 2C was performed to give the target compound a pale yellow solid with a yield of 74.68%.

[0046] The NMR data are as follows: 1H NMR (400MHz, DMSO-d6) δ10.03(s,1H),7.60(s,4H),7.04(t,J=6.1Hz,1H),3.75(d,J=6.1Hz,2H),1.39(s,9H),1.28(s,12H).

[0047] The structural formula is as follows:

[0048] Example 3 Preparation of 3-amino-N-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborhecyclopentan-2-yl)phenyl)propionamide (compound 2B)

[0049] Using 4-aminophenylboronic acid pinacol ester and Boc-β-alanine, a reaction similar to that used to synthesize compound 2C was performed to give the target compound a pale yellow solid with a yield of 76.26%.

[0050] The NMR data are as follows: 1 H NMR(400MHz,DMSO-d6)δ10.03(s,1H),7.64–7.57(m,4H),6.86(t,J=5.8Hz,1 H),3.22(q,J=6.9Hz,2H),2.48(d,J=7.1Hz,2H),1.37(s,9H),1.28(s,12H).

[0051] The structural formula is as follows:

[0052] Example 4 Preparation of 5-amino-N-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborhecyclopentan-2-yl)phenyl)pentanamide (compound 2D)

[0053] Using 4-aminophenylboronic acid pinacol ester and Boc-5-aminopentanoic acid, a reaction similar to that used to synthesize compound 2C was performed to give the target compound a pale yellow solid with a yield of 78.58%.

[0054] The NMR data are as follows: 1 H NMR (400MHz, DMSO-d6) δ9.95 (s, 1H), 7.62–7.56 (m, 4H), 6.77 (t, J = 5.7Hz, 1H), 2.92 (q, J = 6.6Hz, 2H), 2.30 (t, J=7.4Hz, 2H), 1.55 (p, J=7.2Hz, 2H), 1.45–1.38 (m, 2H), 1.36 (s, 9H), 1.27 (s, 12H).

[0055] The structural formula is as follows:

[0056] Example 5 Preparation of 6-amino-N-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborhexacyclopentan-2-yl)phenyl)hexanoamide (compound 2E)

[0057] Using 4-aminophenylboronic acid pinacol ester and tert-butoxycarbonyl 6-aminohexanoic acid, a reaction similar to that used to synthesize compound 2C was performed to give the target compound as a pale yellow solid g, with a yield of 76.57%.

[0058] The NMR data are as follows: 1 H NMR (400MHz, DMSO-d6) δ10.03 (s, 1H), 7.64–7.57 (m, 4H), 6.75d, J = 6.4Hz, 1H), 2.89 (d ,J=6.6Hz,2H),2.31(t,J=7.5Hz,2H),1.61–1.54(m,2H),1.37(s,13H),1.28(s,12H).

[0059] The structural formula is as follows:

[0060] Example 6 Preparation of 7-amino-N-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborhexacyclopentan-2-yl)phenyl)heptamide (compound 2F)

[0061] Using 4-aminophenylboronic acid pinacol ester and BOC-7-aminoheptanoic acid, a reaction similar to that used to synthesize compound 2C was performed to give the target compound a pale yellow solid with a yield of 77.65%.

[0062] The NMR data are as follows: 1 H NMR (400MHz, DMSO-d6) δ10.03 (s, 1H), 7.63–7.57 (m, 4H), 6.75d, J = 6.4Hz, 1H), 2.90 (d, J=6.6Hz,2H),2.31(t,J=7.5Hz,2H),1.58(t,J=7.5Hz,2H),1.36(s,15H),1.28(s,12H).

[0063] The structural formula is as follows:

[0064] Example 7 Preparation of 8-amino-N-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborhecyclopentan-2-yl)phenyl)octamide (compound 2G)

[0065] Using 4-aminophenylboronic acid pinacol ester and BOC-8-aminooctanoic acid, a reaction similar to that used to synthesize compound 2C was performed to give the target compound a pale yellow solid with a yield of 78.28%.

[0066] The NMR data are as follows: 1 H NMR (400MHz, DMSO-d6) δ10.02 (s, 1H), 7.63–7.56 (m, 4H), 6.75d, J = 6.4Hz, 1H), 2.89 (q, J=6.6Hz,2H),2.30(t,J=7.5Hz,2H),1.58(t,J=7.5Hz,2H),1.35(s,21H),1.28(s,12H).

[0067] The structural formula is as follows:

[0068] Example 8 Preparation of 12-amino-N-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborhecyclopentan-2-yl)phenyl)dodecanoamide (compound 2H)

[0069] Using 4-aminophenylboronic acid pinacol ester and 12-(BOC-amino)dodecanoic acid, a reaction similar to that used to synthesize compound 2C was performed to give the target compound a pale yellow solid with a yield of 78.74%.

[0070] The NMR data are as follows: 1 H NMR (400MHz, DMSO-d6) δ10.10(s,1H),7.96(s,1H),7.65–7.58(m,4H),6.74(s,2H),3.70(t,J=6.2Hz,2H),3.64–3.58 (m,2H),3.51(s,5H),3.46(s,2H),3.05(t,J=6.2Hz,4H),2.57(d,J=5.4Hz,3H),1.37(d,J=2.8Hz,15H),1.28(s,12H).

[0071] The structural formula is as follows:

[0072] Example 9 Preparation of tert-butyl carbamate (compound 2I) of 2-(2-(3-oxo-3-((4-(4,4,5,5-tetramethyl-1,3,2-dioxaborhecyclopentan-2-yl)phenyl)amino)propyl)ethoxy)ethyl)carbamate

[0073] Using 4-aminophenylboronic acid pinacol ester and N-tert-butoxycarbonyl-polyethylene glycol-carboxylic acid, a reaction similar to that used to synthesize compound 2C was employed to give the target compound a pale yellow solid with a yield of 80.68%.

[0074] The NMR data are as follows: 1 H NMR (400MHz, DMSO-d6) δ10.10(s,1H),7.96(s,1H),7.65–7.58(m,4H),6.74(s,2H),3.70(t,J=6.2Hz,2H),3.64–3.58 (m,2H),3.51(s,5H),3.46(s,2H),3.05(t,J=6.2Hz,4H),2.57(d,J=5.4Hz,3H),1.37(d,J=2.8Hz,15H),1.28(s,12H).

[0075] The structural formula is as follows:

[0076] Example 10: Preparation of N-(3-aminopropyl)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborhecyclopentan-2-yl)benzamide (compound 2J)

[0077] Using 4-carboxyphenylboronic acid pinacol ester and N-tert-butoxycarbonyl-1,3-propanediamine as raw materials, the target compound was obtained as a white solid with a yield of 88% through a reaction similar to that used to synthesize compound 2C.

[0078] The NMR data are as follows: 1 H NMR(400MHz, DMSO-d6)δ8.53(t,J=5.6Hz,1H),7.88–7.83(m,2H),7.77–7.70(m,3H), 3.27(q,J=6.6Hz,2H),3.08(q,J=6.6Hz,2H),1.82(s,2H),1.38(s,9H),1.28(s,12H).

[0079] The structural formula is as follows:

[0080] Example 11 Preparation of 4-(2-((3r,5r,7r)-adamantane-1-yl)acetamyl)-N-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborhexacyclopentan-2-yl)phenyl)butyramide (compound 4C)

[0081] Step 1: Preparation of 4-amino-N-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborhecyclopentan-2-yl)phenyl)butyramide

[0082] The (4-oxo-4-((4-(4,4,5,5-tetramethyl-1,3,2-dioxaborhexacyclopentan-2-yl)phenyl)amino)tert-butyl)carbamate (compound 2C) (500 mg, 1.28 mmol) and trifluoroacetic acid (2.94 mL, 3.84 mmol) from Example 1 were added to 20 mL of dichloromethane (CH2Cl2) and reacted overnight at room temperature. After the reaction was completed as monitored by TLC, the product was concentrated under vacuum to obtain the target product. 371 mg of brown liquid, yield: 100%. The target product did not require purification and could be used directly in the next step. Its structural formula is shown below:

[0083]

[0084] Step 2: Preparation of 4-(2-((3r, 5r, 7r)-adamantane-1-yl)acetamityl)-N-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborhecyclopentan-2-yl)phenyl)butyramide

[0085] Compounds 4-amino-N-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborhecyclopentan-2-yl)phenyl)butyramide (500 mg, 1.64 mmol), 1-adamantaneacetic acid (383 mg, 1.97 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) (565.7 mg, 2.95 mmol), and 1-hydroxy-7-azobenzotriazole (HOAT) (403.4 mg, 2.95 mmol), along with N-methylmorpholine (0.83 mL, 8.19 mmol), were added to 20 mL of dimethyl sulfoxide (DMSO) and reacted overnight at room temperature. After the reaction was complete as detected by TLC, the reaction solution was poured into ice water and extracted with dichloromethane. The organic phase was collected, concentrated, and the target product was obtained by column chromatography. 570 mg brown solid, yield: 66.8%.

[0086] The NMR data are as follows: 1 H NMR (400MHz, DMSO-d6) δ10.02(s,1H),7.74–7.69(m,1H),7.63–7.56(m,4H),3.06(q,J=6.7Hz,2H),2.33(t,J=7.5Hz, 2H),1.92–1.88(m,3H),1.82(s,2H),1.70(t,J=7.3Hz,2H),1.65(d,J=12.4Hz,3H),1.59–1.53(m,9H),1.28(s,12H).

[0087] The structural formula is as follows:

[0088] Example 12 Preparation of 2-((3r, 5r, 7r)-adamantane-1-yl)-N-(2-oxo-2-((4-(4,4,5,5-tetramethyl-1,3,2-dioxaborhecyclopentan-2-yl)phenyl)amino)ethyl)acetamide (compound 4A)

[0089] Using compound 2A, 1-adamantaneacetic acid from Example 2 as a starting material, the target compound was obtained as a white solid with a yield of 68% through a reaction similar to that used to synthesize compound 4C.

[0090] The NMR data are as follows: 1 H NMR (400MHz, DMSO-d6) δ10.08(s,1H),7.80(t,J=5.7Hz,1H),7.60(q,J=8.5Hz,4H),3.75 (d,J=6.3Hz,2H),1.84(s,3H),1.60(d,J=12.2Hz,4H),1.58–1.51(m,10H),1.28(s,12H).

[0091] The structural formula is as follows:

[0092] Example 13 Preparation of 3-(2-((3r,5r,7r)-adamantane-1-yl)acetamyl)-N-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborhecyclopentan-2-yl)phenyl)acrylamide (compound 4B)

[0093] Using compound 2B, 1-adamantaneacetic acid from Example 3 as a starting material, the target compound was obtained as a white solid with a yield of 69% through a reaction similar to that used to synthesize compound 4C.

[0094] The NMR data are as follows: 1 H NMR (400MHz, DMSO-d6) δ10.08(s,1H),7.80(t,J=5.7Hz,1H),7.60(q,J=8.5Hz,4H),3.30(d,J=6 .3Hz,2H),2.53(s,2H),1.84(s,3H),1.60(d,J=12.2Hz,4H),1.56–1.49(m,10H),1.28(s,12H).

[0095] The structural formula is as follows:

[0096] Example 14 Preparation of (3r, 5r, 7r)-N-(4-oxo-4-((4-(4,4,5,5-tetramethyl-1,3,2-dioxaborhecyclopentan-2-yl)phenyl)amino)butyl)adamantane-1-carboxamide (compound 4D)

[0097] Using compound 2C, 1-adamantanecarboxylic acid from Example 1 as a starting material, the target compound was obtained as a white solid with a yield of 68% through a reaction similar to that used to synthesize compound 4C.

[0098] The NMR data are as follows: 1 H NMR(400MHz, DMSO-d6)δ9.97(s,1H),7.63–7.57(m,4H),7.33(t,J=5.7Hz,1H),3.34(q,J=6 .6Hz,2H),2.31(t,J=7.3Hz,2H),1.98(d,J=2.9Hz,5H),1.68–1.62(m,10H),1.28(s,12H).

[0099] The structural formula is as follows:

[0100] Example 15: Preparation of N-(3-(2-((3r, 5r, 7r)-adamantane-1-yl)acetamityl)propyl)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborhexacyclopentane-2-yl)benzamide (compound 4E)

[0101] Using compound 2J, 1-adamantaneacetic acid from Example 10 as a starting material, the target compound was obtained through a reaction similar to that used to synthesize compound 4C. The compound was a white solid with a yield of 70%.

[0102] The NMR data are as follows: 1 H NMR(400MHz, DMSO-d6)δ8.53(t,J=5.6Hz,1H),7.88–7.83(m,2H),7.77–7.70(m,3H),3.27(q,J=6.6Hz,2H),3.08 (q,J=6.6Hz,2H),1.93–1.88(m,3H),1.82(s,2H),1.64(dd,J=8.9,5.4Hz,5H),1.59–1.53(m,9H),1.31(s,12H).

[0103] The structural formula is as follows:

[0104] Example 16 Preparation of 5-(2-((3r, 5r, 7r)-adamantane-1-yl)acetamyl)-N-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborhecyclopentan-2-yl)phenyl)pentanamide (compound 4F)

[0105] Using compound 2D, 1-adamantaneacetic acid from Example 4 as a starting material, the target compound was obtained as a white solid with a yield of 70% through a reaction similar to that used to synthesize compound 4C.

[0106] The NMR data are as follows: 1 H NMR (400MHz, DMSO-d6) δ10.01(s,1H),7.76–7.62(m,1H),7.63–7.56(m,4H),3.06(q,J=6.7Hz,2H),2.32(t ,J=7.5Hz,2H),1.90–1.87(m,3H),1.82(s,2H),1.65(d,J=12.0Hz,5H),1.60–1.53(m,11H),1.28(s,12H).

[0107] The structural formula is as follows:

[0108] Example 17 Preparation of (3r, 5r, 7r)-N-(5-oxo-5-((4-(4,4,5,5-tetramethyl-1,3,2-dioxaborhecyclopentan-2-yl)phenyl)amino)pentyl)adamantane-1-carboxamide (compound 4G)

[0109] Using compound 2D, 1-adamantanecarboxylic acid from Example 4 as a starting material, the target compound was obtained as a white solid with a yield of 71% through a reaction similar to that used to synthesize compound 4C.

[0110] The NMR data are as follows: 1 H NMR (400MHz, DMSO-d6) δ9.97(s,1H),7.63–7.57(m,4H),7.33(t,J=5.7Hz,1H),3.04(q,J=6.6Hz,2H),2.31(t,J=7.3 Hz,2H),1.95(d,J=2.9Hz,3H),1.68–1.62(m,10H),1.54(dd,J=9.1,6.1Hz,2H),1.42(q,J=7.1Hz,2H),1.28(s,12H).

[0111] The structural formula is as follows:

[0112] Example 18 Preparation of 6-(2-((3r,5r,7r)-adamantane-1-yl)acetamyl)-N-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborhexacyclopentan-2-yl)phenyl)hexamamide (compound 4H)

[0113] Using compound 2E, 1-adamantaneacetic acid from Example 5 as a starting material, the target compound was obtained as a white solid with a yield of 72% through a reaction similar to that used to synthesize compound 4C.

[0114] The NMR data are as follows: 1 H NMR(400MHz, DMSO-d6)δ9.97(s,1H),7.61(dd,J=11.7,3.1Hz,5H),3.01(q,J=6.6Hz,2H),2.30(t,J=7.4Hz,2H), 1.89(s,3H),1.79(d,J=3.3Hz,2H),1.63(d,J=12.0Hz,5H),1.58–1.50(m,11H),1.43–1.37(m,2H),1.28(s,12H).

[0115] The structural formula is as follows:

[0116] Example 19 Preparation of 7-(2-((3r,5r,7r)-adamantane-1-yl)acetamyl)-N-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborhexacyclopentan-2-yl)phenyl)heptamide (4I)

[0117] Using compound 2F, 1-adamantaneacetic acid from Example 6 as a starting material, the target compound was obtained as a white solid with a yield of 72% through a reaction similar to that used to synthesize compound 4C.

[0118] The NMR data are as follows: 1 H NMR (400MHz, DMSO-d6) δ9.98 (s, 1H), 7.66–7.57 (m, 5H), 3.00 (q, J = 6.4Hz, 2H), 2.30 (t, J = 7.4Hz, 2H), 1.90 (d ,J=4.6Hz,3H),1.79(s,2H),1.64(d,J=12.4Hz,4H),1.59–1.52(m,12H),1.38(t,J=6.7Hz,2H),1.28(s,14H).

[0119] The structural formula is as follows:

[0120] Example 20 Preparation of 8-(2-((3r,5r,7r)-adamantane-1-yl)acetamyl)-N-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborhexacyclopentan-2-yl)phenyl)octamide (compound 4J)

[0121] Using compound 2G, 1-adamantaneacetic acid from Example 7 as a starting material, the target compound was obtained as a white solid with a yield of 69% through a reaction similar to that used to synthesize compound 4C.

[0122] The NMR data are as follows: 1 H NMR (400MHz, DMSO-d6) δ9.97(s,1H),7.66–7.56(m,5H),3.00(q,J=6.5Hz,2H),2.30(t,J=7.4Hz,2H),1.89(q,J=3.7,3.2H z,3H),1.79(d,J=1.9Hz,2H),1.63(d,J=12.0Hz,5H),1.55(dd,J=12.3,3.3Hz,11H),1.37(t,J=6.6Hz,2H),1.28(s,16H).

[0123] The structural formula is as follows:

[0124] Example 21 Preparation of 11-(2-((3r,5r,7r)-adamantane-1-yl)acetamyl)-N-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborhecyclopentan-2-yl)phenyl)undecanoamide (compound 4K)

[0125] Using compound 2H,1-adamantaneacetic acid from Example 8 as a starting material, the target compound was obtained as a white solid with a yield of 70% through a reaction similar to that used to synthesize compound 4C.

[0126] The NMR data are as follows: 1 H NMR (400MHz, DMSO-d6) δ9.97(s,1H),7.61(dd,J=7.8,3.4Hz,5H),3.00(q,J=6.3Hz,2H),2.31(t,J=7.4Hz,2H),1.91(d ,J=5.0Hz,3H),1.80(s,2H),1.68–1.61(m,5H),1.59–1.53(m,11H),1.36(t,J=6.6Hz,2H),1.28(s,12H),1.24(s,12H).

[0127] The structural formula is as follows:

[0128] Example 22 Preparation of 3-(2-(2-((3r,5r,7r)-adamantane-1-yl)acetamityl)ethoxy)ethoxy)N-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborhexacyclopentan-2-yl)phenyl)acrylamide (compound 4L)

[0129] Using compound 2I,1-adamantaneacetic acid from Example 9 as a starting material, the target compound was obtained as a white solid with a yield of 72% through a reaction similar to that used to synthesize compound 4C.

[0130] The NMR data are as follows: 1 H NMR (400MHz, DMSO-d6) δ10.07(s,1H),7.69(t,J=5.8Hz,1H),7.65–7.57(m,4H),3.69(t,J=6.2Hz,2H),3.52–3.44(m,8H),2.57(t, J=6.2Hz,2H),2.50(p,J=1.9Hz,2H),1.89(t,J=3.1Hz,3H),1.81(s,2H),1.64(d,J=11.7Hz,4H),1.58–1.52(m,10H),1.28(s,12H).

[0131] The structural formula is as follows:

[0132] Example 23 Preparation of tert-butyl 2-(((5-bromopyridin-3-yl)methyl)(methyl)amino)ethyl)(methyl)carbamate (compound 6A)

[0133] 5-Bromopyridin-3-carboxaldehyde (400 mg, 2.15 mmol), methyl (2-(methylamino)ethyl)carbamate tert-butyl ester (607 mg, 3.23 mmol), and glacial acetic acid (738 μM, 12.90 mmol) were added to dichloroethane, followed by the addition of sodium triacetoxyborohydride (911 mg, 4.30 mmol) in portions at 0 °C. The reaction solution was allowed to react overnight at room temperature. After the reaction was complete as monitored by TLC, the pH was adjusted to 7-8 with the addition of saturated sodium bicarbonate solution, followed by extraction with dichloromethane. The organic phase was collected. The target compound was obtained by column chromatography. 400 mg of the solution was a pale yellow oily liquid, yielding 51.9%.

[0134] The NMR data are as follows: 1H NMR(400MHz, DMSO-d6)δδ8.58(d,J=2.3Hz,1H),8.47(d,J=1.9Hz,1H),7.93(t,J=2.0Hz,1H),3.53(s,2H),3. 27(d,J=8.6Hz,2H),2.82–2.69(m,3H),2.45(d,J=6.6Hz,2H),2.18(d,J=12.6Hz,3H),1.35(d,J=28.7Hz,9H).

[0135] The structural formula is as follows:

[0136] Example 24 Preparation of tert-butyl carbamate (compound 6B)

[0137] Using 5-bromopyridine-3-carboxaldehyde as a starting material, the target compound was obtained as a white solid with a yield of 53% through a reaction similar to that used to synthesize compound 6A.

[0138] The NMR data are as follows: 1 H NMR (400MHz, DMSO-d6) δ8.58(d,J=2.3Hz,1H),8.47(d,J=1.7Hz,1H),7.93(t,J=2.0Hz,1H),6.42(s,2H),3 .53(s,2H),3.26(d,J=6.2Hz,2H),2.80–2.71(m,3H),2.45(d,J=6.6Hz,2H),1.36(dd,J=23.1,5.7Hz,9H).

[0139] The structural formula is as follows:

[0140] Example 25 Preparation of tert-butyl (2-(((5-bromopyridin-3-yl)methyl)amino)ethyl)(methyl)carbamate (compound 6C)

[0141] Using 5-bromopyridine-3-carboxaldehyde as a starting material, the target compound was obtained as a white solid with a yield of 54% through a reaction similar to that used to synthesize compound 6A.

[0142] The NMR data are as follows: 1H NMR (400MHz, DMSO-d6) δ8.56(d,J=2.3Hz,1H),8.50(d,J=1.7Hz,1H),8.00(t,J=2.0Hz,1H),3.74(s,2H),3 .23(t,J=6.6Hz,2H),2.78(s,3H),2.60(t,J=6.6Hz,2H),1.91(s,3H),1.45–1.30(dd,J=23.1,5.7Hz,9H).

[0143] The structural formula is as follows:

[0144] Example 26 Preparation of tert-butyl (2-(((6-bromopyridin-2-yl)methyl)(methyl)amino)ethyl)(methyl)carbamate (compound 6D)

[0145] Using 6-bromopyridine-2-carboxaldehyde as a starting material, the target compound was obtained as a white solid with a yield of 56% through a reaction similar to that used to synthesize compound 6A.

[0146] The NMR data are as follows: 1 H NMR (400MHz, DMSO-d6) δ7.79–7.68(m,1H),7.54–7.41(m,2H),3.61(s,2H),3.19(t,J=6.7Hz,2H),2 .78(s,3H),2.50(td,J=4.9,4.3,2.4Hz,2H),2.25(d,J=10.6Hz,3H),1.38(dd,J=23.1,5.7Hz,9H).

[0147] The structural formula is as follows:

[0148] Example 27 Preparation of tert-butyl 2-(((2-bromopyridin-4-yl)methyl)(methyl)amino)ethyl)(methyl)carbamate (compound 6E)

[0149] Using 2-bromopyridine-4-carboxaldehyde as a starting material, the target compound was obtained as a white solid with a yield of 56% through a reaction similar to that used to synthesize compound 6A.

[0150] The NMR data are as follows: 1H NMR (400MHz, DMSO-d6) δ8.31(dd,J=5.0,3.6Hz,1H),7.55(d,J=3.9Hz,1H),7.36(dd,J=6.4,5.1Hz,1H),3.54 (s,2H),3.27(q,J=6.6Hz,2H),2.77(s,3H),2.45(d,J=6.5Hz,2H),2.19(s,3H),1.36(dd,J=23.1,5.7Hz,9H).

[0151] The structural formula is as follows:

[0152] Example 28 Preparation of tert-butyl 2-(((5-bromopyridin-3-yl)methyl)(methyl)amino)ethyl)(methyl)carbamate (compound 6F)

[0153] Using 4-bromopyridine-3-carboxaldehyde as a starting material, the target compound was obtained as a white solid with a yield of 56% through a reaction similar to that used to synthesize compound 6A.

[0154] The NMR data are as follows: 1 H NMR (400MHz, DMSO-d6) δ8.67(s,1H),8.49(d,J=4.9Hz,1H),7.47(d,J=4.9Hz,1H),3.59(s,2H),3.30(s, 2H), 2.78 (d, J = 12.5Hz, 3H), 2.51 (dd, J = 6.4, 4.6Hz, 2H), 2.25 (d, J = 10.9Hz, 3H), 1.34 (d, J = 24.2Hz, 9H).

[0155] The structural formula is as follows:

[0156] Example 29 Preparation of (2-(5-bromo-N-methylnicotinamide)ethyl)(methyl)carbamate (compound 6G)

[0157] 5-Bromonicotinic acid (500 mg, 2.48 mmol), tert-butyl(2-aminoethyl)carbamate (475.3 mg, 2.73 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) (855.0 mg, 4.46 mmol), 1-hydroxy-7-azobenzotriazole (HOAT) (607.1 mg, 4.46 mmol), and N-methylmorpholine (1.36 mL, 12.40 mmol) were added to 20 mL of dimethyl sulfoxide (DMSO) and reacted overnight at room temperature. After the reaction was complete as detected by TLC, the reaction solution was poured into ice water and extracted with dichloromethane. The organic phase was collected, concentrated, and the target product was obtained by column chromatography. 420 mg of the product was a grayish-white liquid, yield 52.8%.

[0158] The NMR data are as follows: 1 H NMR (400MHz, DMSO-d6) δ8.94(d,J=1.8Hz,1H),8.84(d,J=2.3Hz,1H),8.74(s,1H),8.39(t,J= 2.1Hz, 1H), 6.92 (t, J = 5.8Hz, 1H), 3.29 (d, J = 6.4Hz, 2H), 3.11 (q, J = 6.1Hz, 2H), 1.37 (s, 9H).

[0159] The structural formula is as follows:

[0160] Example 30 Preparation of 4-(2-((3r, 5r, 7r)-adamantane-1-yl)acetamityl)-N-(4-(5-((methyl(2-(methylamino)ethyl)amino)methyl)pyridin-3-yl)phenyl)butyramide (compound 8C) Step 1: Preparation of (2-((5-(4-(4-(2-((3r, 5r, 7r)-adamantane-1-yl)acetamityl)butyramide)phenyl)pyridin-3-yl)methyl)(methyl)amino)ethyl)(methyl)carbamate tert-butyl ester (compound 7C)

[0161] Compound 4C (170 mg, 0.52 mmol) from Example 11, compound 6A (188 mg, 0.52 mmol) from Example 23, and sodium carbonate (223 mg, 2.08 mmol) were added to a 4:1 mixture of dioxane and water, and the mixture was purged with nitrogen five times. Then, [1,1'-bis(diphenylphosphine)ferrocene]palladium dichloride dichloromethane complex (37.6 mg, 0.05 mmol) was added, and the mixture was purged with nitrogen five times. The mixture was heated at 100 °C for 4 hours, concentrated under vacuum, extracted with ethyl acetate, and the combined organic layers were purified by column chromatography with a mobile phase of dichloromethane:methanol = 20:1 to obtain the product, a grayish-white solid, in 58% yield.

[0162] The NMR data are as follows: 1 H NMR (400MHz, DMSO-d6) δ10.10(s,1H),8.75(d,J=2.3Hz,1H),8.43(d,J=1.9Hz,1H),8.0 5(t,J=5.8Hz,1H),7.91(t,J=2.0Hz,1H),7.75–7.65(m,4H),3.88(d,J=5.8Hz,2H),3.59 (s,2H),3.29(m,2H),2.75(d,J=17.9Hz,3H),2.46(s,2H),2.22(d,J=14.0Hz,3H),1.92 (d,J=5.2Hz,5H),1.66(d,J=12.1Hz,3H),1.60(d,J=2.8Hz,9H),1.31(d,J=20.7Hz,9H).

[0163] The structural formula is as follows:

[0164] Step 2: Preparation of 4-(2-((3r,5r,7r)-adamantane-1-yl)acetamityl)-N-(4-(5-((methyl(2-(methylamino)ethyl)amino)methyl)pyridin-3-yl)phenyl)butyramide

[0165] Compound 7C (150 mg, 0.24 mmol) was added to a 1 M hydrochloric acid-ethanol solution. The reaction was stirred at room temperature for 8 hours. After TLC analysis, the crude product was obtained by concentration under vacuum. The crude product was washed with dry ethyl acetate, filtered, and dried to give the final product. It was pale yellow, 117 mg, yield 93%.

[0166] The NMR data are as follows: 1 H NMR(400MHz,DMSO-d6)δ10.11(s,1H),8.77(s,1H),8.47(s,1H),7.97(s,1H),7.74(d ,J=9.3Hz,3H),7.69(d,J=8.4Hz,2H),4.09(s,1H),3.60(s,2H),3.17(s,2H),3.08(d, J = 6.7 Hz, 3H), 2.77 (t, J = 6.0 Hz, 2H), 2.36 (d, J = 6.3 Hz, 4H), 2.17 (s, 3H), 1.91 (s, 3H), 1.83 (s, 2H), 1.75–1.70 (m, 2H), 1.65 (d, J = 12.5 Hz, 3H), 1.58 (d, J = 10.2 Hz, 9H). HRMS m / z [M+H] + 532.3643, calculated value: 532.3651.

[0167] The structural formula is as follows:

[0168] Example 31 Preparation of 2-((3r,5r,7r)-adamantane-1-yl)-N-((4-((5-((methyl(2-(methylamino)ethyl)amino)methyl)pyridin-3-yl)phenyl)amino)-2-oxoethyl)acetamide (compound 8A)

[0169] Using compound 6A from Example 23 and compound 4A from Example 12 as raw materials, the target compound was obtained as a white solid through a reaction similar to that used to synthesize compound 8C, with a yield of 57%.

[0170] The NMR data are as follows: 1 H NMR (400MHz, DMSO-d6) δ10.10(s,1H),8.76(d,J=2.3Hz,1H),8.45(d,J=1.9Hz,1H) ,8.06(t,J=5.8Hz,1H),7.94(t,J=2.2Hz,1H),7.71(s,4H),3.89(d,J=5.8Hz,2H),3 .58(s,2H),2.59(t,J=6.4Hz,2H),2.45(t,J=6.2Hz,2H),2.26(s,3H),2.17(d,J=4. 7Hz,3H),1.93(d,J=5.8Hz,5H),1.66(d,J=12.1Hz,3H),1.60(d,J=2.9Hz,9H).HRMS m / z[M+H]+504.3331, calculated value: 504.3338.

[0171] The structural formula is as follows:

[0172] Example 32 Preparation of 3-(2-((3r,5r,7r)-adamantane-1-yl)acetamityl)-N-(4-(5-((methyl(2-(methylamino)ethyl)amino)methyl)pyridin-3-yl)phenyl)acrylamide (compound 8B)

[0173] Using compound 6A from Example 23 and compound 4B from Example 13 as raw materials, the target compound was obtained as a white solid through a reaction similar to that used to synthesize compound 8C, with a yield of 59%.

[0174] The NMR data are as follows: 1H NMR (400MHz, DMSO-d6) δ10.10(s,1H),8.76(d,J=2.3Hz,1H),8.45(d,J=1.9Hz,1H) ,8.06(t,J=5.8Hz,1H),7.94(t,J=2.2Hz,1H),7.71(s,4H),3.89(d,J=5.8Hz,2H),3 .58(s,2H),2.59(t,J=6.4Hz,2H),2.45(t,J=6.2Hz,2H),2.26(s,3H),2.17(d,J=4. 7Hz,3H),1.93(d,J=5.8Hz,5H),1.66(d,J=12.1Hz,3H),1.60(d,J=2.9Hz,9H).HRMS m / z[M+H]+518.3491, calculated value: 518.3495.

[0175] The structural formula is as follows:

[0176] Example 33: Preparation of (3r, 5r, 7r)-N-(4-((4-((5-((methyl(2-(methylamino)ethyl)amino)methyl)pyridin-3-yl)phenyl)amino)-4-oxobutyl)adamantane-1-carboxamide (compound 8D)

[0177] Using compound 6A from Example 23 and compound 4D from Example 14 as raw materials, the target compound was obtained as a white solid through a reaction similar to that used to synthesize compound 8C, with a yield of 56%.

[0178] The NMR data are as follows: 1 H NMR (400MHz, DMSO-d6) δ10.07(s,1H),8.75(d,J=2.3Hz,1H),8.44(d,J=1.9Hz,1H),7.9 3(d,J=2.2Hz,1H),7.74(d,J=8.8Hz,2H),7.68(d,J=8.5Hz,2H),7.37(d,J=5.5Hz,1H),3 .57(s,2H),3.11(q,J=6.6Hz,3H),2.62(t,J=6.3Hz,3H),2.48–2.42(m,3H),2.32(t,J=7.4Hz,2H),2.28(s,4H),2.16(d,J=4.5Hz,4H),1.94(s,3H),1.75(t,J=4.5Hz,8H).HRMS m / z[M+H]+518.3489, calculated value: 518.3495.

[0179] The structural formula is as follows:

[0180] Example 34 Preparation of 5-(2-((3r, 5r, 7r)-adamantane-1-yl)acetamyl)-N-(4-(5-((methyl(2-(methylamino)ethyl)amino)methyl)pyridin-3-yl)phenyl)pentanamide (compound 8E): Using compound 6A from Example 23 and compound 4F from Example 16 as starting materials, the target compound was obtained as a white solid with a yield of 57% through a reaction similar to that used in the synthesis of compound 8C.

[0181] The NMR data are as follows: 1 H NMR (400MHz, DMSO-d6) δ10.03(s,1H),8.76(d,J=2.2Hz,1H),8.45(d,J=1.9Hz,1H),7.94(t,J=2 .2Hz,1H),7.73(d,J=8.8Hz,2H),7.71–7.65(m,3H),3.58(s,2H),3.05(q,J=6.7Hz,2H),2.65(t, J = 6.3Hz, 2H), 2.47(q, J = 2.6Hz, 2H), 2.34(t, J = 7.3Hz, 2H), 2.30(s, 3H), 2.17(d, J = 4.6Hz, 4H), 1.90(s, 3H), 1.81(s, 2H), 1.64(d, J = 12.0Hz, 4H), 1.60–1.53(m, 10H), 1.44(q, J = 7.3Hz, 2H). HRMS m / z [M+H] + 546.3810, calculated value: 546.3808.

[0182] The structural formula is as follows:

[0183] Example 35: Preparation of (3r, 5r, 7r)-N-(5-((4-((5-((methyl(2-(methylamino)ethyl)amino)methyl)pyridin-3-yl)phenyl)amino)-5-oxopentyl)adamantane-1-carboxamide (compound 8F)

[0184] Using compound 6A from Example 23 and compound 4G from Example 17 as raw materials, the target compound was obtained as a white solid through a reaction similar to that used to synthesize compound 8C, with a yield of 53%.

[0185] The NMR data are as follows: 1H NMR (400MHz, DMSO-d6) δ10.09(s,1H),8.76(d,J=2.2Hz,1H),8.46(d,J=1.8Hz,1H),7.96(d,J=2.2Hz,1H),7.7 5(d,J=8.8Hz,2H),7.68(d,J=8.7Hz,2H),7.38(t,J=5.7Hz,1H),3.59(s,2H),3.06(q,J=6.6Hz,2H),2.76(t,J= 6.2Hz, 2H), 2.53(d, J = 6.3Hz, 2H), 2.35(d, J = 6.3Hz, 5H), 2.17(s, 3H), 1.95(d, J = 4.2Hz, 3H), 1.75(d, J = 2.9Hz, 6H), 1.70–1.60(m, 7H), 1.56(q, J = 7.5Hz, 2H), 1.45(q, J = 7.3Hz, 2H).HRMSm / z[M+H]+532.3643, calculated value: 532.3651.

[0186] The structural formula is as follows:

[0187] Example 36: Preparation of N-(3-(2-((3r,5r,7r)-adamantane-1-yl)acetamido)propyl)-4-((methyl(2-(methylamino)ethyl)amino)methyl)pyridin-3-yl)benzamide (compound 8G)

[0188] Using compound 6A from Example 23 and compound 4E from Example 15 as raw materials, the target compound was obtained as a white solid through a reaction similar to that used to synthesize compound 8C, with a yield of 52%.

[0189] The NMR data are as follows: 1 H NMR(400MHz, DMSO-d6)δ8.84(d,J=2.3Hz,1H),8.59–8.51(m,2H),8.04(t,J=2.2Hz,1H),8 .00–7.95(m,2H),7.88–7.82(m,2H),7.73(t,J=5.7Hz,1H),3.60(s,2H),3.32(d,J=6.6Hz ,3H),3.10(q,J=6.7Hz,2H),2.63(t,J=6.3Hz,2H),2.47(t,J=6.3Hz,2H),2.28(s,3H),2.18(s,3H),1.90(s,3H),1.83(s,2H),1.65(dd,J=12.7,5.1Hz,5H),1.60–1.53(m,9H).HRMS m / z[M+H]+532.3649, calculated value: 532.3651.

[0190] The structural formula is as follows:

[0191] Example 37 Preparation of 4-(2-((3r,5r,7r)-adamantane-1-yl)acetamyl)-N-(4-(5-((2-aminoethyl)(methyl)amino)methyl)pyridin-3-yl)phenyl)butyramide (compound 8H)

[0192] Using compound 6B from Example 24 and compound 4C from Example 11 as raw materials, the target compound was obtained as a white solid through a reaction similar to that used to synthesize compound 8C, with a yield of 57%.

[0193] The NMR data are as follows: 1 H NMR (400MHz, DMSO-d6) δ10.08(s,1H),8.76(d,J=2.3Hz,1H),8.46(d,J=1.9Hz,1H),7.9 5(t,J=2.3Hz,1H),7.74(d,J=8.9Hz,3H),7.71–7.66(m,2H),3.58(s,2H),3.08(q,J=6. 7Hz, 3H), 2.70(t, J = 6.5Hz, 3H), 2.41(t, J = 6.4Hz, 2H), 2.16(d, J = 4.4Hz, 4H), 1.91(s, 3H), 1.83(s, 2H), 1.72(t, J = 7.3Hz, 2H), 1.65(d, J = 11.8Hz, 4H), 1.60–1.54(m, 9H). HRMS m / z[M+H]+518.3488, calculated value: 518.3495. The structural formula is shown below:

[0194] Example 38 Preparation of 4-(2-((3r,5r,7r)-adamantane-1-yl)acetamyl)-N-(4-(5-((2-(methylamino)ethyl)amino)methyl)pyridin-3-yl)phenyl)butyramide (8I)

[0195] Using compound 6C from Example 25 and compound 4C from Example 11 as raw materials, the target compound was obtained as a white solid through a reaction similar to that used to synthesize compound 8C, with a yield of 54%.

[0196] The NMR data are as follows: 1H NMR (400MHz, DMSO-d6) δ10.07(s,1H),8.73(d,J=2.2Hz,1H),8.46(d,J=1.9Hz,1H),7 .98(d,J=2.3Hz,1H),7.73(d,J=8.5Hz,3H),7.68(d,J=8.7Hz,2H),3.77(s,2H),3.08( q,J=6.6Hz,3H),2.58(t,J=4.2Hz,5H),2.35(t,J=7.5Hz,2H),2.27(s,3H),1.91(s,3H),1.83(s,2H),1.72(t,J=7.2Hz,2H),1.65(d,J=12.3Hz,3H),1.60–1.53(m,9H).HRMS m / z[M+H]+518.3489, calculated value: 518.3495.

[0197] The structural formula is as follows:

[0198] Example 39 Preparation of 5-(2-((3r,5r,7r)-adamantane-1-yl)acetamyl)-N-(4-(5-((2-aminoethyl)(methyl)amino)methyl)pyridin-3-yl)phenyl)pentanamide (compound 8J)

[0199] Using compound 6B from Example 24 and compound 4F from Example 16 as raw materials, the target compound was obtained as a white solid through a reaction similar to that used to synthesize compound 8C, with a yield of 52%.

[0200] The NMR data are as follows: 1 H NMR (400MHz, DMSO-d6) δ10.05(s,1H),8.76(d,J=2.3Hz,1H),8.46(d,J=1.9Hz,1H),7.94(t,J=2.2Hz,1H),7.74 (d,J=8.8Hz,2H),7.69(td,J=7.6,6.4,4.1Hz,3H),3.57(s,2H),3.05(q,J=6.6Hz,2H),2.70(td,J=6.4,2.2Hz, 3H), 2.42(dd,J=6.4,3.9Hz,2H), 2.35(t,J=7.4Hz,2H), 2.16(d,J=4.5Hz,4H), 1.89(d,J=4.1Hz,3H), 1.81(s,2H), 1.64(d,J=12.3Hz,5H), 1.59–1.53(m,9H), 1.45(q,J=7.0Hz,2H).HRMSm / z[M+H]+532.3644, calculated value: 532.3651.

[0201] The structural formula is as follows:

[0202] Example 40 Preparation of 6-(2-((3r,5r,7r)-adamantane-1-yl)acetamyl)-N-(4-(5-((methyl(2-(methylamino)ethyl)amino)methyl)pyridin-3-yl)phenyl)hexamamide (compound 8K): Using compound 6A from Example 23 and compound 4H from Example 18 as starting materials, the target compound was obtained as a white solid with a yield of 57% through a reaction similar to that used in the synthesis of compound 8C.

[0203] The NMR data are as follows: 1 H NMR (400MHz, DMSO-d6) δ10.11(s,1H),8.76(d,J=2.2Hz,1H),8.47(s,1H),7.98(s,1H),7.75(d, J=8.4Hz,2H),7.71(s,3H),3.60(s,2H),3.02(q,J=6.5Hz,2H),2.84(t,J=6.2Hz,2H),2.57(t,J =6.0Hz,2H),2.41(d,J=1.1Hz,3H),2.33(t,J=7.3Hz,2H),2.16(d,J=3.7Hz,4H),1.89(s,3H),1.80(s,2H),1.67–1.59(m,5H),1.57–1.52(m,9H),1.45–1.39(m,2H),1.30(t,J=7.6Hz,2H).HRMS m / z[M+H]+560.3955, calculated value: 560.3964.

[0204] The structural formula is as follows:

[0205] Example 41 Preparation of 7-(2-((3r,5r,7r)-adamantane-1-yl)acetamyl)-N-(4-(5-((methyl(2-(methylamino)ethyl)amino)methyl)pyridin-3-yl)phenyl)heptamide (compound 8L): Using compound 6A from Example 23 and compound 4I from Example 19 as starting materials, the target compound was obtained as a white solid with a yield of 57% through a reaction similar to that used in the synthesis of compound 8C.

[0206] The NMR data are as follows: 1H NMR (400MHz, DMSO-d6) δ10.03(s,1H),8.75(d,J=2.2Hz,1H),8.45(d,J=1.9Hz,1H),7.93(t,J=2.2Hz,1H),7. 73(d,J=8.9Hz,2H),7.68(d,J=8.8Hz,2H),7.63(d,J=5.9Hz,1H),3.57(s,2H),3.02(t,J=6.3Hz,2H),2.61(t ,J=6.3Hz,2H),2.46(dd,J=6.3,4.0Hz,2H),2.33(t,J=7.4Hz,2H),2.27(s,3H),2.17(d,J=4.4Hz,4H),1.90(s,3H),1.81(s,2H),1.64(d,J=12.6Hz,5H),1.58–1.54(m,9H),1.39(d,J=6.8Hz,2H),1.34–1.29(m,4H).HRMS m / z[M+H]+574.4115, calculated value: 574.4121.

[0207] The structural formula is as follows:

[0208] Example 42 Preparation of 8-(2-((3r,5r,7r)-adamantane-1-yl)acetamyl)-N-(4-(5-((methyl(2-(methylamino)ethyl)amino)methyl)pyridin-3-yl)phenyl)octamide (compound 8M) Using compound 6A from Example 23 and compound 4J from Example 20 as starting materials, the target compound was obtained as a white solid with a yield of 57% through a reaction similar to that used in the synthesis of compound 8C.

[0209] The NMR data are as follows: 1H NMR (400MHz, DMSO-d6) δ10.03(s,1H),8.75(d,J=2.3Hz,1H),8.45(d,J=1.9Hz,1H),7.94(d,J=2.3Hz,1 H),7.73(d,J=8.6Hz,2H),7.67(d,J=8.7Hz,2H),7.64(d,J=6.4Hz,1H),3.58(s,2H),3.04–2.99(m,2H), 2.64(t,J=6.3Hz,2H),2.45(dd,J=6.6,4.0Hz,2H),2.33(d,J=7.4Hz,2H),2.29(s,3H),2.16(d,J=4.3Hz,4H),1.88(s,3H),1.80(s,2H),1.62(d,J=12.5Hz,5H),1.57–1.52(m,9H),1.39(d,J=8.7Hz,2H).HRMS m / z[M+H]+588.4269, calculated value: 588.4277.

[0210] The structural formula is as follows:

[0211] Example 43 Preparation of 12-(2-((3r,5r,7r)-adamantane-1-yl)acetamyl)-N-(4-(5-((methyl(2-(methylamino)ethyl)amino)methyl)pyridin-3-yl)phenyl)dodecanoic acid (compound 8N)

[0212] Using compound 6A from Example 23 and compound 4K from Example 21 as raw materials, the target compound was obtained as a white solid through a reaction similar to that used to synthesize compound 8C, with a yield of 56%.

[0213] The NMR data are as follows: 1H NMR (400MHz, DMSO-d6) δ10.00(s,1H),8.75(d,J=2.3Hz,1H),8.45(d,J=1.9Hz,1H),7.93(d,J=2.3Hz,1H),7 .73(d,J=8.8Hz,2H),7.67(d,J=8.7Hz,2H),7.60(d,J=5.9Hz,1H),3.57(s,2H),2.99(q,J=6.5Hz,2H),2.63( t,J=6.3Hz,2H),2.45(q,J=2.6Hz,2H),2.33(d,J=7.2Hz,2H),2.29(s,3H),2.16(d,J=4.4Hz,4H),1.89(s,3H),1.79(s,2H),1.67–1.59(m,5H),1.58–1.53(m,9H),1.35(d,J=8.7Hz,2H),1.28(s,4H),1.24(s,10H).HRMS m / z[M+H]+644.4895, calculated value: 644.4903.

[0214] The structural formula is as follows:

[0215] Example 44 Preparation of 3-(2-(2-(2-((3r,5r,7r)-adamantane-1-yl)acetamamido)ethoxy)ethoxy)N-(4-(5-((methyl(2-(methylamino)ethyl)amino)methyl)pyridin-3-yl)phenyl)propionamide (compound 8O)

[0216] Using compound 6A from Example 23 and compound 4L from Example 22 as raw materials, the target compound was obtained as a white solid through a reaction similar to that used to synthesize compound 8C, with a yield of 55%.

[0217] The NMR data are as follows: 1H NMR(400MHz,DMSO-d6)δ10.12(s,1H),8.76(s,1H),8.46(s,1H),7.95(s,1H),7.72 (q,J=8.3Hz,5H),3.72(t,J=6.2Hz,2H),3.58(s,2H),3.52(s,9H),3.16(d,J=9.9Hz ,3H),2.67(t,J=6.3Hz,3H),2.60(d,J=5.9Hz,2H),2.31(s,3H),2.17(d,J=4.6Hz, 3H),1.89(s,3H),1.81(s,2H),1.63(d,J=11.3Hz,3H),1.55(d,J=12.6Hz,9H).HRMS m / z[M+H]+650.4271, calculated value: 650.4281.

[0218] The structural formula is as follows:

[0219] Example 45 Preparation of 4-(2-((3r, 5r, 7r)-adamantane-1-yl)acetamyl)-N-(4-((6-((methyl(2-(methylamino)ethyl)amino)methyl)pyridin-2-yl)phenyl)butyramide (compound 8P) Using compound 6D from Example 26 and compound 4C from Example 11 as starting materials, the target compound was obtained as a white solid with a yield of 54% through a reaction similar to that used in the synthesis of compound 8C.

[0220] The NMR data are as follows: 1 H NMR(400MHz,DMSO-d6)δ10.10(s,1H),8.02(d,J=8.5Hz,2H),7.72–7.69(m,3H),7.61(d,J =7.9Hz,3H),7.38(d,J=7.3Hz,1H),3.71(s,2H),3.08(q,J=6.6Hz,2H),2.83(t,J=6.2Hz, 2H), 2.63–2.57(m,6H), 2.35(t,J=7.4Hz,2H), 2.27(d,J=10.0Hz,3H), 1.91(d,J=4.1Hz,3H), 1.83(s,2H), 1.72(t,J=7.3Hz,2H), 1.65(d,J=12.3Hz,3H), 1.56(d,J=2.8Hz,9H).HRMS m / z[M+H]+532.3645, calculated value: 532.3651.

[0221] The structural formula is as follows:

[0222] Example 46 Preparation of 4-(2-((3r,5r,7r)-adamantane-1-yl)acetamyl)-N-(4-((methyl(2-(methylamino)ethyl)amino)methyl)pyridin-2-yl)phenyl)butyramide (compound 8Q)

[0223] Using compound 6E from Example 27 and compound 4C from Example 11 as raw materials, the target compound was obtained as a white solid through a reaction similar to that used to synthesize compound 8C, with a yield of 55%.

[0224] The NMR data are as follows: 1 H NMR (400MHz, DMSO-d6) δ10.08(s,1H),8.55(d,J=5.0Hz,1H),8.03(d,J=8.5Hz,2H),7.83( s,1H),7.71(d,J=8.6Hz,2H),7.66(s,1H),7.25(d,J=4.8Hz,1H),3.57(s,2H),3.11–3.06( m,2H), 2.65–2.61(m,3H), 2.35(t,J=7.4Hz,2H), 2.30(s,3H), 2.18(d,J=5.3Hz,4H), 1.91(s,3H), 1.83(s,2H), 1.72(t,J=7.2Hz,2H), 1.65(d,J=12.3Hz,3H), 1.60–1.53(m,9H).HRMS m / z[M+H]+532.3644, calculated value: 532.3651.

[0225] The structural formula is as follows:

[0226] Example 47 Preparation of 4-(2-((3r,5r,7r)-adamantane-1-yl)acetamyl)-N-(4-((methyl(2-(methylamino)ethyl)amino)methyl)pyridin-3-yl)phenyl)butyramide (compound 8R)

[0227] Using compound 6F from Example 28 and compound 4C from Example 11 as raw materials, the target compound was obtained as a white solid through a reaction similar to that used to synthesize compound 8C, with a yield of 57%.

[0228] The NMR data are as follows: 1H NMR (400MHz, DMSO-d6) δ10.06(s,1H),8.50(d,J=5.0Hz,1H),8.37(s,1H),7.74(t,J=5.5 Hz,1H),7.71–7.66(m,2H),7.56(d,J=5.0Hz,1H),7.52(d,J=5.0Hz,2H),3.47(d,J=3.1H z,2H),3.09(q,J=6.6Hz,4H),2.54(d,J=6.4Hz,3H),2.37–2.33(m,5H),2.04(s,3H),1.90(s,3H),1.83(s,2H),1.75–1.71(m,2H),1.65(d,J=12.4Hz,3H),1.59–1.55(m,9H).HRMS m / z[M+H]+532.3645, calculated value: 532.3651.

[0229] The structural formula is as follows:

[0230] Example 48 Preparation of 5-(4-(4-(2-((3r,5r,7r)-adamantane-1-yl)acetamido)butamido)phenyl)-N-methyl-N-(2-(methylamino)ethyl)nicotinamide (compound 8S)

[0231] Using compound 6G from Example 29 and compound 4C from Example 11 as raw materials, the target compound was obtained as a white solid through a reaction similar to that used to synthesize compound 8C, with a yield of 54%.

[0232] The NMR data are as follows: 1 ¹H NMR (400MHz, DMSO-d⁶) δ 10.08 (s, 1H), 8.93 (s, 1H), 8.57–8.52 (m, 1H), 8.09 (s, 1H), 7.74 (s, 5H), 3.56 (s, 2H), 3.17 (s, 1H), 3.08 (q, J = 6.7Hz, 3H), 2.77 (s, 2H), 2.38–2.33 (m, 4H), 2.11 (s, 3H), 1.91 (s, 3H), 1.82 (s, 2H), 1.72 (t, J = 7.3Hz, 2H), 1.65 (d, J = 12.4Hz, 3H), 1.59–1.54 (m, 9H). HRMS m / z [M+H] + 546.3435, calculated value: 546.3444.

[0233] The structural formula is as follows:

[0234] Example 49: Experiment on the inhibition of proliferation of two tumor cell lines by compound 8A-8S

[0235] MDA-MB-435 breast cancer cell line and HCC827 non-small cell lung cancer cell line were purchased from the American Type Culture Collection (ATCC). RPMI 1640 medium and fetal bovine serum (FBS) were purchased from GIBICO, USA. Penicillin and streptomycin were purchased from Takara Bio Inc., Dalian. Cell culture dishes and 96-well plates were purchased from Corning, Inc. Centrifuge tubes of various sizes were purchased from BD, Inc. MTT reagent was purchased from Donjindo Chemical Research Institute, Japan.

[0236] HCC827 non-small cell lung cancer cell line and MDA-MB-435 breast cancer cell line were cultured in a conventional high-glucose RPMI 1640 or DMEM complete medium containing 10% fetal bovine serum (FBS), 100 IU / mL penicillin, and 100 μg / mL streptomycin in an incubator at 37°C and 5% CO2.

[0237] To investigate the effects of a novel compound on the proliferation of MDA-MB-435 breast cancer cell line and HCC827 non-small cell lung cancer cell line, cells in logarithmic growth phase were collected and their concentrations adjusted to 1000–2000 cells / mL in a single-cell suspension. 100 μL of this suspension was seeded into 96-well plates. The compound stock solution (10 mM / L dissolved in DMSO) was diluted to different concentrations using a three-fold dilution method. 100 μL of this solution was added to each well of the 96-well plate, with the highest concentration being 10 μM and the lowest being 0.0015 μM. Two replicates were set for each drug concentration. Cells were treated with 0.1% DMSO medium and pure medium as negative controls. Cells were cultured for 3 days. The activity of mitochondrial succinate dehydrogenase was measured using the MTT assay, and the half-maximal inhibitory concentration (IC50) of the novel compound against tumor cells was calculated. 50 The values ​​are shown in Table 1. In Table 1, letter A represents IC. 50 Values ​​>1 μM to ≤10 μM; the letter B indicates IC 50 Values ​​>10μM to ≤20μM; the letter C indicates IC 50 Value >20μM.

[0238] Table 1. Inhibitory effects of some compounds on the proliferation of two tumor cell lines (MTT assay)

[0239]

[0240] As shown in Table 1, several tested compounds, including 8A, 8C, 8J, 8L, 8M, 8N, and 8R, exhibited strong inhibitory activity against the proliferation of two tumor cell lines, with IC50 values ​​of [missing data]. 50Value ≤10μM.

[0241] Example 50: The inhibitory effect of compound 8C on MDA-MB-435 and HCC827 cells was dose-dependent.

[0242] The cell lines, reagents, and consumables were sourced from the same sources as in Example 49; the remaining reagents, such as PI, were purchased from Sigma (USA).

[0243] The novel compound (8C) was used to further investigate its inhibitory activity against the MDA-MB-435 breast cancer cell line and the HCC827 non-small cell lung cancer cell line. Cells in the logarithmic growth phase were collected, and a single-cell suspension was adjusted to a concentration of 1000–2000 cells / mL. 100 μL of this suspension was seeded into each well of a 96-well plate. The compound stock solution (10 mM / L dissolved in DMSO) was diluted to different concentrations using a three-fold dilution method. 100 μL of this solution was added to each well of the 96-well plate, with the highest concentration being 10 μM and the lowest being 0.0015 μM. Two replicates were set for each drug concentration. Cells were treated with medium containing 0.1% DMSO and pure medium as negative controls. The drug-containing medium was changed every 3 days. The previously reported PRMT6 inhibitors EPZ020411 and Cpd.a25 were used as positive controls. After 6 days of continuous treatment, the activity of mitochondrial succinate dehydrogenase in cells was detected by MTT assay, and the half-maximal inhibitory concentration (IC50) of the new compound against tumor cells was calculated. 50 )value.

[0244] from Figure 1 It can be seen that the inhibitory activity of compound 8C on MDA-MB-435 breast cancer cells and HCC827 non-small cell lung cancer cells increases with increasing concentration.

[0245] Example 51: Colony formation experiment of compound 8C on MDA-MB-435 and HCC827 cells.

[0246] The cell lines, reagents, and consumables were sourced exactly the same as in Example 50.

[0247] To investigate the inhibitory effect of the new compound 8C on colony formation in MDA-MB-435 breast cancer cells and HCC827 non-small cell lung cancer cells, tumor cells in the logarithmic growth phase were injected with 3–4 × 10⁻⁶ cells per cell line. 3 Cells were seeded at a concentration of [number] cells / well in 6-well plates. The next day, after cell attachment, the supernatant was aspirated, and 2 mL of prepared culture medium containing different concentrations of the compound was added. Cells were also treated with medium containing 0.1% DMSO as a negative control. The drug-containing medium was changed every 3 days. After 6 consecutive days of treatment, the medium was discarded, and the cells were fixed with 4% paraformaldehyde solution for 20 min, washed with deionized water, stained with crystal violet for 15 min, washed, and air-dried.

[0248] from Figure 2 It can be seen that after treating cells with 0, 5, and 10 μM compound 8C for 6 days, the colony-forming ability of MDA-MB-435 and HCC827 cells was significantly weakened, and the number and size of cells decreased with increasing drug concentration, showing a certain concentration dependence.

[0249] Example 52 Effect of compound 8C on PRMT6 protein expression in MDA-MB-435 and HCC827 cells

[0250] The cell lines, reagents, and consumables were sourced from the same sources as in Example 49; the remaining cell lysis buffers and other reagents were purchased from Sigma (USA).

[0251] To investigate the degradation effect of the new compound 8C on PRMT6 protein in MDA-MB-435 and HCC827 cells, tumor cells in the logarithmic growth phase were subjected to a concentration of 3–4 × 10⁻⁶ cells. 3 Cells were seeded at a concentration of [number] cells / well in 6-well plates. The following day, after cell attachment, the supernatant was aspirated, and 2 mL of prepared culture medium containing different concentrations of the compound was added. Cells were treated with medium containing 0.1% DMSO as a negative control. The drug-containing medium was changed every 3 days. After 6 consecutive days of treatment, the cell suspension and adherent cells were collected and lysed for 1 hour in RIPA buffer containing the mixture and a phosphatase inhibitor (1 / 1000). The cell lysates were then centrifuged at 13000 rpm for 30 minutes at 4°C, and the supernatant was collected to determine protein concentration using the BCA method. Equal protein samples were separated on an SDS-PAGE gel and transferred to a nitrocellulose (NC) filter membrane. The membrane was then incubated with the appropriate primary and secondary antibodies. Specific protein bands were detected by chemiluminescence.

[0252] from Figure 3 It can be seen that MDA-MB-435 and HCC827 cells were treated with compound 8C at concentrations of 1.25, 2.5, 5, 10, and 20 μM for 6 days, respectively, and the changes in PRMT6 protein levels were then detected by Western blotting. Compound 8C significantly induced the degradation of PRMT6 protein in both MDA-MB-435 and HCC827 cells, and the effect was dose-dependent. Figure 4 As can be seen, MDA-MB-435 and HCC827 cells were treated with 20 μM compound 8C for 0.5, 1, 3, 5, and 7 days, respectively, and the changes in PRMT6 protein levels were detected by Western blotting. Compound 8C significantly induced the degradation of PRMT6 protein in MDA-MB-435 and HCC827 cells, and the effect was time-dependent.

[0253] Example 53: Compound 8C degrades PRMT6 protein via ubiquitination.

[0254] To investigate the potential mechanism by which compound 8C degrades PRMT6, MDA-MB-435 cells were co-treated with the proteasome inhibitor MG132 and the lysosomal inhibitor chloroquine, and then subjected to Western blotting assays along with compound 8C. Figure 5 As shown in (A) and (B), MG132 reversed the degradation of compound 8C by inhibiting the cellular UPS pathway, while pretreatment with the lysosomal inhibitor chloroquine did not affect PRMT6 degradation, indicating that the proteasome plays a central role in the HyT-mediated degradation mechanism. Then, to investigate the competition between the PRMT6 inhibitor and compound 8C for the PRMT6 target, MDA-MB-435 cells were co-treated with the PRMT6 inhibitor in EPZ020411 cells and subjected to Western blotting assays along with compound 8C. Figure 5 As shown in (C), the degradation of compound 8C was slightly reversed under the same concentrations of EPZ020411 and compound 8C.

[0255] Example 53 Compound 8C induces apoptosis in MDA-MB-435 and HCC827 cells.

[0256] The cell lines, reagents, and consumables were sourced exactly the same as in Example 50.

[0257] The effects of compounds on apoptosis were studied using flow cytometry: cells in the logarithmic growth phase were collected, and the cell suspension concentration was adjusted to 2–10 × 10⁶ cells per well. 5 Two cells were seeded into 6-well plates, 2 mL per well; after incubation at 37°C and 5% CO2 for 24 hours, the supernatant was carefully aspirated, and 2 mL of the prepared drug-containing culture medium was added to each well according to the concentration gradient. A control group without drug was also set up. The cells were incubated at 37°C and 5% CO2 for 72 hours. Then the cells were processed as follows: (1) The cells in each well were collected, numbered sequentially according to concentration, and loaded into flow cytometry tubes. The cells were centrifuged at 2000 r / min for 3 minutes at 4°C, and the supernatant was discarded; (2) The cells were resuspended in 3 mL of PBS, centrifuged in the same manner, and the PBS was discarded; (3) The cells were stained with annexin V-fluorescein isothiocyanate (FITC) apoptosis detection kit (Roche, Indianapolis, IN, USA), mixed, and incubated at room temperature in the dark for 20 minutes; the cells were then detected by flow cytometry.

[0258] from Figure 6 As can be seen, MDA-MB-435 and HCC827 cells were treated with 0, 10, and 20 μM of compound 8C for 72 hours, respectively, and then the changes in cell apoptosis were detected by flow cytometry. Compound 8C can significantly induce apoptosis in tumor cells, and the effect is dose-dependent.

Claims

1. An arginine methyltransferase 6 inhibitor, characterized in that, It has the structure shown in Equation I: （I）； Where Z represents pyridine, and the connection method of pyridine is selected from... , or ; R1 and R2 are independently either H or CH3; X is -CH2- or carbonyl, and Y is -CO-NH- or -NH-CO-. Linkers are aliphatic chains with a length of 1-11 carbons; n is 0 or 1.

2. An arginine methyltransferase 6 inhibitor, characterized in that, The arginine methyltransferase 6 inhibitor is selected from one or more compounds shown in the following structural formulas: 。 3. A method for preparing the arginine methyltransferase 6 inhibitor according to claim 2, characterized in that, include: The first and second raw materials are subjected to a first condensation reaction under alkaline conditions to obtain a first intermediate product. The structural formula of the first raw material is as follows: Y1 is an amino or carboxyl group, and the structural formula of the second raw material is: X1 is an amino or carboxyl group, and n1 is any positive integer from 1 to 11; The first intermediate product reacts under first acidic conditions to give a second intermediate product; The second intermediate product and the third raw material undergo a second condensation reaction under alkaline conditions to obtain the third intermediate product, wherein the structural formula of the third raw material is as follows: n is 0 or 1; The fourth and fifth raw materials were subjected to a reductive amination reaction under second acidic conditions to obtain a fourth intermediate product. The fourth raw material was 5-bromopyridine-3-carboxaldehyde, and the fifth raw material had the following structural formula: R1 and R2 are each independently H or CH3; The third intermediate and the fourth intermediate were subjected to a palladium-mediated coupling reaction under alkaline conditions to obtain the fifth intermediate; The fifth intermediate was reacted under third acidic conditions to obtain the arginine methyltransferase 6 inhibitor.

4. The preparation method according to claim 3, characterized in that, The condensing agents used in the first and second condensation reactions are 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 1-hydroxy-7-azobenzotriazole; the base used is N-methylmorpholine; the molar ratio of the first raw material, the second raw material, the base, the 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and the 1-hydroxy-7-azobenzotriazole is 1:(1~3):(3~10):(1~3):(1~3); the molar ratio of the second intermediate product, the third raw material, the base, the 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and the 1-hydroxy-7-azobenzotriazole is 1:(1~3):(3~10):(1~3):(1~3).

5. The preparation method according to claim 3, characterized in that, The acids used in the first acidic condition and the third acidic condition are each trifluoroacetic acid or hydrochloric acid independently; the molar ratio of the first intermediate product to the acid is 1:(3~10), and the molar ratio of the fifth intermediate product to the acid is 1:(3~10).

6. The preparation method according to claim 3, characterized in that, The solvents for the reductive amination reaction are dichloromethane and dichloroethane; the acid used in the second acidic condition is glacial acetic acid; and the reductive amination agent used is sodium triacetoxyborohydride.

7. The preparation method according to claim 3, characterized in that, The palladium is any one of [1,1'-bis(diphenylphosphine)ferrocene]palladium dichloride dichloromethane complex, palladium acetate, or tetra(triphenylphosphine)palladium; the base used in the coupling reaction is any one of sodium carbonate, potassium carbonate, or cesium carbonate; the solvent used in the coupling reaction is a mixture of dioxane and water in a volume ratio of (5:1) to (1:1); the molar ratio of the third intermediate, the fourth intermediate, the palladium, and the base is 1:(1~2):(0.05~0.15):(1~5).

8. The preparation method according to claim 3, characterized in that, The reductive amination reaction is carried out at a temperature of 0°C, the coupling reaction is carried out at a temperature of 90-110°C, and the reaction temperatures for the remaining reaction conditions are 5-25°C.

9. A pharmaceutical composition, characterized in that, It includes the arginine methyltransferase 6 inhibitor as described in claim 1 or 2 and its pharmaceutically acceptable adjuvant.

10. Use of the arginine methyltransferase 6 inhibitor of claim 1 or 2 and the pharmaceutical composition of claim 9 in the preparation of a medicament for the prevention and / or treatment of diseases related to abnormal PRMT6 expression; The disease is a tumor, specifically breast cancer or lung cancer.

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