CN110950841A

CN110950841A - Synthesis and application of novel triazole compounds

Info

Publication number: CN110950841A
Application number: CN201911152555.7A
Authority: CN
Inventors: 张华�; 刘凯璐; 闫薛; 朱孔凯
Original assignee: University of Jinan
Current assignee: University of Jinan
Priority date: 2019-11-22
Filing date: 2019-11-22
Publication date: 2020-04-03

Abstract

The invention discloses a novel triazole compound shown in figure 1 and application thereof as a protein arginine methyltransferase 5 inhibitor, and discloses application of the compound in preventing and/or treating cancer-related diseases caused by abnormal activity of protein arginine methyltransferase 5.

Description

Synthesis and application of novel triazole compounds

Technical Field

The invention relates to the field of medicinal chemistry and pharmacotherapeutics, in particular to synthesis and application of a novel triazole compound.

Background

Protein arginine methyltransferases (PRMTs) -catalyzed methylation of protein arginine is an important post-translational modification that uses S-adenosylmethionine (SAM) as a methyl donor to methylate and modify the nitrogen atom of the protein arginine side chain. The substrates of PRMTs are glycine and arginine rich proteins. The 9 human PRMTs identified (PRMT 1-9) were further subdivided into type I, type II and type III, depending on the status of the methylated arginine. Type I PRMT catalyzes the formation of monomethyl arginine (MMA) and asymmetric dimethyl arginine (aDMA), including PRMT1, PRMT2, PRMT3, PRMT4, PRMT6 and PRTM 8; type ii PRMT catalyzes the formation of MMA and symmetric dimethylarginine (sDMA), including PRMT5 and PRMT 9; type iii PRMT catalyzes the formation of MMA, including PRMT 7. PRMT5 belongs to type ii PRMT, transferring a methyl group from the SAM to the guanidine nitrogen of an arginine residue to produce MMA and sDMA. Human tissue proteomics analysis showed that PRMT1, -4 and-5 expression was more prevalent compared to PRMT2, -3, -6, -7 and-8. PRMT5 mediates various cellular processes such as transcription, RNA metabolism, signal transduction, and cell differentiation through symmetric dimethylation of multiple substrate proteins in the nucleus and cytoplasm.

Interestingly, increasing research has shown that PRMT5 is an attractive target for cancer therapy, which is overexpressed or upregulated in a variety of human cancers, including leukemia, lymphoma, breast cancer, lung cancer, bladder cancer, gastric cancer, pancreatic cancer, prostate cancer, colon cancer, multiple myeloma, liver cancer, melanoma, head and neck cancer, thyroid cancer, renal cell carcinoma, glioblastoma, or testicular cancer. Selective inhibition of PRMT5 was reported to block B cell transformation. In addition, PRMT5 controls melanoma growth through the SKI/SOX10 regulatory axis. PRMT5 is considered to be an effective target for the treatment of Mantle Cell Lymphoma (MCL), glioblastoma, multiple myeloma, and mixed leukemia (MLL) -acute myeloid leukemia.

LLY-283, Sinefungin and A9145C are reported to be SAM binding site inhibitors, EPZ015666 and its analogue GSK3326595 are substrate binding site inhibitors, EPZ015666 is the first PRMT5 inhibitor with potent activity in both in vitro and in vivo models of mantle cell lymphoma. In addition, clinical trials for GSK3326595 in phase i for the treatment of solid and non-hodgkin's lymphoma are ongoing. Although there are more than 10 PRMT5 inhibitors reported, most exhibit low potency or lack in vivo activity. Therefore, there is a need to find more effective PRMT5 inhibitors.

In view of the above, there is an urgent need in the art to develop more novel arginine methyltransferase 5 inhibitors.

Disclosure of Invention

In order to solve the problems, the invention provides a novel triazole compound, a preparation method thereof and application thereof in preventing and/or treating cancer or related diseases.

The invention is realized by the following technical scheme.

In a first aspect of the invention, triazole compounds with novel structures are provided, and the general formula is shown in figure 1, wherein R is 3-nitrophenyl, 3-aminophenyl, 4-aminophenyl, 3- (cyclobutylamino) phenyl and 3- ((tetrahydro-2H-pyran-4-yl) amino) phenyl.

Among them, a preferred structure is shown in fig. 2.

A more preferred configuration is shown in figure 3.

The preparation method of the compound related to the invention has a preparation route shown in figure 5.

Preferably, the cancer to be prevented and/or treated is leukemia, lymphoma, breast cancer, lung cancer, bladder cancer, stomach cancer, pancreatic cancer, prostate cancer, colon cancer, multiple myeloma AML, liver cancer, melanoma, head and neck cancer, thyroid cancer, renal cell carcinoma, glioblastoma or testicular cancer.

Preferably, the related disease is a disease associated with an abnormal arginine methyltransferase 5 activity.

Has the advantages that: the compound has stronger inhibitory activity and selectivity on PRMT 5; the preparation method of the compound is simple and feasible; the compound has stronger tumor cell proliferation inhibition activity, and the targeting of PRMT5 in cells is confirmed; the raw materials are easy to obtain.

Drawings

FIG. 1 is a new class of triazole compounds.

FIG. 2 shows a preferred novel triazole compound.

More preferred novel triazole compounds are shown in FIG. 3.

FIG. 4 shows the structural design of compounds C _1 to C _ 6.

FIG. 5 is a scheme for preparing compounds C _1 to C _ 6.

FIG. 6A test of the enzyme inhibitory activity of compounds C _1 to C _6 by means of radioisotopes.

Figure 7 the selectivity of compound C _4 for PRMTs was assessed by radioisotope methods.

FIG. 8 Effect of Compounds C _1 to C _6 on Z-138 cell proliferation.

FIG. 9 dose curve of Compound C _4 for inhibition of Z-138 cell proliferation.

FIG. 10 Effect of Compound C _4 on symmetric dimethylation of Z-138 cells.

Detailed Description

The following examples are given for the detailed implementation and specific operation of the present invention, but the scope of the present invention is not limited to the following examples.

Example 1: and designing the structures of the compounds C _ 1-C _ 6.

Obtaining compounds C _ 1-C _6 by using a traditional structure optimization mode, wherein the structure design is shown in figure 4, wherein R₁Nitro, amino, cyclobutylamino and (tetrahydro-2H-pyran-4-yl) amino.

Example 2: and (3) preparing compounds C _ 1-C _ 6.

The compounds C _ 1-C _6 are synthesized by starting from 3-or 4-bromonitrobenzene, the 3-or 4-bromonitrobenzene and trimethylsilylacetylene are subjected to Sonogashaaira coupling reaction, and then potassium carbonate is used for deprotection to obtain 1-ethynyl-3-nitrobenzene or 1-ethynyl-4-nitrobenzene (an intermediate 3); treating 2- (oxido-2-ethylmethyl) -1,2,3, 4-tetrahydroisoquinoline (compound 4) with sodium azide or ammonia gas in methanol to give 1-amino-3- (3, 4-dihydroisoquinolin-2 (1H) -yl) propan-2-ol or 1-azido-3- (3, 4-dihydroisoquinolin-2 (1H) -yl) propan-2-ol (intermediate 5); preparing compounds C _1 and 6 by using the intermediate 3 and the intermediate 5 as raw materials by a click chemistry method; and reducing C _1 and C _6 by hydrogen to obtain C _2 and C _5, and reacting C _2 and C _5 with ketone to obtain C _3, C _4 and C _ 6. The specific preparation route is shown in figure 5, wherein (a): trimethylsilylacetylene, methylidene iodide, bis (triphenylphosphine) palladium chloride, triethylamine, N, N-dimethylformamide, microwave irradiation, 120 ℃; (b): potassium carbonate, methanol, room temperature; (c) the method comprises the following steps 5 a: refluxing sodium azide and ethanol; 5 b: sealing with nitrogen and methanol at 80 deg.C; (d): copper sulfate pentahydrate, sodium ascorbate, tetrahydrofuran/water; (e): palladium on carbon, hydrogen, methanol; (f): sodium cyanoborohydride, ketone, methanol.

1- (3, 4-dihydroisoquinolin-2 (1H) -yl) -3- (4- (3-nitrophenyl) -1H-1,2, 3-triazol-1-yl) propan-2-ol (C _ 1): yellow solid (152 mg, 80%).¹H NMR (300 MHz, CDCl₃) δ 8.64 (s, 1H), 8.22(d, J = 7.5 Hz, 1H), 8.17 (s, 2H), 7.60 (t, J = 8.0 Hz, 1H), 7.22-7.06 (m,3H), 7.00 (d, J = 6.0 Hz, 1H), 4.73 - 4.61 (m, 1H), 4.45 (m, 1H), 4.30 (m,1H), 3.89 (d, J = 14.7 Hz, 1H), 3.69 (d, J = 14.7 Hz, 1H), 3.07 - 2.88 (m,3H), 2.87 - 2.78 (m, 1H), 2.74 (m, 1H), 2.59 - 2.39 (m, 1H).¹³C NMR (151MHz, DMSO-d6) delta 148.9, 144.5, 133.1, 131.8, 131.1, 128.9, 126.9, 124.2, 122.8,119.8, 67.6, 61.8, 56.5, 54.9, 51.6. formula is C₂₀H₂₁N₅O₃。

1- (4- (3-aminophenyl) -1H-1,2, 3-triazol-1-yl) -3- (3, 4-dihydroisoquinolin-2 (1H) -yl) propan-2-ol (C _ 2): yellow solid (78 mg, 86%).¹H NMR (400 MHz, CD₃OD) δ 8.21 (s, 1H), 7.19- 6.98 (m, 7H), 6.70 (m, 1H), 4.60 (m, 1H), 4.41 (m, 1H), 4.37 - 4.29 (m,1H), 3.80 (s, 2H), 2.92 (s, 4H), 2.70 (m, 2H).¹³C NMR (151 MHz, DMSO-d6) δ149.8, 149.0, 147.4, 128.9, 126.9, 126.6, 126.0, 120.5, 119.1, 114.4, 67.5,61.6, 56.3, 54.5, 51.5. HRMS (ESI+) m/z+: 350.1973 [M+ H]⁺Molecular formula is C₂₀H₂₄N₅O。

1- (3, 4-dihydroisoquinolin-2 (1H) -yl) -3- (4- (3- ((tetrahydro-2H-pyran-4-yl) amino) phenyl) -1H-1,2, 3-thiazol-1-yl) propan-2-ol (C _ 3): yellow solid (37 mg, 43%).¹H NMR (300 MHz, CDCl₃)δ 7.96 (s, 1H), 7.29 - 7.03 (m, 6H), 6.99 (d, J = 6.0 Hz, 1H), 6.58 (d, J =7.8 Hz, 1H), 4.59 (m, 1H), 4.40 (m, 1H), 4.22 (m, 1H), 4.00 (d, J = 11.6 Hz,2H), 3.81 (m, 1H), 3.67 - 3.50 (m, 4H), 2.92 (m, 3H), 2.79 - 2.67 (m, 1H),2.67 - 2.56 (m, 1H), 2.42 (m, 1H), 2.06 (m, 2H), 1.48 (m, 2H).¹³C NMR (151MHz, DMSO-d6) delta 148.5, 147.2, 132.0, 129.9, 128.9, 126.9, 122.5, 113.3,112.7, 109.3, 66.5, 55.3, 48.3, 33.3 HRMS (ESI +) m/z +: 434.2546 minSub-formula is C₂₅H₃₂N₅O₂。

1- (4- (3- (cyclobutylamino) phenyl) -1H-1,2, 3-triazol-1-yl) -3- (3, 4-dihydroisoquinolin-2 (1H) -yl) propan-2-ol (C _ 4): yellow solid (42 mg, 52%).¹H NMR (400 MHz, CD₃OD) δ 8.24 (s,1H), 7.15 (m, 4H), 7.07 (m, 3H), 6.60 (d, J = 6.8 Hz, 1H), 4.65 (m, 1H), 4.54- 4.41 (m, 1H), 4.36 (s, 1H), 4.07 - 3.91 (m, 1H), 3.83 (s, 2H), 2.96 (m,4H), 2.71 (d, J = 8.8 Hz, 2H), 2.44 (s, 2H), 1.87 (m, 4H).¹³C NMR (151 MHz,CD₃OD) δ 148.3, 147.9, 133.5, 130.8, 129.2, 128.2, 126.2, 125.6, 121.9,114.1, 113.1, 109.7, 67.0, 60.7, 55.8, 54.3, 51.2, 48.7, 30.4, 28.0, 14.65.HRMS (ESI+) m/z+: 404.2439 [M + H]⁺Molecular formula is C₂₄H₃₀N₅O。

1- (4- (4-aminophenyl) -1H-1,2, 3-triazol-1-yl) -3- (3, 4-dihydroisoquinolin-2 (1H) -yl) propan-2-ol (C _ 5): yellow solid (74 mg, 83%).¹H NMR (400 MHz, CD₃OD) δ 8.11 (s, 1H), 7.58- 7.51 (m, 2H), 7.14 - 7.06 (m, 3H), 7.02 (d, J = 5.1 Hz, 1H), 6.79 - 6.73(m, 2H), 4.61 (m, 1H), 4.41 (m, 1H), 4.30 (m, 1H), 3.72 (s, 2H), 2.91 (t, J =5.6 Hz, 2H), 2.87 - 2.79 (m, 2H), 2.68 - 2.54 (m, 2H).¹³C-NMR (151 MHz, DMSO-d6) δ 149.5, 147.1, 131.8, 129.8, 128.9, 126.9, 126.6, 126.0, 122.3, 113.9,113.4, 110.9, 72.8, 60.7, 54.5, 51.5. HRMS (ESI+) m/z+: 350.1973 [M + H]⁺Molecular formula is C₂₀H₂₄N₅O。

1- (3, 4-dihydroisoquinolin-2 (1H) -yl) -3- (4- (4- ((tetrahydro-2H-pyran-4-yl) amino) phenyl) -1H-1,2, 3-triazol-1-yl) propan-2-ol (C _ 6): yellow solid (35 mg, 42%).¹H NMR (400 MHz,CD₃OD) δ 8.13 (s, 1H), 7.59 (m, 2H), 7.11 (m, 4H), 6.74 (m, 2H), 4.60 (m,1H), 4.41 (m, 2H), 3.97 (m, 2H), 3.89 (m, 2H), 3.55 (m, 3H), 3.00 (m, 4H),2.80 (m, 2H), 2.02 (m, 2H), 1.52 (m, 2H).¹³C NMR (151 MHz, DMSO-d6) δ 147.9,128.9, 126.9, 126.7, 113.0, 66.5, 65.4, 48.3, 35.9, 33.3. HRMS (ESI+) m/z+:434.2546 [M + H]⁺Molecular formula is C₂₅H₃₂N₅O₂。

Example 3: inhibitory activity of the compounds C _ 1-C _6 on PRMT 5.

The compounds were tested for enzyme inhibitory activity by means of radioisotopes. The specific experimental steps are as follows:

(1) preparing 1x experiment buffer (modified Tris-HCl buffer);

(2) diluting the compound to the desired concentration in a 96-well plate;

(3) preparing a protein solution, and using 1x experiment buffer solution;

(4) adding a substrate into 1x experiment buffer solution to prepare a substrate solution;

(5) will 2³H]Preparation of [ 2 ], [ SAM ] by adding to 1X test buffer³H]-a SAM solution;

(6) adding SAM to 1x experiment buffer solution to prepare cold SAM solution;

(7) removing 10. mu.L of the protein solution into a 96-well plate containing the compound;

(8) incubation for 15 minutes at room temperature;

(9) add 10. mu.L of substrate solution to each well;

(10) to each well was added 10. mu.L of³H]-the SAM solution initiates the reaction;

(11) incubation at room temperature for 240 min;

(12) add 10. mu.L of cold SAM solution to each well to stop the reaction;

(13) transferring 40 mu L of reaction mixed solution to a GF/B plate, and washing for 3 times by using triple distilled water in vacuum;

(14) reading data on a Microbeta liquid scintillation/luminescence counter;

(15) the inhibition rate was calculated according to the formula% Inh = (maximum signal-compound signal)/(maximum signal-minimum signal) × 100, the maximum signal being obtained from the reaction of the enzyme and the substrate and the minimum signal being obtained from the substrate. Data were plotted after processing using GraphPadPrism 5.0. EPZ015666 was used as a positive control. The experimental results are shown in fig. 6, and the results show that the compound C _4 has greater PRMT5 inhibitory activity.

Example 4: selectivity of compound C _ 4.

The selectivity of the compound C _4 to other PRMTs was evaluated by a radioisotope method, and the results of the experiment are shown in FIG. 7, which shows the IC of the compound C _4 to PRMT1, PRMT4 and PRMT6₅₀Values were all greater than 200 μ M, indicating that it is a selective PRMT5 inhibitor.

Example 5: effect of Compounds C _1 to C _6 on cell proliferation.

Cell culture: the culture medium for Z-138 cell culture is RPMI 1640 + 10% fetal bovine serum, and 100U/mL penicillin and 100. mu.g/mL streptomycin are added to the culture medium to prevent bacterial contamination, and 5% CO is added at 37 deg.C₂The cells are cultured under the saturated humidity condition, and the cells for experiments are all in the exponential growth phase.

And (3) detecting cell proliferation activity: adjusting the cell concentration to 1X 10⁵Performing inoculation on a 24-well culture plate with 1 mL of each well volume, establishing a control group and an experimental group, adding DMSO into the control group, adding PRMT5 small molecule inhibitor into the experimental group to make the final concentration reach 0-200 mu M, and placing at 37 ℃ and 5% CO₂The cells were cultured in an incubator, the amount of viable cells was measured every four days using CellTiter-Glo kit, and data was recorded on day 8. The experimental result is shown in figure 8, and the result shows that the compound shows effective concentration-dependent anti-Z-138 cell proliferation activity, the compound C _4 has the strongest proliferation inhibition effect on Z-138 cells, and IC₅₀2.60. + -. 0.81. mu.M, the relevant dose curve is shown in FIG. 9.

Example 6: effect of compound C _4 on cell symmetric dimethylation.

Exponentially growing Z-138 cells were incubated with different concentrations of compound C _4 for 4 days, the cells were collected and dissolved in 100 μ L of lysis buffer, total cell lysates were separated by 4-12% polyacrylamide gel electrophoresis and transferred onto PVDF membranes, then blocked with blocking solution (5% skim milk, 1% tween 20 PBS buffer), then primary antibodies [ SDMA (CST, 13222s), GAPDH (CST, 5174) and SmD3 (Sigma, HPA001170-100UL) ] were added to the blot, incubated overnight at 4 ℃, the next day, washed three times with PBST, incubated with anti-rabbit secondary antibody (HRP labeled) for 2 hours, and finally the signal was detected using the ChemiScope3400 imaging system (Clinx Science Instruments). The experimental results are shown in fig. 10, and it is known that different concentrations of compound C _4 have different inhibitory effects on the symmetric dimethylated arginine substrate SmD3 in Z-138 cells, and show that the concentration-dependent inhibition intensity of SmD3me2S is shown. Since SmD3 is one of the substrate proteins of PRMT5, it can be concluded that Compound C _4 targets PRMT5 in the cell.

Claims

1. A general formula of the novel triazole compound is shown in figure 1, wherein R is 3-nitrophenyl, 3-aminophenyl, 4-aminophenyl, 3- (cyclobutylamino) phenyl and 3- ((tetrahydro-2H-pyran-4-yl) amino) phenyl.

2. The compound of claim 1, wherein the compound of figure 3.

3. A process for the preparation of a compound according to claim 1 or 2, wherein the desired product is prepared by the route of figure 5, wherein R is₂Is nitro or amino, R₃Is azido or amino.

4. Use of a compound according to claim 1 for the prophylaxis and/or treatment of cancer or a related disease.

5. The use according to claim 4, wherein the cancer to be prevented and/or treated is leukemia, lymphoma, breast cancer, lung cancer, bladder cancer, stomach cancer, pancreatic cancer, prostate cancer, colon cancer, multiple myeloma AML, liver cancer, melanoma, head and neck cancer, thyroid cancer, renal cell carcinoma, glioblastoma or testicular cancer.

6. The use according to claim 4, wherein the related disease is a disease caused by abnormal activity of protein arginine methyltransferase 5.