A thiophene-containing pyrimidine compound, a preparation method thereof, and its application in preparing a drug with anticancer effect

Abstract

The present invention relates to the field of biomedicine technology, and specifically discloses a thiophene-containing pyrimidine compound, a preparation method thereof, and its use in the preparation of drugs with anti-cancer effects. The thiophene-containing pyrimidine compound has a chemical structure represented by formula (I). Studies have shown that the thiophene-containing pyrimidine compound has an inhibitory effect on PLK1 protein kinase; therefore, using it as an active ingredient in the preparation of drugs with anti-cancer effects has important application value. In particular, in existing anti-cancer experiments, the thiophene-containing pyrimidine compound has a better effect on non-small cell lung cancer than the known compound BI2536, showing a more significant anti-non-small cell lung cancer effect.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to a thiophene-containing pyrimidine compound, a preparation method thereof, and an application thereof in preparing a drug with anti-cancer effect. Background Art

[0002] Cancer is a massive global health burden, affecting nearly every region and socioeconomic level. It is the second leading cause of death worldwide, and conquering cancer remains a significant challenge for modern medicine. According to statistics, there are currently over 200 types of cancer worldwide. Lung cancer, with the fastest growing incidence and mortality rates, is one of the most threatening malignancies to human health and life. Non-small cell lung cancer (NSCLC) accounts for approximately 80-85% of all lung cancers. Most patients are diagnosed with NSCLC in the advanced stage, with local or distant organ metastases. The five-year survival rate after surgical treatment is only 10-15%. Therefore, inhibiting and treating NSCLC has become a major focus.

[0003] PLK1 (Polo-like Kinase 1) belongs to the Polo-like kinase family, a class of serine / threonine kinases widely present in eukaryotic cells. PLK1 expression is very low in most normal tissues (including the kidney, liver, brain, lung, and pancreas). In contrast, PLK1 is highly expressed in a variety of malignant tumors, including lung cancer, colon cancer, melanoma, ovarian cancer, prostate cancer, and breast cancer. Its expression level is closely correlated with tumor proliferation, invasion, and migration, making it a potential therapeutic target for inhibiting tumor metastasis. To date, no PLK1 inhibitors are marketed, and over twenty drugs are under investigation, most of which are in preclinical research. Patent Publication No. WO2006018182A1 discloses compound BI2536; it is the first potent and selective PLK1 inhibitor to induce all the characteristics of PLK1 inhibition, inhibiting PLK1 enzyme activity at low nanomolar concentrations. The compound effectively caused mitotic arrest and induced apoptosis in human cancer cell lines with diverse tissue origins and tumor genomic characteristics. Cells arrested in prometaphase, accumulated phospho-histone H3, and contained abnormal mitotic spindles. BI2536 also inhibited the growth of human tumor xenografts in nude mice and induced regression of large tumors under a well-tolerated intravenous dosing regimen. However, studies have found that even short-term use of BI2536 can cause grade 3-4 myelosuppression, neutropenia, and even sepsis in patients.

[0004] Therefore, targeting PLK1 and developing more compounds that have inhibitory effects on PLK1 will provide more options for the screening of anti-cancer compounds and have important application value for the successful development of more effective anti-cancer drugs. Summary of the Invention

[0005] In order to overcome any technical problems existing in the prior art, the present invention first provides a thiophene-containing pyrimidine compound, a preparation method thereof, and an application thereof in preparing a drug having an anti-cancer effect.

[0006] The above technical problems to be solved by the present invention are achieved by adopting the following technical solutions:

[0007] The present invention first provides a thiophene-containing pyrimidine compound having the formula (I):

[0008] The chemical structural formula shown;

[0009]

[0010] Wherein, R in formula (I) is selected from nitrogen-containing groups or halogens.

[0011] Preferably, the nitrogen-containing group is a nitro group.

[0012] Preferably, the halogen is fluorine.

[0013] Most preferably, the thiophene-containing pyrimidine compound is selected from the following structures

[0014] Compound:

[0015]

[0016] The present invention also provides a method for preparing the above-mentioned thiophene-containing pyrimidine compound, which

[0017] Include

[0018] The steps are as follows:

[0019] (1) a compound represented by formula (II) and 2-amino-4,5,6,7-tetrahydrobenzo[b]

[0020] Thiophene-3-

[0021] Methyl formate reacts to prepare an intermediate compound;

[0022] (2) reacting the intermediate compound with 4-(4-methyl-1-piperazinyl)aniline to obtain a compound having the structure shown in formula (I);

[0023]

[0024] Wherein, R in formula (II) is selected from nitrogen-containing groups or halogens.

[0025] Preferably, the nitrogen-containing group is a nitro group.

[0026] Preferably, the halogen is fluorine.

[0027] Preferably, the reaction in step (2) is carried out at 100-140° C. for 16-24 hours.

[0028] Most preferably, the reaction in step (2) is carried out at 120° C. for 20 h.

[0029] The present invention also provides a use of the above-mentioned thiophene-containing pyrimidine compound in the preparation of a drug with anti-cancer effect.

[0030] Preferably, the cancer is lung cancer.

[0031] Preferably, the lung cancer is non-small cell lung cancer.

[0032] Preferably, the thiophene-containing pyrimidine compound is used in the preparation of a drug having the effect of inhibiting the growth of cancer cells.

[0033] Preferably, the cancer cells are A549 and / or PC-9 human non-small cell lung cancer cells.

[0034] The present invention also provides a use of the above-mentioned thiophene-containing pyrimidine compound in the preparation of a drug having an inhibitory effect on PLK1 protein kinase.

[0035] Beneficial effects: The present invention provides a thiophene-containing pyrimidine compound with a novel structure; studies have shown that the thiophene-containing pyrimidine compound has an inhibitory effect on PLK1 protein kinase; therefore, using it as an active ingredient in the preparation of drugs with anti-cancer effects has important application value.

[0036] In particular, in existing anti-cancer experiments, the thiophene-containing pyrimidine compound has a better anti-non-small cell lung cancer effect than the known compound BI2536, showing a more significant anti-non-small cell lung cancer effect; therefore, it has more important application value as an active ingredient for the preparation of drugs with anti-non-small cell lung cancer effects.

[0037] In addition, the present invention also provides a novel method for preparing thiophene-containing pyrimidine compounds. The preparation method has simple reaction steps and mild reaction conditions and is suitable for large-scale industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1It is the hydrogen nuclear magnetic resonance spectrum of methyl 2-((2-((4-(4-methylpiperazin-1-yl)phenyl)amino)-5-nitropyrimidin-4-yl)amino)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylate of the present invention.

[0039] Figure 2 It is the hydrogen nuclear magnetic resonance pattern of methyl 2-((5-fluoro-2-((4-(4-methylpiperazin-1-yl)phenyl)amino)pyrimidin-4-yl)amino)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylate of the present invention. DETAILED DESCRIPTION

[0040] The present invention is further explained below with reference to the following examples, but the examples do not limit the present invention in any form.

[0041] The reaction process of the present invention is shown in flow charts (V) and (VIII):

[0042]

[0043] The compound represented by formula (IV) in the above reaction process is an intermediate compound;

[0044] The compound represented by the formula (VII) is also referred to as the thiophene-containing pyrimidine compound of the present invention.

[0045] Example 1 Preparation of methyl 2-((2-((4-(4-methylpiperazin-1-yl)phenyl)amino)-5-nitropyrimidin-4-yl)amino)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylate

[0046] (1) 4.0 mmol of 2,4-dichloro-5-nitropyrimidine and 4.2 mmol of potassium carbonate were added to 30 ml of acetone, stirred, and reacted for 10 minutes. Then, 4.0 mmol of 2-amino-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylic acid methyl ester was added and monitored by TLC. After the reaction was completed, the reaction solution was poured into 100 mL of water and extracted with ethyl acetate (100 mL × 3). The mixture was allowed to stand for separation. The organic phase was washed with saturated brine (50 mL × 3), then dried over anhydrous magnesium sulfate, filtered, and the ethyl acetate was removed under reduced pressure to obtain the intermediate 2-((2-chloro-5-nitropyrimidin-4-yl)amino)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylic acid methyl ester;

[0047] (2) 2.0 mmol of the intermediate obtained in step (1) and 4-(4-methyl-1-piperazinyl)aniline were dissolved in ethylene glycol methyl ether, and the mixture was reacted at 120° C. for 20 hours under the catalysis of hydrochloric acid. The reaction solution was extracted with ethyl acetate, and the organic phase was dried over anhydrous sodium sulfate, desolvated, and separated and purified by column chromatography to obtain the methyl 2-((2-((4-(4-methylpiperazin-1-yl)phenyl)amino)-5-nitropyrimidin-4-yl)amino)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylate;

[0048] The obtained brown solid was identified by nuclear magnetic resonance spectroscopy and high-resolution mass spectrometry, and the identification results were: 1 H NMR(400MHz,DMSO-d6)δ:2.32(s,2H,CH2),2.83(s,4H,2xCH2),3.69(s,3H,CH3O),3.76(s,3H,CH3O),3.84(s,6H,2xCH3 O),6.77(s,1H,ArH),7.21(s,1H,ArH),9.14(s,1H,Pyrimidine-H),10.51(s,1H,NH),12.91(s,1H,NH).HRMS(ESI)calcd for C 22 H 23 N5O7S(M+H + )502.1391found387.1205.

[0049] From the above identification results, it can be seen that the obtained 2-((2-((4-(4-methylpiperazin-1-yl)phenyl)amino)-5-nitropyrimidin-4-yl)amino)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylic acid methyl ester has the structural formula The yield of the method described in this example was calculated to be 40.3%.

[0050] Example 2 Preparation of methyl 2-((5-fluoro-2-((4-(4-methylpiperazin-1-yl)phenyl)amino)pyrimidin-4-yl)amino)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylate

[0051] (1) 4.0 mmol of 2,4-dichloro-5-fluoropyrimidine and 4.2 mmol of potassium carbonate were added to 30 ml of acetone, stirred, and reacted for 10 minutes. Then, 4.0 mmol of 2-amino-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylic acid methyl ester was added and monitored by TLC. After the reaction was completed, the reaction solution was poured into 100 mL of water and extracted with ethyl acetate (100 mL × 3). The mixture was allowed to stand for separation. The organic phase was washed with saturated brine (50 mL × 3), then dried over anhydrous magnesium sulfate, filtered, and the ethyl acetate was removed under reduced pressure to obtain the intermediate 2-(2-chloro-5-fluoropyrimidin-4-ylamino)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylic acid methyl ester.

[0052] (2) 2.0 mmol of the intermediate obtained in step (1) and 4-(4-methyl-1-piperazinyl)aniline were dissolved in ethylene glycol methyl ether, and the mixture was reacted at 120° C. for 20 hours under the catalysis of hydrochloric acid. The reaction solution was extracted with ethyl acetate, and the organic phase was dried over anhydrous sodium sulfate, desolvated, and separated and purified by column chromatography to obtain the methyl 2-((5-fluoro-2-((4-(4-methylpiperazin-1-yl)phenyl)amino)pyrimidin-4-yl)amino)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylate;

[0053] The obtained white solid was identified by nuclear magnetic resonance spectroscopy and high-resolution mass spectrometry, and the identification results were: 1 H NMR(400MHz,DMSO-d6)δ:1.70(s,4H,2xCH2),2.41(s,2H,CH2),2.68(s,4H,2xCH2),3.66(s,3H,CH3O),3.69(s,3H,CH3O),3.75(s, 3H,CH3O),3.84(s,3H,CH3O),6.76(s,1H,ArH),7.19(s,1H,ArH),9.13(s,1H,Pyrimidine-H),10.52(s,1H,NH),13.03(s,1H,NH).

[0054] From the above identification results, it can be seen that the obtained white solid is methyl 2-((5-fluoro-2-((4-(4-methylpiperazin-1-yl)phenyl)amino)pyrimidin-4-yl)amino)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylate, whose structural formula is The yield of the method described in this example was calculated to be 40.6%.

[0055] Example 3 Antitumor Activity Study

[0056] The in vitro antitumor activity of the compounds of the present invention was verified using the following method. The following results indicate that the compounds of the present invention are useful for treating non-small cell lung cancer. The specific verification method is as follows:

[0057] The MTT method was used to detect the in vitro antitumor activity of the thiophene-containing pyrimidine compounds prepared in Examples 1 and 2. The logarithmic phase cells were collected and the concentration of the cell suspension was adjusted to 3×10 3 -4×10 3 100 μmol / mL was inoculated into a 96-well plate and incubated for 12-24 hours. After the cells adhered, different concentrations of the drug were added, with a total of 6 concentration gradients of 0.1, 0.3, 1, 3, 10, and 30 μmol / L, and four replicates were performed for each concentration. The plate was placed in a 37°C, 5% CO2 incubator and timed incubation was started. 48 hours after drug addition, the 96-well plate was removed and 20 μL of 5 mg / mL MTT solution was added to each well. The cells were incubated at 37°C for another 4 hours. The supernatant was then carefully aspirated and 200 μL of DMSO was added to each well. The cells were shaken for 10 minutes to dissolve the crystals. The absorbance (OD) of each well was measured on an enzyme-linked immunosorbent assay (ELISA) at a wavelength of 570 nm. The data were statistically analyzed, with the OD value (570 nm) on the vertical axis and the treatment time on the horizontal axis to plot the inhibitory effect of the drug on cell growth. The inhibition rate was calculated using the following formula: Inhibition rate = (1 - OD value of the drug-treated group / OD value of the control group) × 100%. GraphPad Prism software was used to calculate the half-maximal inhibitory concentration (IC) 50 .

[0058] The verification results are shown in Table 1:

[0059] Table 1 Antitumor activity of target compounds

[0060]

[0061] The reference substance (BI2536) in Table 1 above and Table 2 below is the compound disclosed in the Examples of patent application publication number WO2006018182A1: 4-[[(7R)-8-cyclopentyl-7-ethyl-5,6,7,8-tetrahydro-5-methyl-6-oxo-2-pteridinyl]amino]-3-methoxy-N-(1-methyl-4-piperidinyl)benzamide. This compound is a novel, potent, and highly selective PLK1 inhibitor. BI2536 is currently in Phase I / II clinical studies.

[0062] Ia in Table 1 above and Table 2 below refers to methyl 2-((2-((4-(4-methylpiperazin-1-yl)phenyl)amino)-5-nitropyrimidin-4-yl)amino)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylate prepared in Example 1. Ib refers to methyl 2-((5-fluoro-2-((4-(4-methylpiperazin-1-yl)phenyl)amino)pyrimidin-4-yl)amino)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylate prepared in Example 2.

[0063] The above-mentioned in vitro experimental results show that the thiophene-containing pyrimidine compounds of formula (I) have a strong inhibitory effect on non-small cell lung cancer cells, and the effects are better than the inhibitory activity of the control drug (BI2536); in particular, the inhibitory effect on PC-9 non-small cell lung cancer cells is significantly or even significantly higher than the inhibitory activity of the control drug (BI2536).

[0064] The above in vitro experimental results show that Ib has a significantly higher inhibitory effect on non-small cell lung cancer cells than Ia; this indicates that in the thiophene-containing pyrimidine compounds of formula (I), the compounds in which the R group is replaced by fluorine have a more significant anti-non-small cell lung cancer effect than the compounds obtained by replacing the R group by nitro.

[0065] Example 4 PLK1 enzyme inhibition activity experiment

[0066] Compounds were prepared as 10 mM stocks in DMSO and diluted to 100X based on the starting concentration. 100 nL of each compound dilution was then added to a 384-well plate. 5 μL of 1× kinase buffer (50 mM HEPES pH 7.5, 10 mM MgCl2, 2 mM DTT, 1 mM EGTA, and 0.01% Tween-20) containing Plk1 (final concentration 15 nM) was added to each well, and the mixture was incubated at room temperature for 10 minutes. 5 μL of substrate solution containing Light-TopoIIa (final concentration 50 nM) and ATP (final concentration 5 μM) in 1× kinase buffer was added to each well and incubated at room temperature for 30 minutes. 10 μL of detection solution (Europium anti-phospho-TopoIIa antibody (final concentration 2 nM, EDTA (final concentration 20 mM) in 1× assay buffer) was then added, mixed, and incubated at room temperature for 60 minutes. All samples were then analyzed using an Envision 2104 multifunctional reader (excitation wavelength 320 nm, emission wavelengths 665 nm and 615 nm). The ratio of RFU at 665 nm to that at 615 nm was calculated and converted to a percentage inhibition value. Inhibition ratio = (maximum ratio - sample ratio) / (maximum ratio - minimum ratio) * 100. Dose-response curves were then drawn and IC50 values ​​were calculated using the XLfit application in Excel software.

[0067] Table 2 below shows the in vitro enzyme inhibition activity results of the compounds of the present invention.

[0068] Table 2 PLK1 enzyme activity of compounds

[0069]

[0070] The in vitro experimental results above demonstrate that the thiophene-containing pyrimidine compounds of formula (I) of the present invention can achieve an inhibition rate of over 90% against PLK1 at a concentration of 1 μM. Therefore, the kinase activity assay results demonstrate that the thiophene-containing pyrimidine compounds of formula (I) of the present invention possess significant in vitro PLK1 protein kinase inhibitory activity.

Claims

1. A thiophene-containing pyrimidine compound, characterized in that: Having the chemical structure shown in formula (I); （Ⅰ）； Wherein, R in formula (I) is selected from nitro or fluorine.

2. The method for preparing thiophene-containing pyrimidine compounds according to claim 1, characterized in that: The following steps are included: (1) reacting a compound represented by formula (II) with methyl 2-amino-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylate to prepare an intermediate compound; (2) reacting the intermediate compound with 4-(4-methyl-1-piperazinyl)aniline to obtain a compound having the structure shown in formula (I); (II); Wherein, R in formula (II) is selected from nitro or fluorine.

3. The preparation method according to claim 2, characterized in that In step (2), the reaction is carried out at 100-140°C for 16-24 hours.

4. Use of the thiophene-containing pyrimidine compound according to claim 1 in the preparation of a drug having an anti-non-small cell lung cancer effect.

5. Use of the thiophene-containing pyrimidine compound according to claim 1 in the preparation of a drug having an inhibitory effect on PLK1 protein kinase.

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