Pyrimidine compound containing thiophene, preparation method of pyrimidine compound and application of pyrimidine compound in preparation of medicine with anti-cancer effect

Abstract

The invention relates to the technical field of biological medicines, and particularly discloses a thiophene-containing pyrimidine compound, a preparation method thereof and application of the thiophene-containing pyrimidine compound in preparation of a medicine with an anti-cancer effect. The thiophene-containing pyrimidine compound has a chemical structural formula as shown in a formula (I). Researches show that the thiophene-containing pyrimidine compound has an inhibition effect on PLK1 protein kinase; therefore, the compound has important application value when being used as an active ingredient for preparing the medicine with the anti-cancer effect. Particularly, in anticancer experiments, the non-small cell lung cancer resisting effect of the thiophene-containing pyrimidine compound is superior to that of a known compound BI2536; and a more obvious non-small cell lung cancer resisting effect is shown.

Description

Technical Field

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

[0002] Cancer is a huge global health burden, affecting almost every region and socioeconomic level. It is also the second leading cause of death in the world, and conquering cancer remains a huge challenge for modern medicine. According to statistics, there are more than 200 types of cancer in the world. Among them, lung cancer has the fastest growing incidence and mortality rate, and is one of the malignant tumors that poses the greatest threat to people's health and life. Non-small-cell lung cancer (NSCLC) accounts for about 80-85% of all lung cancers. Most patients are diagnosed with NSCLC in the middle and late stages, with local or distant organ metastasis, and the 5-year survival rate after surgical treatment is only 10-15%. Therefore, inhibiting and treating non-small-cell lung cancer has become the focus of attention.

[0003] PLK1 (Polo-like Kinase 1) belongs to the Polo-like kinase family and is a class of serine / threonine kinases widely present in eukaryotic cells. PLK1 is expressed at very low levels in most normal tissues (including kidney, liver, brain, lung and pancreas). On the contrary, it is highly expressed in a variety of malignant tumors such as lung cancer, colon cancer, melanoma, ovarian cancer, prostate cancer, breast cancer, and its expression level is closely related to tumor proliferation, invasion, migration, etc. It has been considered as a potential therapeutic target that can inhibit the metastasis of malignant tumors. So far, no PLK1 has been marketed, and there are more than 20 drugs under development, most of which are in the preclinical research stage. The invention patent with publication number WO2006018182A1 discloses the compound BI2536; BI2536 is the first potent and selective PLK1 inhibitor that can induce all the characteristics of PLK1 inhibition, and it can inhibit the activity of PLK1 enzyme at low nanomolar concentrations. The compound can effectively cause mitotic arrest and induce apoptosis in human cancer cell lines with different tissue origins and tumor genomic characteristics. Cells arrest in prometaphase, accumulate phospho-histone H3, and contain abnormal mitotic spindles. At the same time, BI2536 inhibits the growth of human tumor xenografts in nude mice and induces large tumor regression under a well-tolerated intravenous dose regimen. However, the study found that even short-term use of BI2536 in patients can cause grade 3-4 bone marrow suppression, neutropenia, and even sepsis.

[0004] Therefore, taking PLK1 as the target, developing more compounds that have inhibitory effects on PLK1 will provide more options for the screening of anti-cancer compounds, which has 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 and a preparation method thereof and an application thereof in preparing a drug having an anti-cancer effect.

[0006] The above technical problem to be solved by the present invention is achieved by adopting the following technical solution:

[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 compounds of any structure:

[0016]

[0017] The present invention also provides a method for preparing the above-mentioned thiophene-containing pyrimidine compound, wherein

[0018] Include

[0019] Follow these steps:

[0020] (1) reacting a compound having a structure represented by formula (II) with 2-amino-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylic acid methyl ester to prepare an intermediate compound;

[0023] (2) reacting the intermediate compound with 4-(4-methyl-1-piperazinyl)aniline to obtain a compound having a structure represented by formula (I);

[0024]

[0025] Wherein, R in formula (II) is selected from nitrogen-containing groups or halogens.

[0026] Preferably, the nitrogen-containing group is a nitro group.

[0027] Preferably, the halogen is fluorine.

[0028] Preferably, the reaction in step (2) is carried out at 100-140° C. for 16-24 h.

[0029] Most preferably, the reaction in step (2) is carried out at 120° C. for 20 h.

[0030] The present invention also provides an application of the above thiophene-containing pyrimidine compound in the preparation of a drug with anti-cancer effect.

[0031] Preferably, the cancer is lung cancer.

[0032] Preferably, the lung cancer is non-small cell lung cancer.

[0033] Preferably, the thiophene-containing pyrimidine compound is used in the preparation of a drug having an effect of inhibiting the growth of cancer cells.

[0034] Preferably, the cancer cells are A549 and / or PC-9 human non-small cell lung cancer cells.

[0035] The present invention also provides a use of the above thiophene-containing pyrimidine compound in the preparation of a drug having an inhibitory effect on PLK1 protein kinase.

[0036] Beneficial effects: The present invention provides a thiophene-containing pyrimidine compound with a completely new 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 for preparing a drug with anti-cancer effect has important application value.

[0037] In particular, in the anti-cancer experiments, the thiophene-containing pyrimidine compound has better anti-non-small cell lung cancer effect than the known compound BI2536; it shows a more significant anti-non-small cell lung cancer effect; therefore, it has more important application value to use it as an active ingredient in the preparation of drugs with anti-non-small cell lung cancer effect.

[0038] 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

[0039] Figure 1It is the hydrogen nuclear magnetic resonance spectrum of 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 of the present invention.

[0040] Figure 2 It is the hydrogen nuclear magnetic resonance hydrogen pattern of 2-((5-fluoro-2-((4-(4-methylpiperazin-1-yl)phenyl)amino)pyrimidin-4-yl)amino)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylic acid methyl ester of the present invention. DETAILED DESCRIPTION

[0041] The present invention is further explained below with reference to the examples, but the examples do not limit the present invention in any form.

[0042] The reaction process of the present invention is shown in flow charts (V) and (VIII):

[0043]

[0044] The compound of the structure shown in formula (IV) in the above reaction process is an intermediate compound;

[0045] The compound represented by the formula (VII) is also referred to as the thiophene-containing pyrimidine compound of the present invention.

[0046] 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

[0047] (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, extracted with ethyl acetate (100 mL×3), allowed to stand for separation, and 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;

[0048] (2) dissolving 2.0 mmol of the intermediate obtained in step (1) and 4-(4-methyl-1-piperazinyl)aniline in ethylene glycol methyl ether, reacting at 120° C. for 20 hours under the catalysis of hydrochloric acid, extracting the reaction solution with ethyl acetate, drying the organic phase with anhydrous sodium sulfate, desolventizing, and separating and purifying 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;

[0049] 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.

[0050] 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%.

[0051] 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

[0052] (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, extracted with ethyl acetate (100 mL×3), allowed to stand for separation, and 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;

[0053] (2) dissolving 2.0 mmol of the intermediate obtained in step (1) and 4-(4-methyl-1-piperazinyl)aniline in ethylene glycol methyl ether, reacting at 120° C. for 20 hours under the catalysis of hydrochloric acid, extracting the reaction solution with ethyl acetate, drying the organic phase with anhydrous sodium sulfate, desolventizing, and separating and purifying 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;

[0054] 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).

[0055] 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, and its structural formula is The yield of the method described in this example was calculated to be 40.6%.

[0056] Example 3 Antitumor Activity Study

[0057] The in vitro antitumor activity of the compounds of the present invention was verified by the following method. The following effects indicate that the compounds of the present invention can be used to treat non-small cell lung cancer. The specific verification method is as follows:

[0058] 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 cells in the logarithmic phase were collected and the concentration of the cell suspension was adjusted to 3×10 3 -4×10 3 / mL was inoculated in a 96-well plate and incubated for 12-24h. After the cells adhered to the wall, different concentrations of drugs were added, and 6 concentration gradients of 0.1, 0.3, 1, 3, 10, and 30μmol / l were set, with 4 replicates for each concentration. Place in a 37℃, 5% CO2 incubator and start timed culture. 48h after adding the drug, take out the 96-well plate, add 20μL of 5mg / mL MTT solution to each well, and continue to culture at 37℃ for 4h. Then, carefully discard the supernatant in the well, add 200μL DMSO to each well, and shake for 10min to dissolve the crystals. Select 570nm wavelength to measure the absorbance value (OD value) of each well on an enzyme-linked immunosorbent assay. Finally, data statistics were performed, with OD value (570nm) as the vertical axis and treatment time as the horizontal axis to depict the inhibitory effect of the drug on cell growth. The inhibition rate was calculated according to the following formula: inhibition rate = (1-OD value of the drug-added group / OD value of the control group) × 100%. GraphPad Prism software was used to calculate the half inhibition IC 50 .

[0059] The verification results are shown in Table 1:

[0060] Table 1 Antitumor activity of target compounds

[0061]

[0062] The reference substance (BI2536) in Table 1 above and Table 2 below is a compound disclosed in the example of patent application No. 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, which is a novel, potent, and highly selective PLK1 inhibitor. Currently, BI2536 has been used in clinical Phase I / II studies.

[0063] 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.

[0064] The above 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); especially the inhibitory effect on PC-9 non-small cell lung cancer cells, the inhibitory activity is significantly or even much higher than the inhibitory activity of the control drug (BI2536).

[0065] The above in vitro experimental results show that the inhibitory effect of Ib on non-small cell lung cancer cells is significantly higher than that of 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.

[0066] Example 4 PLK1 enzyme inhibition activity experiment

[0067] Compounds were prepared as 10mM DMSO stocks and diluted to 100X in the experiment according to the starting concentration of the experiment. Then 100nL of each compound dilution was added to a 384-well plate, and 5μL of 1× kinase buffer (50mM HEPES pH7.5, 10mM MgCl2, 2mM DTT, 1mM EGTA and 0.01% Tween-20) containing Plk1 (final concentration 15nM) was added to each well, and the mixture was incubated at room temperature for 10 minutes. 5μL of substrate solution containing ULight-TopoIIa (final concentration 50nM) and ATP (final concentration 5μM) was added to each well in 1× kinase buffer, incubated at room temperature for 30 minutes, and then 10μL of detection solution (Europium-anti-phospho-TopoIIa antibody final concentration 2nM, EDTA final concentration 20mM in 1× detection buffer) was added, mixed, and incubated at room temperature for 60 minutes. All samples were then analyzed using an Envision2104 multifunctional reader (excitation wavelength 320 nm, emission wavelength 665 nm and 615 nm). The ratio of RFU at 665 nm and at 615 nm was calculated, and the ratio value was converted into an inhibition percentage value. Inhibition rate = (maximum ratio - sample ratio) / (maximum ratio - minimum ratio) * 100. The dose-response curve was then drawn, and the IC50 value was then calculated using the XLfit application in Excel software.

[0068] Table 2 below shows the in vitro enzyme inhibition activity results of the compounds of the present invention.

[0069] Table 2 PLK1 enzyme activity of compounds

[0070]

[0071] The above in vitro experimental results show that the thiophene-containing pyrimidine compound of the present invention with the structure of formula (I) can achieve an inhibition rate of more than 90% on PLK1 at a concentration of 1 μM. Therefore, the results of the kinase activity assay show that the thiophene-containing pyrimidine compound of the present invention with the structure of formula (I) has good 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); Wherein, R in formula (I) is selected from nitrogen-containing groups or halogens.

2. The thiophene-containing pyrimidine compound according to claim 1, characterized in that: The nitrogen-containing group is a nitro group.

3. The thiophene-containing pyrimidine compound according to claim 1, characterized in that: The halogen is fluorine.

4. The thiophene-containing pyrimidine compound according to claim 1, characterized in that: A compound selected from any of the following structures:

5. The method for preparing a thiophene-containing pyrimidine compound according to any one of claims 1 to 4, characterized in that: The following steps are included: (1) a compound represented by the structure of formula (II) and 2-amino-4,5,6,7-tetrahydrobenzo[b]thiophene-3- Methyl formate is reacted to prepare an intermediate compound; (2) reacting the intermediate compound with 4-(4-methyl-1-piperazinyl)aniline to obtain a compound having a structure represented by formula (I); Wherein, R in formula (II) is selected from nitrogen-containing groups or halogens.

6. The preparation method according to claim 5, characterized in that: The nitrogen-containing group is a nitro group.

7. The preparation method according to claim 5, characterized in that: The halogen is fluorine.

8. The preparation method according to claim 5, characterized in that: In step (2), the reaction is carried out at 100-140° C. for 16-24 hours.

9. Use of the thiophene-containing pyrimidine compound according to any one of claims 1 to 4 in the preparation of a drug having an anti-cancer effect.

10. Use of the thiophene-containing pyrimidine compound according to any one of claims 1 to 4 in the preparation of a drug having an inhibitory effect on PLK1 protein kinase.

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