CN103059030B - Pyrimidine compound with effect of adhesion kinase inhibition and preparation method and application thereof - Google Patents

Abstract

The present invention provides a pyrimidine compound, the structure of which is shown in the following formulas (I) and (II), wherein, R is substituted phenyl or substituted pyridyl; R ₁ is -NO ₂ , -Br, -COOH or -OCH ₃ ; n is an integer of 1-3; X is -Br or Cl; R ₈ , R ₉ and R ₁₀ are the same or different -H, -OCH ₃ , -COOMe, -Br, -COOEt, - _CH2COOMe , _-NO2 or ( _CH2 ) _1-4OH . The compound described in the present invention is a compound with FAK inhibitory effect, the compound can effectively enter tumor cells, have a good inhibitory effect on tumor cells and can retain better. The invention also provides the preparation method of the compound and the application of the compound in the preparation of tumor suppressors.

Description

A pyrimidine compound with focal adhesion kinase inhibitory effect and its preparation method and application

technical field

The present invention relates to the field of medicinal chemistry, specifically to the field of radiopharmaceutical chemistry, in particular to a series of pyrimidine derivatives with focal adhesion kinase inhibitory effect.

Background technique

Focal Adhesion Kinase (FAK) is a non-receptor tyrosine protein kinase ^[1] , which was identified in 1992 as a highly phosphorylated protein related to the oncogene v-src, located in the normal Integrin-rich focal adhesion regions of cells. FAK is an important cytoskeletal protein and a key molecule of various signaling pathways in cells, and plays an important role in cell survival, proliferation, migration and invasion ^[2-7] . FAK plays a very important role in the occurrence and development of tumors, so it may be used as a marker molecule for tumor invasion and prognosis of malignant tumors, and FAK may become an effective target for tumor treatment and may become a tumor diagnosis and new targets for therapy. Therefore, FAK has great potential to play a role in tumor-specific diagnosis, targeted molecular therapy, and tumor prognosis assessment. Currently, the main FAK inhibitors are TAC544 and TAE226 from Novartis, PF-562,271 and PF-573,228 from Pfizer, GSK2256098 from GlaxoSmithKline, Y15 from Sigma and CFAK-C4 from CureFAKtor Pharmaceuticals ^[8] .

At present, the commonly used inspection methods for the diagnosis of malignant tumors have certain limitations, or have false positives, or have poor specificity, or low sensitivity; B-ultrasound, CT, and nuclear magnetic resonance are based on the morphological changes of the disease, and it is difficult to detect them in the early stage of cancer. Provides diagnostic information in the absence of structural morphological changes. The most prominent advantage of radionuclide-labeled compound imaging and targeted molecular therapy is its functional imaging and targeted molecular therapy advantages, which can reflect the blood flow, receptor density and activity, metabolism, and functional changes of organs or tissues. It has unique advantages in chemical process and is one of the most advanced technologies for clinical diagnosis and treatment of diseases.

Based on the above background technology, it is necessary to develop a FAK inhibitor that can be labeled with radionuclides, so as to play a greater role in promoting tumor molecular imaging agents and malignant tumor radioimmunotherapy.

Contents of the invention

One object of the present invention is to provide a pyrimidine compound with FAK inhibitory effect, which can effectively enter tumor cells, have a good inhibitory effect on tumor cells and can retain better.

Another object of the present invention is to provide pharmaceutically acceptable salts of the pyrimidine compounds.

Another object of the present invention is to provide a preparation method of the pyrimidine compounds.

Another object of the present invention is to provide applications of the pyrimidine compounds.

Above-mentioned purpose of the present invention is achieved through the following technical solutions:

A pyrimidine compound is provided, the structure of which is shown in the following formula (I):

Wherein, R is substituted phenyl or substituted pyridyl; R ₁ is -NO ₂ , -Br, -COOH or -OCH ₃ ; n is an integer of 1-3.

In a preferred solution of the present invention, R in formula (I) is a substituted phenyl group, n=1, and its structure is shown in formula (I-1):

Wherein, R ₂ , R ₃ , R ₄ and R ₅ are the same or different -H, -COOMe, Br, -COPh or (CH ₂ ) _1-4 OH.

Among the compounds represented by the above formula (I-1) of the present invention, further preferred compounds include:

Compound 1, the structure is as follows:

Compound 2, the structure is as follows:

In another preferred solution of the present invention, R in formula (I) is a substituted pyridyl group, n=1, and its structure is shown in formula (I-2):

Wherein, R ₆ and R ₇ are the same or different -H, -COOMe, -Br, -NO ₂ or (CH ₂ ) _1-4 OH.

Among the compounds represented by the above formula (I-2), a further preferred compound is compound 3, which has the following structure:

The present invention also provides a pyrimidine compound, the structure of which is shown in the following formula (II):

Wherein, X is -Br or Cl; R ₁ is -NO ₂ , -Br, -COOH or -OCH ₃ ; n is an integer from 1 to 3; R ₈ , R ₉ and R ₁₀ are the same or different -H , -OCH ₃ , -COOMe, -Br, -COOEt, -CH ₂ COOMe, -NO ₂ or (CH ₂ ) _1-4 OH.

In another preferred version of the present invention, X in formula (II) is Br, R ₁ is -COOH or -OCH, R ₈ , R ₉ and R ₁₀ are the same or different -H, -OCH ₃ , - Br or -COOEt.

Among the compounds represented by the above formula (II) of the present invention, the following compounds are further preferred:

Compound 4 has the following structure:

Compound 5 has the following structure:

Compound 6 has the following structure:

Compound 7 has the following structure:

Compound 8 has the following structure:

Compound 9 has the following structure:

Compound 10 has the following structure:

Compound 11 has the following structure:

Compound 12 has the following structure:

Compound 13 has the following structure:

Compound 14 has the following structure:

The invention also provides a preparation method of the compound.

Wherein, the compound preparation method represented by formula (I-1) comprises the following steps:

As shown in the following synthetic route, 10mmol of 2-chloro-7H-pyrrole[2,3-d]pyrimidine, 30mmol of anhydrous potassium phosphate, a small amount of CuI and trans-1,2-cyclohexanediamine and 10ml of dioxane Add it into a three-neck flask, then slowly add 12 mmol of compound A, and stir and reflux at 110°C for 5 h under the protection of N ₂ . Detect the reaction with a thin-layer chromatographic plate. After the reaction is over, filter the solid, and wash the solid with a small amount of dioxane for 3 times, then add saturated NaHCO ₃ solution, wash the organic phase with 3×10mL saturated brine, dry with anhydrous MgSO , and filter , spin-dried dioxane, and passed through a silica gel column with a developer ethyl acetate:petroleum ether=1:10-1:2, and finally obtained a pale white solid B. Add compound B, compound C, concentrated hydrochloric acid, and n-butanol into the digestion tank, seal it well, and react at 140°C for 24 hours. Pour the reaction solution into a 100ml flask, add 50ml of water, adjust the pH to 6-7 with saturated sodium carbonate solution, extract with 3×50ml of ethyl acetate, collect the organic phase, spin dry the ethyl acetate, and wash the solid with water three times. Then wash with n-hexane for 3 times, filter, collect the filter cake, and use the developer ethyl acetate:petroleum ether=1:10-1:1 to pass through a silica gel column to finally obtain compound D. That is, the compound represented by the formula (I-1) of the present invention. The synthetic route is as follows, wherein each substituent of R1-R5 is as defined above:

The preparation method of the compound represented by formula (I-2) of the present invention can be completed by referring to the preparation method of the compound of formula (I-1).

The compound preparation method represented by formula (II) of the present invention comprises the following steps:

As shown in the following synthetic route, 10 mmol of B', 12 mmol of compound A', 30 mmol of potassium carbonate, 25 ml of isopropanol and 25 ml of water were stirred overnight at room temperature. The reaction was detected with a thin-layer chromatography plate. After the reaction was completed, the solid was filtered and rinsed with a small amount of ethanol for 3 times. The solid was collected and put into a beaker, and 25ml of water was added to stir at room temperature and filtered to obtain compound E. Dissolve 10 mmol of compound E in dioxane, add compound C, 15 mmol p-toluenesulfonic acid, a small amount of DMF, and stir at 100°C overnight. The reaction was detected with a thin-layer chromatographic plate. After the reaction was completed, the solid was filtered and washed with a small amount of ethyl acetate for 3 times to obtain a gray solid. Add 20ml of water, and extract with 3×10ml of ethyl acetate, collect the organic phase, spin dry, and finally compound F is the compound represented by formula (II) of the present invention. The synthetic route is as follows, and each substituent is defined as before:

All raw material compounds and preparations used in the above preparation methods are existing products.

The present invention also provides the pharmaceutically acceptable salts of the pyrimidine compounds.

The salt is an addition salt formed by the pyrimidine compound and an acid, and the acid can be hydrochloric acid, hydrobromic acid, sulfuric acid, citric acid, tartaric acid, lactic acid, pyruvic acid, acetic acid, maleic acid, methanesulfonic acid acid, p-toluenesulfonic acid or arginine.

The above-mentioned addition salts of the compounds of the present invention can use various acids described above and each compound of the present invention as raw materials, and acid addition salts can be prepared by referring to existing methods.

The addition salt formed by the pyrimidine compound and acid provided by the present invention can be used in the preparation of medicines in different dosage forms according to different physical and chemical properties.

The invention also provides the application of the compound in the preparation of tumor cell inhibitors.

Through preliminary cytotoxicity experiments and radionuclide labeling experiments, the compound of the present invention has significant FAK inhibitory effect, and has a good inhibitory effect on tumor cells; the results of biodistribution experiments show that the compound of the present invention can effectively enter tumor cells, and Can stay better.

The tumor cells are liver cancer cells as an example. Among the 14 compounds synthesized so far, the inhibitory effect of 5 compounds on the growth of HepG2 liver cancer cells has been measured. Four compounds have good cytotoxic effects: Compound No. 1 has a strong growth inhibitory effect on HepG2 liver cancer cells, and can inhibit the growth of more than 80% of the cells at a concentration of 1 μmol. The data at a concentration of 5 μmol in the figure may be due to manipulation, etc. Other reasons lead to wrong experimental results; Compound No. 3 has a strong growth inhibitory effect on HepG2 liver cancer cells only at a concentration of 10umol, and other concentrations have no obvious effect; Compound No. 5 has a significant inhibitory effect on HepG2 liver cancer cells at a concentration of 2umol, which can inhibit 40 % of the cell growth, 5umol can inhibit the growth of more than 90% of the cells; No. 13 compound has a growth inhibitory effect on the cells in the range of 5-50umol, and has a good dose-effect relationship.

Description of drawings

Fig. 1 is the effect of compound 1 of the present invention at different concentrations on the growth of HepG2 liver cancer cells.

Fig. 2 is the effect of compound 3 of the present invention at different concentrations on the growth of HepG2 liver cancer cells.

Fig. 3 is the effect of compound 5 of the present invention at different concentrations on the growth of HepG2 liver cancer cells.

Fig. 4 is the effect of compound 13 of the present invention at different concentrations on the growth of HepG2 liver cancer cells.

Detailed ways

Example 1

The synthesis of compound 1, the synthetic route is as follows:

Specific steps include:

1.5g (10mmol) 2-chloro-7H-pyrrole[2,3-d]pyrimidine, 3g anhydrous potassium phosphate, a small amount of CuI and trans-1,2-cyclohexanediamine and 10ml dioxane were added to Then slowly add 3.2 g (12 mmol) of 3-bromobenzophenone into the three-necked flask, and stir and reflux for 5 h at 110° C. under the protection of N ₂ . Detect the reaction with a thin-layer chromatographic plate. After the reaction is over, filter the solid, and wash the solid with a small amount of dioxane for 3 times, then add saturated NaHCO ₃ solution, wash the organic phase with 3×10mL saturated brine, dry with anhydrous MgSO , and filter , spin-dried dioxane, and passed through a silica gel column with a developer ethyl acetate:petroleum ether=1:10-1:2, and finally obtained a pale white solid A with a yield of 75.5%. Add 300mg of compound A, 600mg of p-bromoaniline, 3ml of concentrated hydrochloric acid, and 4ml of n-butanol into the digestion tank, seal it well, and react at 140°C for 24h. Pour the reaction solution into a 100ml flask, add 50ml of water, adjust the pH to 6-7 with saturated sodium carbonate solution, extract with 3×50ml of ethyl acetate, collect the organic phase, spin dry the ethyl acetate, and wash the solid with water three times. Then wash with n-hexane for 3 times, filter, collect the filter cake, and pass through a silica gel column with a developer ethyl acetate:petroleum ether=1:10-1:1, and finally obtain 130 mg of solid, which is compound 1 of the present invention. NMR spectrum: 6.7785(s,1H);7.4534(d,2H);7.6146(t,2H);7.7279(t,1H);7.8269(m,5H);7.9714(d,2H);8.1716(d, 2H); 8.8671(s,1H); 9.7563(s,1H).

Example 2

The synthesis of compound 2, the synthetic route is as follows:

Concrete synthetic steps include:

Add 3.5g of 2,5-dibromobenzoic acid and 20ml of methanol into a 100ml single-necked bottle, stir slowly at room temperature and add 10ml of concentrated sulfuric acid, stir for 10 minutes after adding the concentrated sulfuric acid and start heating to reflux, react for 8 hours, and detect the reaction with a thin-layer chromatography plate. After the reaction, excess methanol was spun to dry, 50ml of water was added, and filtered to obtain a white solid with a yield greater than 85%. 1.5g (10mmol) 2-chloro-7H-pyrrole[2,3-d]pyrimidine, 3g anhydrous potassium phosphate, a small amount of CuI and trans-1,2-cyclohexanediamine and 10ml dioxane were added to In the three-necked flask, add 3.0 g of methyl 2,5-dibromobenzoate, and stir and reflux at 110° C. for 6 h. Detect the reaction with a thin-layer chromatographic plate. After the reaction is over, filter the solid, and wash the solid with a small amount of dioxane for 3 times, then add saturated NaHCO ₃ solution, wash the organic phase with 3×10mL saturated brine, dry with anhydrous MgSO , and filter , spin-dried dioxane, and passed through a silica gel column with a developer ethyl acetate:petroleum ether=1:10-1:1, and finally obtained a pale white solid with a yield of 75.5%. Add 300mg of compound B, 500mg of anisidine, 3ml of concentrated hydrochloric acid, and 4ml of n-butanol into the digestion tank, seal it well, and react overnight at 140°C. Pour the reaction solution into a 100ml flask, add 50ml of water, adjust the pH to 6-7 with saturated sodium carbonate solution, extract with 3×50ml of ethyl acetate, collect the organic phase, spin dry the ethyl acetate, and wash the solid with water three times. Rinse with n-hexane for 3 times, filter, collect the filter cake, pass through a silica gel column with a developer ethyl acetate:petroleum ether=1:10-1:1, and finally isolate and obtain 79 mg of the product, which is compound 2 of the present invention. NMR spectrum: 3.812(s,3H);3.970(s,3H);6.675(t,1H);6.93(q,2H);7.261(d,1H);7.378(m,2H);7.585(d, 1H);8.167(q,2H);8.272(t,1H);8.376(t,1H);8.600(m,1H).

Example 3

The synthesis of compound 3, the synthetic route is as follows:

Concrete synthetic steps include:

1.5g (10mmol) 2-chloro-7H-pyrrole[2,3-d]pyrimidine, 3g anhydrous potassium phosphate, a small amount of CuI and trans-1,2-cyclohexanediamine and 10ml dioxane were added to Then slowly add 4.5g of 2,5-dibromopyridine into the three-neck flask, and stir and reflux at 110°C for 5h. Detect the reaction with a thin-layer chromatographic plate. After the reaction is over, filter the solid, and wash the solid with a small amount of dioxane for 3 times, then add saturated NaHCO ₃ solution, wash the organic phase with 3×10mL saturated brine, dry with anhydrous MgSO , and filter , spin-dried dioxane, and passed through a silica gel column with a developer ethyl acetate:petroleum ether=1:10-1:2, and finally obtained 950 mg of a light white solid. Add 300mg of compound C, 600mg of anisidine, 3ml of concentrated hydrochloric acid, and 4ml of n-butanol into the digestion tank, seal it well, and react overnight at 140°C. Pour the reaction solution into a 100ml flask, add 50ml of water, adjust the pH to 6-7 with saturated sodium carbonate solution, extract with 3×50ml of ethyl acetate, collect the organic phase, spin dry the ethyl acetate, and wash the solid with water three times. Rinse with n-hexane for 3 times, filter, collect the filter cake, and pass through a silica gel column with a developer ethyl acetate:petroleum ether=1:10-1:1, and finally obtain 70 mg of solid, which is compound 3 of the present invention. NMR spectrum: 6.617(d,4H);6.944(t,2H);7.528(d,2H);7.903(d,1H);8.115(s,3H);8.528(d,2H); 8.565(s, 1H); 8.622(s,1H).

Example 4

The synthesis of compound 4, the synthetic route is as follows:

Concrete synthetic steps include:

3g (22mmol) of 5-bromo-2,4-dichloropyrimidine, 4.0g of aniline, 4.0g of potassium carbonate, 30ml of water and 30ml of isopropanol were added to a 100ml three-necked flask, and stirred overnight at room temperature. Detect the reaction with a thin-layer chromatographic plate. After the reaction is over, filter the solid, and wash the solid with a small amount of ethanol for 3 times, collect the solid and put it into a 100ml beaker, add 50ml of water to stir at room temperature, filter, and finally get a white solid, dry the solid, and the yield is 90% %above. Dissolve 1g of the light yellow solid with 50ml of dioxane, add 2.5g of 3,4,5-trimethoxyaniline, 1.5g of p-toluenesulfonic acid, a small amount of DMF, and stir at 100°C overnight. The reaction was detected with a thin-layer chromatographic plate. After the reaction was completed, the solid was filtered and washed with a small amount of ethyl acetate for 3 times. Spin-dry to obtain an off-white solid, add 30ml of water, and extract with 3×10ml ethyl acetate, collect the organic phase, and spin-dry to obtain an off-white solid. The developer ethyl acetate:petroleum ether=1:2 was used to pass through the silica gel column, and finally an off-white solid was obtained, namely compound 4 of the present invention, with a yield of 67.7%. NMR spectrum: 1.433(t,3H);3.758(s,6H);3.849(s,3H);4.445(m,2H);6.776(s,2H);7.084(t,1H);7.262(d, 1H);7.436(t,1H);8.071(d,1H);8.207(s,1H);8.743(d,1H);8.764(s,1H).

Example 5

The synthesis of compound 5, the synthetic route is as follows:

Concrete synthetic steps include:

1) Add 3g (22mmol) 5-bromo-2,4-dichloropyrimidine, 4.5g (35mmol) anisidine, 4.0g potassium carbonate, 30ml water and 30ml isopropanol into a 100ml three-necked flask, and stir overnight at room temperature . Use a thin-layer chromatographic plate to detect the reaction. After the reaction is over, filter the solid, and wash the solid with a small amount of ethanol for 3 times, collect the solid and put it in a 100ml beaker, add 50ml of water to stir at room temperature, filter, and finally get a light yellow solid, dry the solid, and the yield More than 90.

2) Take out 1g of the light yellow solid obtained in step 1) and dissolve it in 50ml of dioxane, add 2.5g of 3,4,5-trimethoxyaniline, 1.5g of p-toluenesulfonic acid, a small amount of DMF, 100° C stirred the reaction overnight. The reaction was detected with a thin-layer chromatographic plate. After the reaction was completed, the solid was filtered and washed with a small amount of ethyl acetate for 3 times. Spin-dry to obtain a light yellow solid, add 30ml of water, and extract with 3×10ml ethyl acetate, collect the organic phase, and spin-dry to obtain a light yellow solid. Use the developer ethyl acetate:petroleum ether=1:2 to pass through the silica gel column, and finally obtain an off-white solid, that is, compound 5 of the present invention, with a yield of 56.7%. NMR spectrum: 3.658(s,5H);3.813(5H,m);6.728(s,2H);6.884(d,2H);7.212(s,1H);7.409(d,2H);8.053(s, 1H).

Example 6

The synthesis of compound 6, the synthetic route is as follows:

Concrete synthetic steps include:

Dissolve 1.5 g of the light yellow solid obtained in step 1) in Example 5 with 50 ml of dioxane, add 2.1 g of 3,4-dimethoxyaniline, 1.5 g of p-toluenesulfonic acid, a small amount of DMF, and stir at 100°C React overnight. The reaction was detected with a thin-layer chromatographic plate. After the reaction was completed, the solid was filtered and washed three times with a small amount of ethyl acetate to obtain a dark red solid. The developer dichloromethane was used to pass through the silica gel column, and finally a light white solid was obtained, namely compound 6 of the present invention, with a yield of 85.2%. NMR spectrum: 3.600(s,3H);3.77(s,3H);6.70(s,3H);6.81(d,1H);6.91(t,3H);7.02(s,1H);7.08(d, 1H); 7.19(s, 1H); 7.36(t, 2H); 8.02(s, 1H).

Example 7

The synthesis of compound 7, the synthetic route is as follows:

Concrete synthetic steps include:

Dissolve 1.5 g of the light yellow solid obtained in step 1) in Example 5 with 50 ml of dioxane, add 1.5 g of anisidine, 1.5 g of p-toluenesulfonic acid, a small amount of DMF, and stir at 100° C. overnight. The reaction was detected with a thin-layer chromatographic plate. After the reaction was completed, the solid was filtered and washed three times with a small amount of ethyl acetate to obtain a dark red solid. Add 20ml of water and extract with 3×10ml of ethyl acetate, collect the organic phase and spin dry to obtain a light red solid. The developer ethyl acetate:petroleum ether=1:2 was used to pass through the silica gel column, and finally a milky white solid was obtained, namely compound 7 of the present invention, with a yield of 78.1%. NMR spectrum: 3.816(q,5H);6.811(t,2H);6.896(q,2H);7.114(s,1H);7.368(q,2H);7.425(m,2H);8.025(s, 1H).

Example 8

The synthesis of compound 8, the synthetic route is as follows:

Concrete synthetic steps include:

Take 2.5 g of anthranilic acid and dissolve it in 50 ml of dioxane, add 1 g of the light yellow solid obtained in step 1) in Example 5, 1.5 g of p-toluenesulfonic acid, a small amount of DMF, and stir at 100° C. overnight. The reaction was detected with a thin-layer chromatographic plate. After the reaction was completed, the solid was filtered and washed with a small amount of ethyl acetate for 3 times to obtain a gray solid. Add 20ml of water, and extract with 3×10ml of ethyl acetate, collect the organic phase, spin dry, and finally obtain a light yellow solid, that is, compound 8 of the present invention, with a yield of 71.5%. NMR spectrum: 3.771(s,3H);6.922(s,2H);7.214(q,2H);7.466(q,3H);7.933(s,1H);8.048(s,1H);8.199(s, 1H); 8.542(s,1H); 9.438(s,1H).

Example 9

The synthesis of compound 9, the synthetic route is as follows:

Concrete synthetic steps include:

1) Take 16g of ethyl m-aminobenzoate, add 15g of 5-bromo-2,4-dichloropyrimidine, 30g of potassium carbonate, 80ml of isopropanol and 80ml of water, and stir overnight at room temperature. Use a thin-layer chromatography plate to detect the reaction. After the reaction is over, filter the solid, and rinse the solid with a small amount of ethanol for 3 times, collect the solid and put it in a 250ml beaker, add 150ml of water to stir at room temperature, filter, and finally get a light yellow solid, dry the solid, and the yield More than 90.

2) Dissolve 2 g of the light yellow solid obtained in step 1) with 50 ml of dioxane, add 3.0 g of anisidine, 1.5 g of p-toluenesulfonic acid, a small amount of DMF, and stir at 100°C overnight. The reaction was detected with a thin-layer chromatographic plate. After the reaction was completed, the solid was filtered and washed with a small amount of ethyl acetate for 3 times to obtain a gray solid. Add 20ml of water, and extract with 3×10ml of ethyl acetate, collect the organic phase, spin dry, and finally obtain a light gray solid, that is, compound 9 of the present invention, with a yield of 81.5%. NMR spectrum: 1.37(s,3H);3.79(s,3H);4.37(s,2H);6.81(s,2H);7.26(t,1H);7.39(q,3H);7.83(s, 1H);7.90(s,1H);7.91(s,1H);7.92(s,1H).

Example 10

The synthesis of compound 10, the synthetic route is as follows:

Concrete synthetic steps include:

Dissolve 2 g of the light yellow solid obtained in step 1) in Example 9 with 50 ml of dioxane, add 3.5 g of 3,4-dimethoxyaniline, 1.5 g of p-toluenesulfonic acid, a small amount of DMF, and stir at 100°C for reaction overnight. The reaction was detected with a thin-layer chromatographic plate. After the reaction was completed, the solid was filtered and washed three times with a small amount of ethyl acetate to obtain a dark gray solid. The solid was washed three times with a small amount of ethyl acetate, 20ml of water was added, and extracted with 3×10ml of ethyl acetate, the organic phase was collected and spin-dried to obtain a light gray solid, namely compound 10 of the present invention, with a yield of 83.6%. NMR spectrum: 1.379(t,3H);3.704(s,3H);3.868(s,3H);4.368(t,2H);6.781(m,1H);6.986(s,1H);7.045(q, 1H);7.261(s,1H);7.398(q,1H);7.823(t,1H);7.922(s,1H);7.945(s,1H);8.113(s,1H).

Example 11

The synthesis of compound 11, the synthetic route is as follows:

Concrete synthetic steps include:

Dissolve 2 g of the light yellow solid obtained in step 1) in Example 9 with 50 ml of dioxane, add 2.5 g of 3,4-dimethoxyaniline, 1.5 g of p-toluenesulfonic acid, a small amount of DMF, and stir at 100°C for reaction overnight. The reaction was detected with a thin-layer chromatographic plate. After the reaction was completed, the solid was filtered and washed three times with a small amount of ethyl acetate to obtain a light yellow solid. The solid was washed three times with a small amount of ethyl acetate, 20ml of water was added, and extracted with 3×10ml of ethyl acetate, the organic phase was collected and spin-dried to obtain a light yellow solid, namely compound 11 of the present invention, with a yield of 65.6%. NMR spectrum: 1.372(s,3H);3.668(s,5H);3.824(d,3H);4.365(t,2H);6.715(s,2H);7.395(t,3H);7.874(q, 1H); 7.944(d, 1H); 8.029(s, 1H); 8.095(s, 1H).

Example 12

The synthesis of compound 12, the synthetic route is as follows:

Concrete synthetic steps include:

1) Add 15g of p-aminobenzoic acid and 100g of absolute ethanol into a 250ml three-neck flask, stir at room temperature, slowly add 16g of concentrated sulfuric acid dropwise, after adding the concentrated sulfuric acid dropwise, stir for 10min, then heat to reflux, react overnight, and use thin layer chromatography Plate detection reaction, after the reaction, the reaction solution from turbid to clear. Remove excess methanol under reduced pressure, add ethyl acetate and saturated aqueous sodium bicarbonate, adjust the pH to 8-9, separate the organic phase, add a small amount of ethyl acetate to the aqueous phase for back extraction, combine the organic phases, and add anhydrous magnesium sulfate Dried, filtered, and spin-dried to obtain ethyl p-aminobenzoate as a white solid.

2) Add 16g of ethyl p-aminobenzoate obtained in step 1), add 15g of 5-bromo-2,4-dichloropyrimidine, 30g of potassium carbonate, 80ml of isopropanol and 80ml of water, and stir overnight at room temperature. Use a thin-layer chromatography plate to detect the reaction. After the reaction is over, filter the solid, and rinse the solid with a small amount of ethanol for 3 times, collect the solid and put it in a 250ml beaker, add 150ml of water to stir at room temperature, filter, and finally get a light yellow solid, dry the solid, and the yield More than 90.

3) Dissolve 2.1g of the light yellow solid obtained in step 2) in 50ml of dioxane, add 2.5g of 3,4,5-trimethoxyaniline, 1.5g of p-toluenesulfonic acid, a small amount of DMF, 100°C The reaction was stirred overnight. The reaction was detected with a thin-layer chromatographic plate. After the reaction was completed, the solid was filtered and washed with a small amount of ethyl acetate for 3 times to obtain a gray solid. 20ml of water was added and extracted with 3×10ml of ethyl acetate, the organic phase was collected and spin-dried to obtain a light white solid, namely compound 12 of the present invention, with a yield of 62.4%. NMR spectrum: 1.318(s,3H);3.575(s,4H);4.290(d,2H);6.969(t,2H);7.870(s,4H);8.310(s,1H);8.871(s, 1H); 9.300(s,1H).

Example 13

The synthesis of compound 13, the synthetic route is as follows:

Concrete synthetic steps include:

1) Add 1.5g (22mmol) 5-bromo-2,4-dichloropyrimidine, 2g (35mmol) m-bromoaniline, 2g potassium carbonate, 20ml water and 20ml isopropanol into a 100ml three-necked flask, stir overnight at room temperature . Use a thin-layer chromatographic plate to detect the reaction. After the reaction is over, filter the solid, and wash the solid with a small amount of ethanol for 3 times, collect the solid and put it in a 100ml beaker, add 50ml of water to stir at room temperature, filter, and finally get a light yellow solid, dry the solid, and the yield More than 90.

2) Dissolve 1 g of the light yellow solid obtained in step 1) with 50 ml of dioxane, add 1.8 g of anisidine, 1.5 g of p-toluenesulfonic acid, a small amount of DMF, and stir at 100°C overnight. The reaction was detected with a thin-layer chromatographic plate. After the reaction was completed, the solid was filtered and washed with a small amount of ethyl acetate for 3 times. Spin-dry to obtain a yellow solid, add 30ml of water, and extract with 3×10ml ethyl acetate, collect the organic phase, and spin-dry to obtain a yellow solid. The developer ethyl acetate:petroleum ether=1:2 was used to pass through a silica gel column, and finally a light yellow solid was obtained, which was compound 13 of the present invention, with a yield of 45.5%. NMR spectrum: 3.824(s,3H);6.874(d,2H);7.138(t,1H);7.260(m,1H);7.374(s,2H);7.436(t,4H);8.107(d, 1H).

Example 14

The synthesis of compound 14, the synthetic route is as follows:

Concrete synthetic steps include:

Dissolve 1 g of the light yellow solid obtained in step 1) in Example 13 with 50 ml of dioxane, add 2.5 g of 3,4,5-trimethoxyaniline, 1 g of p-toluenesulfonic acid, a small amount of DMF, and stir at 50°C for reaction overnight. The reaction was detected with a thin-layer chromatographic plate. After the reaction was completed, the solid was filtered and washed with a small amount of ethyl acetate for 3 times. Spin-dry to obtain a dark yellow solid, add 20ml of water, and extract with 3×10ml ethyl acetate, collect the organic phase, and spin-dry to obtain a yellow solid. The developer ethyl acetate:petroleum ether=1:2 was used to pass through a silica gel column, and finally a yellow solid was obtained, namely compound 14 of the present invention, with a yield of 68.5%. NMR spectrum: 3.655(s,3H);3,767(d,3H);6.665(d,2H);7.192(s,1H);7.383(s,1H);8.061(s,1H).

Example 15

The hydrochloride of compound 1, the synthesis steps are as follows: Dissolve 10 mmol of compound 1 prepared in Example 1 in 10 ml of tetrahydrofuran, pass through dry hydrogen chloride, react at room temperature for 2 hours, a white solid is precipitated, and recrystallized with 20 ml of ether to obtain Pure hydrochloride of compound 1. NMR data: 6.7785(s, 1H); 7.4534(d, 2H); 7.6146(t, 2H); 7.7279(t, 1H); 7.8269(m, 5H); 7.9714(d, 2H); , 2H); 8.8671(s, 1H); 9.7563(s, 1H).

Example 16

The sulfate salt of compound 1, the synthesis steps are as follows: 10 mmol of compound 1 prepared in Example 1 is dissolved in 10 ml of ethanol, 10 mmol of sulfuric acid is added to the above solution, heated to reflux at 100 degrees Celsius until the solution is clear, and slowly lowered to room temperature, The time period from 75 degrees Celsius to room temperature was maintained for 1-2 hours, and a white solid was precipitated, which was the sulfate salt of compound 1, which was recorded as compound 16. NMR data: 6.7785 (s, 1H); 7.4534 (d, 2H); 7.6146 (t, 2H); 7.7279 (t, 1H); 7.8269 (m, 5H); 7.9714 (d, 2H); 8.1716 (d, 2H); 8.8671 (s, 1H); 9.7563 (s, 1H).

Example 17

The hydrochloride salt of compound 2, the synthesis steps are as follows: Dissolve 10 mmol of compound 2 prepared in Example 2 in 10 ml of tetrahydrofuran, pass through dry hydrogen chloride, react at room temperature for 2 hours, a white solid precipitates, and recrystallize with 20 ml of ether to obtain compound 2's pure hydrochloride.

Example 18

The sulfate salt of compound 2, the synthesis steps are as follows: 10 mmol of compound 2 prepared in Example 2 is dissolved in 15 ml of ethanol, 10 mmol of sulfuric acid is added to the above solution, heated to reflux at 100 degrees Celsius until the solution is clear, and slowly lowered to room temperature, The time period from 75 degrees Celsius to room temperature is maintained for 1-1.5 hours, and a white solid is precipitated, which is the sulfate of compound 2.

Cytotoxicity test

experimental method:

HepG2 cells in good growth state and in logarithmic growth phase were spread in 96-well plates and divided into PBS negative control group and drug-dosed group. The dosing group included 12 different dosage groups (0.1, 0.2, 0.5, 1, 2, 5, 10, 20, 50, 100, 200, 500umol/L), and each group had 4 replicate wells. After the experimental intervention, continue to culture for 24 hours in a cell incubator with a volume fraction of 5% CO ₂ and a constant humidity of 37°C, discard the old culture medium, add 100 μl sterile PBS and 10 μl CCK-8 reagent to each well, and shake gently for 10 minutes , placed in a 37°C incubator to continue culturing for 4 hours, then discarded the medium, observed the cell growth state under a microscope, and finally detected the UV absorbance value of each well at 450nm with a microplate reader to detect the changes in cell viability after intervention factors were given. Calculate the cell survival rate according to the formula: cell survival rate (T/C%)=(OD of intervention group/OD of non-intervention cell group)×100.

Summary of experimental results:

Among the 14 compounds synthesized by the present invention, and the inhibitory effect of 5 compounds on the growth of HepG2 liver cancer cells has been measured, 4 compounds have better cytotoxic effect: No. 1 compound has a better effect on HepG2 liver cancer cells. Strong growth inhibitory effect, when the concentration is 1umol, it can inhibit the growth of more than 80% of the cells. The data at the concentration of 5umol may cause errors in the experimental results due to other reasons such as operation (as shown in Figure 1); No. 3 compound at a concentration of 10umol It has a strong growth inhibitory effect on HepG2 liver cancer cells (as shown in Figure 2); Compound No. 5 has a significant inhibitory effect on HepG2 liver cancer cells at 2umol, and can inhibit the growth of about 40% of the cells, and 5umol can inhibit more than 90% of the cells growth (as shown in Figure 3); compound No. 13 has a growth inhibitory effect on cells in the range of 5-50umol, and has a good dose-effect relationship (as shown in Figure 4).

Claims (2)

1. A pyrimidine compound is selected from any one of the following compounds:

compound 4, structure as follows:

compound 5, structure is as follows:

compound 6, structure is as follows:

compound 7, structure as follows:

compound 8, structure as follows:

compound 9, structure as follows:

compound 10, structure as follows:

compound 11, structure as follows:

2 -->

compound 12, structure as follows:

compound 13, structure as follows:

or,

compound 14, structure as follows:

2. an addition salt of a pyrimidine compound of claim 1 with an acid, wherein: the acid includes hydrochloric acid, hydrobromic acid, sulfuric acid, citric acid, tartaric acid, lactic acid, pyruvic acid, acetic acid, maleic acid, methanesulfonic acid, p-toluenesulfonic acid or arginine.