CN110003177B - Benzimidazole compound containing carbamido and application thereof - Google Patents

Abstract

The invention belongs to the technical field of medicines, and relates to a benzimidazole compound containing carbamido, a preparation method thereof and application thereof in preparing anti-tumor medicines. The structural general formulas of the ureido-containing benzimidazole compound, the prodrug thereof, the pharmaceutically active metabolite thereof and the pharmaceutically acceptable salt thereof are as follows: r may be selected from hydrogen, phenyl, 4-chlorophenyl, 4-methylphenyl, 3- (trifluoromethyl) phenyl. R₃、R₅Can be selected from hydrogen and methyl. The compound of the invention has simple and convenient synthesis method, is suitable for industrial production, and is a novel anti-tumor medicament as shown in a biological activity test.

Description

Benzimidazole compounds containing urea group and their applications

technical field

The invention belongs to the technical field of medicine, relates to a benzimidazole compound containing urea group and a preparation method thereof, and also relates to a BRaf kinase, vascular endothelial growth factor receptor-2 (Vascular endothelial growth factor receptor-2, VEGFR-2), Application of platelet-derived growth factor receptor-β (Platelet-derived growth factor receptor-β, PDGFR-β) and T-LAK cell-originated protein kinase (T-LAK cell-originated proteinkinase, TOPK) inhibitors.

Background technique

In recent years, with the continuous elucidation of tumor pathogenesis and the continuous discovery of anti-tumor targets, multi-target inhibition of tumor signal transduction has become an important direction for tumor drug development. Compared with single-target drugs and the combination of multiple single-target drugs, multi-target drugs have more advantages: it can avoid drug interactions, reduce adverse reactions, and have comprehensive therapeutic effects. Among various molecular targets, protein tyrosine kinase (PTK) is one of the most effective and promising antitumor drug targets.

PTKs can be divided into receptor PTKs and non-receptor PTKs according to their structures. Receptor PTKs typically have an extracellular domain that can bind to specific ligands, a transmembrane domain, and an intracellular kinase domain that can selectively bind to and phosphorylate substrates. When the corresponding specific ligand binds to the extracellular domain of the receptor PTK, the cellular structure of the receptor PTK changes to form a dimer, and then the intracellular kinase domain binds to ATP for phosphorylation, resulting in a series of enzymatic catalysis. . Common receptor PTKs are: (1) epidermal growth factor receptor (EGFR; ErbB-1; HER1) family, including ErbB-1 (EGFR), ErbB-2 (HER-2), ErbB- 3 (HER-3) and ErbB-4 (HER-4), these receptors are highly expressed in epithelial cell tumors; (2) Platelet derived growth factor receptor (PDGFR) family, including PDGFRα, PDGFRβ , colony-stimulating factor-1 receptor (CSF-1R) and stem cell growth factor receptor (SCFR; C-Kit), PDGF can act on endothelial cells and stromal cells through a variety of effects, Promote blood vessel formation; (3) Insulin receptor (IR) family, including insulin receptor, insulin-like growth factor receptor (IGFR) and insulin related receptor (IRR) , such receptors are often highly expressed in hematological tumors; (4) the vascular endothelial growth factor receptor (VEGFR) family, including VEGFR-1 (FLT-1), VEGFR-2 (FLK- 1) and VEGFR-3 (FLT-4), VEGF is overexpressed in many tumor tissues, such as liver cancer, lung cancer, breast cancer, etc., and plays a key role in tumor angiogenesis; (5) Fibroblast growth factor is affected by The fibroblast growth factor receptor (FGFR) family, including FGFR-1, FGFR-2, FGFR-3 and FGFR-4, is a pleiotropic growth factor that regulates cell division, proliferation, migration and differentiation. Receptors play an important role in angiogenesis. In addition to the above common receptors PTK, there are also the orginal myosinreceptor kinase (TRK) family, the hepatocyte growth factor receptor (HGFR) family and the leukocyte tyrosine kinase (1eukocyte tyrosine kinase) family. tyrosine kinase, LTK) family, etc., they also play an important role in tumor cell signaling, metastasis and angiogenesis.

Non-receptor PTKs generally have no extracellular structure. They are usually located in the cytoplasm continuously or temporarily, or bind to transmembrane receptors on the inside of the cell membrane, so they are also called cytoplasmic PTKs. And other signaling pathways perform signal transduction functions, mainly including families such as SRC, ABL, JAK, ACK, CSK, FAK, FRK, TEC and SYK.

At present, the listed multi-target tyrosine kinase inhibitors mainly include:

Imatinib mesylate (Gleevec): Imatinib mesylate belongs to the class of 2-phenylaminopyrimidine compounds and is a highly specific tyrosine kinase inhibitor. It can selectively inhibit tyrosine kinases such as bcr-abl, C-kit and platelet-derived growth factor receptor (PDGFR), and its anti-tumor mechanism is as an ATP competitive inhibitor. It blocks the phosphorylation of tyrosine kinases , inhibit bcr-abl expression. Thereby preventing cell proliferation and tumor formation.

Sorafenib (sorafenib, Nexavar): Sorafenib is the first oral multi-target tyrosine kinase inhibitor, the structure is a biaryl urea compound. The tosylate salt of sorafenib is used clinically. It has dual anti-tumor effects. On the one hand, it directly inhibits tumor growth by inhibiting the RAF/MEK/ERK signal transduction pathway, and on the other hand, it blocks the formation of tumor angiogenesis by inhibiting vascular endothelial growth factor and platelet-derived growth factor receptors, and cuts off the nutrient supply to tumor cells. achieve the purpose of suppressing tumors.

Sunitinib (sunitinib, Sutent): Sunitinib is one of the targeted anti-tumor drugs with the most known targets and has broad-spectrum anti-tumor activity. It is an inhibitor of vascular endothelial growth factor receptor, platelet-derived growth factor receptor, stem cell factor receptor, tyrosine kinase, etc.

Lapatinib (Tykerb): Lapatinib is a dual-target tyrosine kinase inhibitor against EGFR/ErbB-2. Lapatinib is a 4-anilinoquinazoline kinase inhibitor in the form of tosylate salt hydrate. It is a reversible multi-targeted tyrosine kinase inhibitor. Its mechanism of action is to inhibit the ATP site of EGFR(ErbB-1)/HERE(ErbB-2) in cells, preventing tumor cell phosphorylation and activation, and through the synergy of EGFR(ErbB-1)/HERE(ErbB-2). Plasma and heterodimers block down-regulating signals, thereby inhibiting the proliferation of breast cancer cells and inducing apoptosis to achieve the purpose of anti-tumor.

Nilotinib (Tasigna): Nilotinib is an anilidine derivative, and its mechanism of action is to selectively inhibit the tyrosine kinase Bcr-Abl and inhibit the stem cell factor receptor Kit and platelet-derived growth factor receptors. Antibody (PDGFR) kinase has an antagonistic effect.

Dasatinib (Sprycel): Dasatinib is a dual inhibitor of tinib ABL and SRC kinases.

Pazopanib (pazopanib, votrient): Pazopanib is a selective multi-target TKI developed by GlaxoSmithKline, UK. All kinds of PTK have inhibitory effect.

The research and development of multi-target tyrosine kinase inhibitor drugs is one of the main research hotspots of anti-tumor drugs. The discovery of such drugs has brought new options and light to the treatment of tumor patients, thus opening up a new field of anti-tumor drug research. Multi-targeted tyrosine kinase inhibitors are specific and effective compared to traditional antitumor drugs. At the same time, it has the advantages of less gastrointestinal reactions and hematological adverse reactions, so it has a broader development prospect.

In addition, recent studies have shown that TOPK is a novel serine-threonine protein kinase identified from the cDNA library of lymphokine-activated killer T cells (T-LAK cells), Scientists found a kinase PBK (PDZ-bindingkinase, PBK) that can bind to the PDZ2 region of hDig in the HeLa cell cDNA library is the same molecule as TOPK, so it is also called PBK/TOPK. T-cell-originatedprotein kinase (TOPK) is highly expressed in many malignant tumors, such as lung cancer, breast cancer, lymphoma, bladder cancer, colon cancer, gastric cancer, liver cancer, pancreatic cancer, prostate cancer, ovarian cancer, etc., and TOPK The degree of expression is related to tumor prognosis, especially in breast cancer and lung cancer. The higher the TOPK expression, the worse the prognosis, but the TOPK expression level in normal tissues is very low, and it is only expressed in the testis and thymus. Therefore, TOPK has become a new target for tumor treatment, and there is currently no drug specifically targeting this target. Therefore, inhibitors that can specifically bind to TOPK have become a research hotspot for new anti-tumor drugs.

SUMMARY OF THE INVENTION

The technical problem to be solved by the present invention is to provide a compound as shown in formula I, its prodrugs and pharmaceutically active metabolites and pharmaceutically acceptable salts thereof, and provide its preparation for prevention and treatment and BRaf kinase, Vascular endothelial growth factor receptor-2 (VEGFR-2), Platelet-derived growth factor receptors-β (PDGFR-β) and T-LAK cell source Application of drugs in diseases related to T-LAK cell-originated protein kinase (TOPK) dysregulation.

in,

R may be selected from hydrogen, phenyl, halogen substituted phenyl, C1-C4 alkyl substituted phenyl, halogen substituted C1-C4 alkyl substituted phenyl.

R ₃ and R ₅ can be selected from H, C1-C4 alkyl.

Further, the present invention preferably has the following compounds of formula I and pharmaceutically acceptable salts thereof:

R may be selected from hydrogen, phenyl, 4-chlorophenyl, 4-methylphenyl, 3-(trifluoromethyl)phenyl.

R ₃ and R ₅ can be selected from hydrogen and methyl.

Further, the present invention preferably the following compounds:

1-{3-{[2-{[(1H-benzimidazol-2-yl)thio]methyl}-3-methyl-pyridin-4-yl}oxy}propyl}-3-benzene base urea;

1-{3-{[2-{[(1H-benzimidazol-2-yl)thio]methyl}-3-methyl-pyridin-4-yl}oxy}propyl}-3-( 4-chlorophenyl)urea;

1-{3-{[2-{[(1H-benzimidazol-2-yl)thio]methyl}-3-methyl-pyridin-4-yl}oxy}propyl}-3-[ 3-(trifluoromethyl)phenyl]urea;

1-{3-{{2-{[(1H-benzimidazol-2-yl)thio]methyl}-3-methyl-pyridin-4-yl}oxy}propyl}-3-( p-tolyl) urea;

"Pharmaceutically acceptable salt" refers to conventional acid or base addition salts formed with suitable non-toxic organic or inorganic acids or organic or inorganic bases that retain the biological potency and properties of the compounds of formula I. Examples of acid addition salts include malate, maleate, sulfamate, hydrochloride, acetate, adipate, alginate, aspartate, benzyl acid salt, benzenesulfonate, p-toluenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, cypionate, digluconate, dodecyl Sulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptanoate, caproate, hydrochlorate, hydrobromide, hydroiodate, 2-Hydroxyethanesulfonate, Lactate, Maleate, Mesylate, 2-Naphthalenesulfonate, Nicotinate, Nitrate, Oxalate, Pamoate, Pectinate, Persulfate, 3-phenylpropionate, picrate, pivalate, propionate, succinate, sulfate, tartrate, thiocyanate, tosylate and undecanoate, etc. . Base salts include ammonium salts, alkali metal salts such as sodium and potassium salts, alkaline earth metal salts such as calcium and magnesium salts, salts of organic bases such as dicyclohexylamine salts, N-methyl-D-glucosamine salts, and salts of amino acids such as arginine, lysine, etc. Also, basic nitrogen-containing groups can be quaternized with reagents such as lower alkyl halides such as methyl, ethyl, propyl and butyl chlorine, bromine and iodide groups; dialkyl sulfates such as dimethyl, diethyl, dibutyl and dipentyl sulfate; long chain halides such as decyl, lauryl, myristyl and hard Chlorine, bromine and iodide of fatty acyl groups; halides of aralkyl groups, such as benzyl and phenethyl bromides, etc. Preferred acids for forming acid addition salts include hydrochloric acid and acetic acid.

"Pharmaceutically acceptable" such as pharmaceutically acceptable carriers, excipients, prodrugs, etc., means pharmacologically acceptable and substantially non-toxic to the patient to whom the particular compound is administered.

"Pharmaceutically active metabolite" refers to a metabolite of a compound of Formula I that is pharmaceutically acceptable and effective.

The present invention also relates to inhibition of BRaf kinase, Vascular endothelial growth factor receptor-2 (VEGFR-2), Platelet-derived growth factor receptors-β (PDGFR-β) and a pharmaceutical composition of T-LAK cell-derived protein kinase, the composition contains a compound of formula I or a derivative or a pharmaceutically acceptable acid addition salt thereof and a pharmaceutically acceptable carrier.

The term "halogen" as used herein includes fluorine, chlorine, bromine or iodine.

The compounds of the present invention can be administered to a patient by a variety of methods, such as orally in capsules or tablets, as sterile solutions or suspensions, and in some cases intravenously as solutions. The free base compounds of the present invention can be formulated and administered in the form of their pharmaceutically acceptable acid addition salts.

The compounds described in the present invention serve as novel structural types of BRaf kinases, Vascular endothelial growth factor receptor-2 (VEGFR-2) and Platelet-derived growth factor receptors (Platelet-derived growth factor receptors). -β, PDGFR-β) and T-LAK cell-originated protein kinase (TOPK) inhibitors, with novel structural types and the ability to act on multiple targets, can be used for treatment or Prevention and BRaf kinase, vascular endothelial growth factor receptor-2 (Vascularendothelial growth factor receptor-2, VEGFR-2) and platelet-derived growth factor receptor-β (Platelet-derived growth factor receptors-β, PDGFR-β) and T -LAK cell-originated protein kinase (TOPK)-related tumor diseases such as small cell lung cancer, squamous cell carcinoma, adenocarcinoma, large cell carcinoma, colorectal cancer, breast cancer, ovarian cancer, renal cell carcinoma , has good application value and development and application prospects.

Detailed ways

The schemes outline the preparative steps for the preparation of compounds of the present invention.

Among them, R, R ₃ and R ₅ are as described above.

The present invention is described in detail by the following examples. It should be understood, however, that the present invention is not limited to the specifically recited examples below.

Example 1: 1-{3-{[2-{[(1H-benzimidazol-2-yl)sulfanyl]methyl}-3-methyl-pyridin-4-yl}oxy}propyl} - Preparation of 3-phenylurea (compound X01)

Step A: Preparation of 2-{[4-(3-Methoxypropoxy)-3-methylpyridin-2-yl]methylthio}-1H-benzimidazole

2-Chloromethyl-4-(3-methoxypropoxy)-3-methylpyridine hydrochloride (0.50 g, 1.88 mmol) was placed in a 125 ml eggplant-shaped flask, and 11 ml of ethanol was added to dissolve it, 1H-benzimidazole-2-thiophenol (0.28 g, 1.88 mmol) and 4 ml NaOH (80 g/L) were added, and the mixture was refluxed at 68° C. for 4 h, and the reaction was completed by TLC monitoring. The reaction solution was poured into a 100-ml beaker, cooled to room temperature naturally, and a white solid was precipitated, which was recrystallized from ethyl acetate and petroleum ether (2:1) to obtain 0.58 g of white needle-like crystals with a yield of 89.3%. m.p.: 115-118°C (literature value: 116-118°C).

Step B: Preparation of 3-{{2-{[(1H-benzimidazol-2-yl)thio]methyl}-3-methylpyridin-4-yl}oxy}propan-1-ol

2-{[4-(3-Methoxypropoxy)-3-methylpyridin-2-yl]methylthio}-1H-benzimidazole (3.00 g, 9.12 mmol) was placed in 100 ml eggplant Into the bottle, add 30 ml of dichloromethane to dissolve it, slowly add boron tribromide (6.80 g, 27.2 mmol) dropwise in an ice-water bath, naturally return to room temperature, react for 5 hours, and monitor the completion of the reaction by TLC. 20 ml of ice water was added to quench the reaction, the pH was adjusted to 8 with saturated aqueous sodium bicarbonate solution, and 2.56 g of white solid was obtained by suction filtration, with a yield of 85.3%. m.p.: 97.0～99.2℃. It was used directly in the next reaction without purification.

Step C: Preparation of 3-{{2-{[(1H-benzimidazol-2-yl)sulfanyl]methyl}-3-methyl-pyridin-4-yl}oxy}propan-1-amine

3-{{2-{[(1H-benzimidazol-2-yl)sulfanyl]methyl}-3-methylpyridin-4-yl}oxy}propan-1-ol (0.5 g, 1.52 mmol) was dissolved in 20 mL of dichloromethane, and 5 mL of thionyl chloride was added dropwise at room temperature. After the addition was completed, the reaction was heated under reflux for 3 h. After the reaction was complete, the remaining thionyl chloride and dichloromethane were evaporated under reduced pressure. The compound was dissolved in 20 mL of DMF, anhydrous potassium carbonate (0.63 g, 4.56 mmol) and potassium phthalimide (0.28 g, 1.52 mmol) were added, and the reaction was stirred at 90 °C for 3 h. After the reaction was completed, the mixture was evaporated under reduced pressure. DMF was removed, the residue was washed with water, filtered with suction, the filter cake was dissolved in 30 mL of absolute ethanol, hydrazine hydrate (5 mL) was added, and the reaction was heated under reflux for 6 h. Methane extraction (20 mL×2 times), combined organic phases, washed with water, washed with saturated brine, dried over anhydrous magnesium sulfate, suction filtered, evaporated to remove the solvent, and the residue was purified by column chromatography (dichloromethane:methanol=60:1) to obtain White solid 0.25g, yield 50.2%.

Step D: 1-{3-{[2-{[(1H-benzimidazol-2-yl)sulfanyl]methyl}-3-methyl-pyridin-4-yl}oxy}propyl}- Preparation of 3-phenylurea (compound X01)

Phenyl isocyanate (0.12 g, 1 mmol) was dissolved in 10 mL of dichloromethane solution, and 3-{{2-{[(1H-benzimidazol-2-yl)sulfanyl]methyl}-3 was added dropwise at room temperature -Methyl-pyridin-4-yl}oxy}propan-1-amine (0.33g, 1mmol) in dichloromethane solution (10mL), after the dropwise addition was completed, the reaction was continued to stir for 3h, after the reaction was complete, 30mL of water was added, Stir for 5 min, separate the layers, wash the organic phase with saturated sodium chloride solution, dry with anhydrous magnesium sulfate, suction filter, concentrate the filtrate, and purify the residue by column chromatography (dichloromethane:methanol=60:1) to obtain a white solid of 0.39 g, yield 86.7%. mp: 104.1-105.5°C; ESI-MS: m/z 448.6[M+H] ⁺ , 470.4[M+Na] ⁺ ; ¹ H NMR (400MHz, DMSO-d ₆ ) δ 12.63 (s, 1H) ,8.41(s,1H),8.25(d,J=5.6Hz,1H),7.56-7.33(m,4H),7.25-7.16(m,2H),7.16-7.09(m,2H),6.97(d , J＝5.7Hz, 1H), 6.92–6.83(m, 1H), 6.25(t, J＝5.8Hz, 1H), 4.70(s, 2H), 4.11(t, J＝6.1Hz, 2H), 3.27 (q,J=6.4Hz,2H),2.24(s,3H),1.93(p,J=6.4Hz,2H).

Example 2: 1-{3-{[2-{[(1H-benzimidazol-2-yl)sulfanyl]methyl}-3-methyl-pyridin-4-yl}oxy}propyl} - Preparation of 3-(4-chlorophenyl)urea (Compound X02)

Referring to the preparation method of Example 01, 0.32 g of white solid was obtained, yield 76.2%, mp: 165.8-167.3°C; ESI-MS: m/z 600.7, 602.0, [M+H] ⁺ ; ¹ H NMR (400MHz, DMSO-d ₆ )δ8.23(d,J=5.6Hz,1H),7.59(dt,J=7.2,3.5Hz,1H),7.45(dd,J=6.1,3.1Hz,1H),7.39-7.32 (m, 2H), 7.23–7.12 (m, 3H), 6.97 (d, J=5.7Hz, 1H), 5.23 (s, 2H), 4.70 (s, 2H), 4.59 (t, J=5.1Hz, 1H), 4.12(t, J＝6.2Hz, 2H), 3.77(d, J＝5.0Hz, 2H), 3.62(t, J＝4.9Hz, 2H), 3.57(q, J＝5.9Hz, 2H) ,3.11(d,J=4.9Hz,2H),2.98(t,J=5.0Hz,2H),2.20(s,3H),1.89(p,J=6.2Hz,2H).

Example 3: 1-{3-{[2-{[(1H-benzimidazol-2-yl)sulfanyl]methyl}-3-methyl-pyridin-4-yl}oxy}propyl} - Preparation of 3-[3-(trifluoromethyl)phenyl]urea (Compound X03)

Referring to the preparation method of Example 01, 0.44 g of white solid was obtained, yield 83%, mp: 96.1-97.2°C; ESI-MS: m/z 516.3 [M+H] ⁺ ; ¹ H NMR (400MHz, DMSO-d ₆ ) δ12.74(s, 1H), 8.87(s, 1H), 8.26(d, J=5.6Hz, 1H), 7.96(d, J=2.0Hz, 1H), 7.53–7.39(m, 4H) ,7.21(d,J＝7.5Hz,1H),7.18–7.08(m,2H),6.98(d,J＝5.8Hz,1H),6.43(t,J＝5.8Hz,1H),4.71(s, 2H), 4.12 (t, J=6.0Hz, 2H), 3.31–3.25 (m, 2H), 2.24 (s, 3H), 1.95 (p, J=6.4Hz, 2H).

Example 4: 1-{3-{{2-{[(1H-benzimidazol-2-yl)sulfanyl]methyl}-3-methyl-pyridin-4-yl}oxy}propyl} - Preparation of 3-(p-tolyl)urea (compound X04)

Referring to the preparation method of Example 01, 0.32 g of white solid was obtained, yield 84.1%, mp: 103.2-104.3°C; ESI-MS: m/z 462.3 [M+H] ⁺ ; ¹ H NMR (400 MHz, DMSO-d ₆ ) δ8.23 (d, J=5.6Hz, 1H), 7.59 (dt, J=7.2, 3.5Hz, 1H), 7.45 (dd, J=6.1, 3.1Hz, 1H), 7.39–7.32 (m, 2H), 7.23–7.12(m, 3H), 6.97(d, J=5.7Hz, 1H), 5.23(s, 2H), 4.70(s, 2H), 4.59(t, J=5.1Hz, 1H), 4.12(t,J＝6.2Hz,2H),3.77(d,J＝5.0Hz,2H),3.62(t,J＝4.9Hz,2H),3.57(q,J＝5.9Hz,2H),3.11( d, J=4.9Hz, 2H), 2.98 (t, J=5.0Hz, 2H), 2.20 (s, 3H), 1.89 (p, J=6.2Hz2H).

The above are only preferred embodiments of the present invention, and are not intended to limit the present invention in other forms. Any person skilled in the art may use the technical content disclosed above to make changes or modifications to equivalent changes. Example. However, any simple modifications, equivalent changes and modifications made to the above embodiments according to the technical essence of the present invention without departing from the content of the technical solutions of the present invention still belong to the protection scope of the technical solutions of the present invention.

Pharmacological Example

Example 5: Inhibitory activity of test compounds on the proliferation of A549, HCT-116 and PC3 cells

(1) Experimental materials

Cell lines: A549, HCT-116, PC3 cells, plated in 96-well plates at a density of 3000/well, 100ul per well, used after 24h.

Target compounds numbered X01 to X04: dissolved in DMSO, diluted with culture medium to prepare five different concentrations of 50 μM, 20 μM, 10 μM, 5 μM, 2 μM and stored at -20°C for later use. The final concentration of DMSO in culture medium is less than 0.1% .

Positive control drug: sorafenib.

MTT: dissolve in PBS to 5 mg/mL and store at -20°C.

(2) Experimental method

Using MTT method, A549, HCT-116 and PC3 cells were selected to evaluate the anti-tumor proliferation activity of the test samples. Cell lines were grown on RPMI 1640 medium containing 10% fetal bovine serum (FBS). Cells were pooled when they had proliferated to 80-90% and then subcultured for no more than 20 passages, then they were acclimated for 24 h before further treatment. The cells were plated in 96-well plates ( ⁸ x 104/mL) and then cultured overnight in a humidified environment containing 5% _CO2 and temperature-controlled at 37°C. After 24 h, various concentrations of representative compounds of the invention were added. After another 24 h of cultivation, MTT (5 mg/mL) was added thereto and the cultivation was continued for 4 h. The culture substrate was removed, the crystals were dissolved in DMSO, and the absorbance was measured at a wavelength of 490 nm using a microplate reader (TECANSPECTRA, WetDar, Germany). According to the formula: cell growth inhibition rate=(1-OD value of drug group/OD value of control group)×100%, calculate the cell growth inhibition rate at the corresponding concentration, and take the different concentrations of the test compound and the inhibition rate to cells as the logarithm Curve, calculate the corresponding _IC50 value of the test compound. Representative compounds of the present invention were determined according to the methods described above, and the results are shown in Table 1:

Table 1

Example 6: Inhibitory activity of test compounds on BRaf, VEGFR-2, PDGFR-β and TOPK kinases

(1) Experimental materials

Kinases: BRaf kinase (wild type), VEGFR-2, PDGFR-beta and TOPK kinases.

Target compounds numbered X01 to X04: dissolve in DMSO, and do the same treatment for the reference substance.

Positive control drug: sorafenib.

Kinase Buffer: Contains 50 mM HEPES, pH 7.5, 10 mM MgCl2, 0.0015% Brij-35 and 2 mM DTT.

Stop Buffer: Contains 100 mM HEPES, pH 7.5, 0.015% Brij-35, 0.2% Coating Reagent #3 and 50 mM ethylenediaminetetraacetic acid (EDTA).

(2) Experimental method

All kinase assays were performed in 50 μL reaction volumes in 96-well plates. Compounds were diluted to 500 [mu]M with DMSO, then 10 [mu]L of compounds were transferred to a new 96-well plate as an intermediate plate, and 90 [mu]L of kinase buffer was added to each well. Transfer 5 µl of each well of the intermediate plate to a 384-well plate. Each well contains the following enzymes and substrates: kinase base buffer, FAM-labeled peptide, ATP and enzyme solution. DMSO wells with substrate, enzyme and no compound served as DMSO controls. Wells containing only substrate without enzyme were used as low controls. Compounds were incubated at room temperature for 10 minutes. Add 10 μL of peptide solution to each well and incubate at 28 °C for the indicated period of time and stop the reaction with 25 μL of stop buffer. Finally, data were collected using the Caliper program, which converts measured data values into inhibition rates.

Inhibition rate (%)=(max-conversion)/(max-min)×100, where “max” represents DMSO control; “min” represents low control.

Representative compounds of the present invention were determined according to the methods described above, and the results are shown in Table 2:

Table 2

Formulation Example

The following formulation examples merely illustrate the scope of protection of the present invention and are not intended to be limiting in any way.

Example 7: Gelatin capsules

Hard gelatin capsules were prepared using:

The above formulations can be modified with reasonable variations provided.

Example 8: Tablets

Tablets were prepared using:

The above components are mixed and compressed into tablets.

Example 9: Tablets

Tablets containing 2.5-1000 mg of active ingredient per tablet are prepared as follows:

The active ingredient, starch and cellulose are passed through a No. 45 mesh U.S. sieve and mixed thoroughly. The polyvinylpyrrolidone solution was mixed with the resulting powder and passed through a No. 14 mesh US sieve. The resulting granules were dried at 50-60°C and passed through a No. 18 mesh US sieve. Sodium carboxymethylcellulose, magnesium stearate and talc, previously passed through a No. 60 mesh U.S. sieve, were added to the granules, followed by mixing and compression on a tablet machine to obtain tablets.

Example 10: Combination Tablets

The active ingredient, starch and cellulose are passed through a No. 45 mesh U.S. sieve and mixed thoroughly. The polyvinylpyrrolidone solution was mixed with the resulting powder and passed through a No. 14 mesh US sieve. The resulting granules were dried at 50-60°C and passed through a No. 18 mesh US sieve. Sodium carboxymethylcellulose, magnesium stearate and talc, previously passed through a No. 60 mesh U.S. sieve, were added to the granules, followed by mixing and compression on a tablet machine to obtain tablets.

From the above description, those skilled in the art can easily understand the essential features of the present invention, and without departing from the spirit and scope of the present invention, various changes and modifications can be made to the present invention to adapt to different applications and conditions.

Claims (10)

1. A compound of formula I, and pharmaceutically acceptable salts of the foregoing compounds:

in, R is selected from hydrogen, phenyl, halogen substituted phenyl, C1-C4 alkyl substituted phenyl, halogen substituted C1-C4 alkyl substituted phenyl; R ₃ and R ₅ are selected from H, C1-C4 alkyl. 2. The compound of formula I according to claim 1, and the pharmaceutically acceptable salts of said compounds: in, R is selected from hydrogen, phenyl, 4-chlorophenyl, 4-methylphenyl, 3-(trifluoromethyl)phenyl. 3. The compound of formula I according to claim 1, and the pharmaceutically acceptable salts of said compounds: in, R ₃ and R ₅ are selected from hydrogen and methyl. 4. The compound of claim 1, and a pharmaceutically acceptable salt, selected from the group consisting of: 1-{3-{{2-{[( 1H -benzimidazol-2-yl)thio]methyl}-3-methylpyridin-4-yl}oxy}propyl}-3-benzene base urea; 1-{3-{{2-{[( 1H -benzimidazol-2-yl)thio]methyl}-3-methylpyridin-4-yl}oxy}propyl}-3-( 4-chlorophenyl)urea; 1-{3-{{2-{[( 1H -benzimidazol-2-yl)thio]methyl}-3-methylpyridin-4-yl}oxy}propyl}-3-[ 3-(trifluoromethyl)phenyl]urea; 1-{3-{{2-{[( 1H -benzimidazol-2-yl)thio]methyl}-3-methylpyridin-4-yl}oxy}propyl}-3-( p-tolyl) urea. 5. A pharmaceutical composition comprising, as an active ingredient, the compound of any one of claims 1-4, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or diluent. 6. the preparation method of the compound of claim 1, its flow process is as follows:

wherein R, R ₃ and R ₅ are as described in claim 1 . 7. Use of the compound according to any one of claims 1 to 4, and a pharmaceutically acceptable salt or the pharmaceutical composition according to claim 5 in the preparation of a medicament for treating tumors. 8. The compound of any one of claims 1-4, and a pharmaceutically acceptable salt or pharmaceutical composition in the preparation of BRaf kinase, vascular endothelial growth factor receptor-2, platelet-derived growth factor receptor-beta or T - Application of LAK cell-derived protein kinase inhibitors. 9. The compound of any one of claims 1-4, and a pharmaceutically acceptable salt or pharmaceutical composition prepared with BRaf kinase, vascular endothelial growth factor receptor-2, platelet-derived growth factor receptor-beta or Application of T-LAK cell-derived protein kinase dysregulation-related disease drugs. 10. The use according to claim 9, wherein the disease related to the deregulation of BRaf kinase, vascular endothelial growth factor receptor-2, platelet-derived growth factor receptor-beta or T-LAK cell-derived protein kinase is lung cancer, liver cancer , melanoma, colon cancer, rectal cancer, breast cancer, ovarian cancer, kidney cancer.