CN110003177B

CN110003177B - Benzimidazole compound containing carbamido and application thereof

Info

Publication number: CN110003177B
Application number: CN201910455053.5A
Authority: CN
Inventors: 胡春; 王旭康; 张传明; 谭孝雨; 曹小龙; 张春旺; 徐文; 李凤荣
Original assignee: Shenyang Pharmaceutical University
Current assignee: Shenyang Pharmaceutical University
Priority date: 2019-05-28
Filing date: 2019-05-28
Publication date: 2021-11-19
Anticipated expiration: 2039-05-28
Also published as: CN110003177A

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 compound containing carbamido and application thereof

Technical Field

The invention belongs to the technical field of medicines, relates to an ureido-containing benzimidazole compound and a preparation method thereof, and also relates to application of the benzimidazole compound serving as inhibitors of BRaf kinase, Vascular endothelial growth factor receptor-2 (VEGFR-2), Platelet-derived growth factor receptor-beta (PDGFR-beta) and T-LAK cell-derived protein kinase (TOPK).

Background

In recent years, with the continuous elucidation of tumor pathogenesis and the continuous discovery of anti-tumor action targets, the multi-target inhibition of tumor signal transduction becomes an important direction for the development of tumor drugs. Compared with the combination of single-target drugs and a plurality of single-target drugs, the multi-target drug has more superiority: can avoid the generation of drug interaction, reduce adverse reaction, have comprehensive treatment effect and the like. Among various molecular targets, Protein Tyrosine Kinase (PTK) is one of the antitumor drug targets with obvious effect and wide prospect.

PTKs can be classified into receptor PTKs and non-receptor PTKs according to their structure. Receptor PTKs typically have an extracellular domain that binds to a particular ligand, a transmembrane domain, and an intracellular kinase domain that selectively binds to and phosphorylates a substrate. When the corresponding specific ligand is combined with the extracellular domain of the receptor PTK, the cellular structure of the receptor PTK is changed to form a dimer, and then the intracellular kinase region is combined with ATP to be phosphorylated, thereby generating a series of enzyme catalysis effects. Common receptor PTKs are: (1) the 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), is highly expressed in epithelial cell tumors; (2) the PDGF family comprises PDGFR alpha, PDGFR beta, colony stimulating factor-1 receptor (CSF-1R) and stem cell growth factor receptor (SCFR; C-Kit), and the PDGF can act on endothelial cells and interstitial cells through various effects to promote angiogenesis; (3) the Insulin Receptor (IR) family, including insulin receptor, insulin-like growth factor receptor (IGFR) and Insulin Related Receptor (IRR), which are often highly expressed in hematological tumors; (4) VEGFR family including VEGFR-1(FLT-1), VEGFR-2(FLK-1) and VEGFR-3(FLT-4), VEGF is over-expressed in many tumor tissues, such as liver cancer, lung cancer, breast cancer, etc., and plays a key role in tumor angiogenesis; (5) the Fibroblast Growth Factor Receptor (FGFR) family comprises FGFR-1, FGFR-2, FGFR-3 and FGFR-4, the FGFR is a pleiotropic growth factor capable of regulating cell division, proliferation, migration and differentiation, and the receptors play an important role in angiogenesis. In addition to the above conventional PTKs, there are the Tropomyosin Receptor Kinase (TRK) family, the Hepatocyte Growth Factor Receptor (HGFR) family, and the Leukocyte Tyrosine Kinase (LTK) family, which also play important roles in signaling, metastasis, angiogenesis, and the like of tumor cells.

Non-receptor PTKs generally have no extracellular structure, they are usually located in the cytosol, either continuously or transiently, or bind to transmembrane receptors inside the cell membrane, and are therefore also referred to as cytosolic PTKs, which perform signal transduction functions through cytokine receptors, T cell receptors and other signaling pathways, mainly including the SRC, ABL, JAK, ACK, CSK, FAK, FRK, TEC and SYK families.

Currently, the multi-target tyrosine kinase inhibitors that have been marketed are mainly:

imatinib mesylate (Gleevec): imatinib mesylate belongs to 2-phenyl aminopyrimidine compounds and is a tyrosine kinase inhibitor with strong specificity. It can selectively inhibit tyrosine kinases such as bcr-abl, C-kit and Platelet Derived Growth Factor Receptor (PDGFR), and its antitumor mechanism is used as ATP competitive inhibitor to block phosphorylation of tyrosine kinase and inhibit bcr-abl expression. Thereby preventing cell proliferation and tumor formation.

Sorafenib (sorafenib, Nexavar): sorafenib is the first oral multi-target tyrosine kinase inhibitor, and the structure of the sorafenib is a diaryl urea compound. The clinical use is the tosylate salt of sorafenib. It has dual anti-tumor effects. On one hand, the growth of the tumor is directly inhibited by inhibiting RAF/MEK/ERK signal transduction pathways, on the other hand, the formation of tumor new vessels is blocked by inhibiting vascular endothelial growth factor and platelet-derived growth factor receptors, and the nutrition supply of tumor cells is cut off, so that the aim of inhibiting the tumor is fulfilled.

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

Lapatinib (lapatinib, Tykerb): lapatinib is a dual-target tyrosine kinase inhibitor against EGFR/ErbB-2. Lapatinib is a 4-anilinoquinazoline kinase inhibitor and exists in the form of tosylate hydrate. It is a reversible, multi-target tyrosine kinase inhibitor. The action mechanism of the anti-tumor cell inhibitor is to inhibit the ATP sites of EGFR (ErbB-1)/HERE (ErbB-2) in the cells, prevent the tumor cells from phosphorylation and activation, and block down-regulation signals through homo-and heterodimers of EGFR (ErbB-1)/HERE (ErbB-2), thereby inhibiting the proliferation of breast cancer cells and inducing apoptosis so as to achieve the aim of resisting tumors.

Nilotinib (nilotinib, Tasigna): nilotinib is an aniline pyrimidine derivative, the action mechanism of the nilotinib is selective inhibition of tyrosine kinase Bcr-Abl, and the nilotinib has antagonism on stem cell factor receptor Kit and Platelet Derived Growth Factor Receptor (PDGFR) kinase.

Dasatinib (dasatinib, Sprycel): dasatinib is a dual inhibitor of the tinib class ABL and SRC kinase.

Pazopanib (pazopanib, votrient): pazopanib is a selective multi-target TKI developed by GlaxoSmithKline in UK, and has inhibitory effect on multiple PTKs such as PDGFR alpha, PDGFR beta, VEGFR-1, VEGFR-2, VEGFR-3 and C-Kit.

Research and development of multi-target tyrosine kinase inhibitor drugs are one of the main research hotspots of the current anti-tumor drugs. The discovery of the drugs brings new selection and brightness for the treatment of tumor patients, thereby opening up a new place for the research of anti-tumor drugs. The multi-target tyrosine kinase inhibitor has specificity and effectiveness compared with the traditional antitumor drugs. Meanwhile, the medicine has the advantages of less gastrointestinal reaction and hematology adverse reaction and the like, so that the medicine has a wider development prospect.

In addition, recent studies have shown that TOPK is a novel serine-threonine protein kinase identified from cDNA libraries of activated lymphokine-activated killer T cells (T-LAK cells), and scientists found in HeLa cell cDNA libraries that a PBK (PDZ-binding kinase, PBK) kinase that binds to the region of hDig PDZ2 as a molecule identical to TOPK, hence the name PBK/TOPK. T-cell-organized protein kinase (TOPK) is highly expressed in many malignant tumors, such as lung cancer, breast cancer, lymphoma, bladder cancer, colon cancer, stomach cancer, liver cancer, pancreatic cancer, prostate cancer, ovarian cancer and the like, and the expression degree of TOPK is related to the prognosis of tumors, particularly breast cancer and lung cancer. Therefore, TOPK becomes a new target for tumor therapy, and no drug specially aiming at the target is on the market at present, so that the inhibitor capable of being specifically combined with TOPK becomes a research hotspot of a novel anti-tumor drug.

Disclosure of Invention

The invention provides a compound shown as a formula I, a prodrug, a pharmaceutically active metabolite and a pharmaceutically acceptable salt thereof, and provides application of the compound in preparing medicaments for preventing and treating diseases related to the imbalance of BRaf kinase, Vascular endothelial growth factor receptor-2 (VEGFR-2), Platelet-derived growth factor receptor-beta (PDGFR-beta) and T-LAK cell-derived protein kinase (T-LAK l-oriented protein kinase, TOPK).

Wherein,

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

R₃、R₅Can be selected from H, C1-C4 alkyl.

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

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

R₃、R₅Can be selected from hydrogen and methyl.

Further, the following compounds are preferred in the present invention:

1- {3- { [2- { [ (1H-benzoimidazol-2-yl) thio ] methyl } -3-methyl-pyridin-4-yl } oxy } propyl } -3-phenylurea;

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

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

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

"pharmaceutically acceptable salt" refers to conventional acid addition salts or base addition salts that retain the biological potency and properties of the compounds of formula I and are formed with suitable non-toxic organic or inorganic acids or organic or inorganic bases. Examples of acid addition salts include malate, maleate, sulfanate, hydrochloride, acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, p-toluenesulfonate, hydrogensulfate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentylpropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oxalate, pamoate, pectinate, persulfate, 3-phenylpropionate, picrate, pivalate, propionate, succinate, sulfate, tartrate, thiocyanate, tosylate, undecanoate, and the like. 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 with organic bases, such as dicyclohexylamine salts, N-methyl-D-glucamine, and salts with amino acids, such as arginine, lysine, and the like, and basic nitrogen-containing groups may be quaternized with such agents as lower alkyl halides, such as methyl, ethyl, propyl, and butyl chlorides, bromides, and iodides; dialkyl sulfates such as dimethyl sulfate, diethyl sulfate, dibutyl sulfate and diamyl sulfate; long chain halides, such as decyl, lauryl, myristyl and stearyl chlorides, bromides and iodides; aralkyl halides such as benzyl and phenethyl bromides and the like. 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 a patient to whom a particular compound is administered.

"pharmaceutically active metabolite" refers to a pharmaceutically acceptable and effective metabolite of a compound of formula I.

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

The term "halogen" as used in the present invention includes fluorine, chlorine, bromine or iodine.

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

The compound of the invention is used as a novel structural type of BRaf kinase, Vascular endothelial growth factor receptor-2 (Vascular endothelial growth factor receptor-2, VEGFR-2) and Platelet-derived growth factor receptor-beta (Platelet-derived growth factor receptors-beta, PDGFR-beta) and T-LAK cell-derived protein kinase (TOPK) inhibitor, has the characteristics of novel structural type, capability of acting on a plurality of targets and the like, can be used for treating or preventing BRaf kinase, Vascular endothelial growth factor receptor-2 (Vascular endothelial growth factor receptor-2, VEGFR-2) and Platelet-derived growth factor receptor-beta (Platelet-derived growth factor receptors-beta, PDGFR-beta) and T-LAK cell-derived protein kinase (T-LAK cell-derived 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 and renal cell carcinoma, and has good application value and development and application prospect.

Detailed Description

The scheme outlines the preparation steps for preparing the compounds of the invention.

Wherein, R, R₃、R₅As previously described.

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 following examples which are specifically set forth.

Example 1: preparation of 1- {3- { [2- { [ (1H-benzimidazol-2-yl) thio ] methyl } -3-methyl-pyridin-4-yl } oxy } propyl } -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 (0.50g, 1.88mmol) was placed in a 125ml eggplant-shaped bottle, 11ml of ethanol was added thereto to dissolve it, 1H-benzimidazole-2-thiophenol (0.28g, 1.88mmol) and 4ml of NaOH (80g/L) were further added thereto, and the mixture was refluxed at 68 ℃ for 4 hours, and the completion of the reaction was monitored by TLC. The reaction solution was poured into a 100ml beaker, cooled naturally to room temperature to precipitate a white solid, and recrystallized from ethyl acetate and petroleum ether (2:1) to give 0.58g of white needle-like crystals with a yield of 89.3%. m.p. 115-118 deg.C (literature value: 116-118 deg.C).

And 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 (3.00g, 9.12mmol) was placed in a 100ml eggplant-shaped bottle, dissolved by adding 30ml of dichloromethane, slowly added boron tribromide (6.80g, 27.2mmol) dropwise in an ice-water bath, allowed to return to room temperature naturally, reacted for 5H, and monitored by TLC for completion of the reaction. 20ml of ice water was added to quench the reaction, the pH was adjusted to 8 with saturated aqueous sodium bicarbonate solution, and 2.56g of a white solid was obtained by suction filtration with a yield of 85.3%. m.p. 97.0-99.2 ℃. The product was used in the next reaction without purification.

And C: preparation of 3- { {2- { [ (1H-benzimidazol-2-yl) thio ] methyl } -3-methyl-pyridin-4-yl } oxy } propan-1-amine

Dissolving 3- { {2- { [ (1H-benzimidazol-2-yl) thio ] methyl } -3-methylpyridin-4-yl } oxy } propan-1-ol (0.5g, 1.52mmol) in 20mL of dichloromethane, dropwise adding 5mL of thionyl chloride at room temperature, after completion of dropwise addition, heating under reflux for reaction for 3 hours, after completion of reaction, evaporating off the remaining thionyl chloride and dichloromethane under reduced pressure, dissolving the residue in 20mL of DMF, adding anhydrous potassium carbonate (0.63g, 4.56mmol) and potassium phthalimide (0.28g, 1.52mmol), stirring at 90 ℃ for reaction for 3 hours, after completion of reaction, evaporating DMF under reduced pressure, washing the residue with water, suction-filtering, dissolving the filter cake in 30mL of anhydrous ethanol, adding hydrazine hydrate (5mL), heating under reflux for reaction for 6 hours, after completion of reaction, evaporating off ethanol under reduced pressure, the residue was extracted with 20mL of water and dichloromethane (20mL × 2 times), the organic phases were combined, washed with water, washed with saturated brine, dried over anhydrous magnesium sulfate, filtered with suction, the solvent was distilled off, and the residue was purified by column chromatography (dichloromethane: methanol: 60:1) to obtain 0.25g of a white solid with a yield of 50.2%.

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

The phenyl isocyanate (0.12g,1mmol) was dissolved in 10mL of dichloromethane solution, and 3- { {2- { [ (1H-benzimidazol-2-yl) thio was added dropwise at room temperature]After the dropwise addition, a dichloromethane solution (10mL) of methyl } -3-methyl-pyridin-4-yl } oxy } propan-1-amine (0.33g, 1mmol) was stirred for 3 hours, after the reaction was completed, 30mL of water was added, stirring was carried out for 5 minutes, liquid separation was carried out, washing was carried out with an organic phase saturated sodium chloride solution, drying was carried out over anhydrous magnesium sulfate, suction filtration was carried out, the filtrate was concentrated, and the residue was purified by column chromatography (dichloromethane: methanol: 60:1) to obtain 0.39g of a white solid with a yield of 86.7%. m.p. 104.1-105.5 ℃; ESI-MS of M/z 448.6[ M + H ]]⁺,470.4[M+Na]⁺；¹H NMR(400MHz,DMSO-d₆)δ12.63(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: preparation of 1- {3- { [2- { [ (1H-benzimidazol-2-yl) thio ] methyl } -3-methyl-pyridin-4-yl } oxy } propyl } -3- (4-chlorophenyl) urea (Compound X02)

According to the preparation method of example 01, 0.32g of white solid is obtained, the yield is 76.2%, and the m.p. is 165.8-167.3 ℃; ESI-MS: M/z 600.7,602.0, [ M + H ]]⁺；¹H NMR(400MHz,DMSO-d₆)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: preparation of 1- {3- { [2- { [ (1H-benzimidazol-2-yl) thio ] methyl } -3-methyl-pyridin-4-yl } oxy } propyl } -3- [3- (trifluoromethyl) phenyl ] urea (Compound X03)

Referring to the preparation method of example 01, 0.44g of white solid was obtained with a yield of 83%, m.p.:96.1-97.2 ℃; ESI-MS of M/z 516.3[ M + H ]]⁺；¹H NMR(400MHz,DMSO-d₆)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: preparation of 1- {3- { {2- { [ (1H-benzimidazol-2-yl) thio ] methyl } -3-methyl-pyridin-4-yl } oxy } propyl } -3- (p-tolyl) urea (Compound X04)

According to the preparation method of example 01, 0.32g of white solid is obtained, the yield is 84.1%, and the m.p. is 103.2-104.3 ℃; ESI-MS of M/z 462.3[ M + H ]]⁺；¹H NMR(400MHz,DMSO-d₆)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 foregoing is directed to preferred embodiments of the present invention, other and further embodiments of the invention may be devised without departing from the basic scope thereof, and the scope thereof is determined by the claims that follow. However, any simple modification, equivalent change and modification of the above embodiments according to the technical essence of the present invention are within the protection scope of the technical solution of the present invention.

Pharmacological examples

Example 5: inhibitory Activity of test Compounds on proliferation of A549, HCT-116, PC3 cells

(1) Experimental Material

Cell line: a549, HCT-116 and PC3 cells were plated at 3000/well density in 96-well plates at 100 ul/well for 24 h.

Number X01 to X04 target compounds: dissolved in DMSO, diluted with culture medium to prepare five different concentrations of 50 μ M, 20 μ M, 10 μ M, 5 μ M and 2 μ M, and stored at-20 deg.C for use, with the final concentration of DMSO in the culture medium being less than 0.1%.

Positive control drug: sorafenib (sorafenib).

MTT: dissolved in PBS at 5mg/mL and stored at-20 ℃.

(2) Experimental methods

A549 cells, HCT-116 cells and PC3 cells were selected by MTT method to evaluate the anti-tumor proliferation activity of the test samples. The cell lines were cultured in RPMI 1640 medium containing 10% bovine serum (FBS). When the cells proliferated to 80-90% they were pooled and subsequently subcultured for no more than 20 passages, and then they were acclimatized for 24h before the next disposal. These cells were plated in 96-well plates (8X 10)⁴/mL) and then in a solution containing 5% CO₂Was incubated overnight in a humidified environment and temperature controlled at 37 ℃. After 24h, various concentrations of representative compounds of the invention were added. After an additional 24h of incubation, MTT (5mg/mL) was added and incubation continued for 4 h. The culture medium was removed, the crystals were dissolved in DMSO, and the absorbance was measured at 490nm using a microplate reader (TECAN SPECTRA, WetDar, Germany). According to the formula: the cell growth inhibition rate is (1-drug group OD value/control group OD value) × 100%, the cell growth inhibition rate under the corresponding concentration is calculated, and the IC corresponding to the tested compound is calculated according to the logarithmic curve of the inhibition rate of the tested compound to the cell and the different concentrations of the tested compound₅₀The value is obtained. Representative compounds of the invention were tested as described above and the results are shown in table 1:

TABLE 1

Example 6: inhibitory Activity of test Compounds against BRaf, VEGFR-2, PDGFR-beta and TOPK kinases

(1) Experimental Material

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

Number X01 to X04 target compounds: dissolved in DMSO and treated the same as the control.

Positive control drug: sorafenib (sorafenib).

Kinase buffer: contains 50mM HEPES, pH7.5,10mM MgCl2, 0.0015% Brij-35 and 2mM DTT.

Stop buffer: contains 100mM HEPES, pH7.5, 0.015% Brij-35, 0.2% coating reagent #3 and 50mM ethylenediaminetetraacetic acid (EDTA).

(2) Experimental methods

All kinase assays were performed in 50 μ L reaction volumes in 96-well plates. Compounds were diluted to 500 μ M with DMSO, then 10 μ L of compound was transferred to a new 96-well plate as an intermediate plate, and 90 μ L of kinase buffer was added to each well. Transfer each well of the 5 microliter 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 containing substrate, enzyme and no compound were used as DMSO controls. Wells containing only substrate without enzyme served as low controls. The compounds were incubated at room temperature for 10 minutes. To each well 10. mu.L of peptide solution was added and incubated at 28 ℃ for the indicated period of time and the reaction was stopped with 25. mu.L of stop buffer. Finally, data was collected using the Caliper program, which converted measured data values to inhibition rates.

Inhibition (%) - (max-conversion)/(max-min) × 100, wherein "max" represents DMSO control; "min" represents low control.

Representative compounds of the invention were tested as described above and the results are shown in table 2:

TABLE 2

Formulation examples

The following formulation examples are merely illustrative of the scope of the invention and are not to be construed as limiting in any way.

Example 7: gelatin capsule

The hard gelatin capsule is prepared by the following steps:

the above formulations can be modified according to the reasonable variations provided.

Example 8: tablet formulation

The preparation of the tablet adopts:

the above ingredients are mixed and compressed into tablets.

Example 9: tablet formulation

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

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

Example 10: combined tablet

From the foregoing description, one skilled in the art can easily ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions.

Claims

1. A compound of formula I, and pharmaceutically acceptable salts of said compound:

wherein,

r is selected from hydrogen, phenyl, halogen substituted phenyl, C1-C4 alkyl substituted phenyl, halogen substituted C1-C4 alkyl substituted phenyl;

R₃、R₅selected from H, C1-C4 alkyl.

2. The compound of claim 1, and pharmaceutically acceptable salts of said compound:

wherein,

r is selected from hydrogen, phenyl, 4-chlorphenyl, 4-methylphenyl and 3- (trifluoromethyl) phenyl.

3. The compound of claim 1, and pharmaceutically acceptable salts of said compound:

wherein,

R₃、R₅selected from hydrogen, methyl.

4. The compound of claim 1, and pharmaceutically acceptable salts, selected from:

1-{3-{{2-{[(1H-benzimidazol-2-yl) thio]Methyl } -3-methylpyridin-4-yl } oxy } propyl } -3-phenylurea;

1-{3-{{2-{[(1H-benzimidazol-2-yl) Sulfur based radicals]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 active ingredient a compound according to any one of claims 1 to 4 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier or diluent.

6. A process for the preparation of the compound of claim 1, which process is as follows:

wherein, R, R₃、R₅As claimed in claim 1.

7. Use of a compound according to any one of claims 1 to 4, and a pharmaceutically acceptable salt or a pharmaceutical composition according to claim 5 for the manufacture of a medicament for the treatment of tumours.

8. Use of a compound according to any one of claims 1 to 4, and a pharmaceutically acceptable salt or pharmaceutical composition thereof, for the preparation of inhibitors of BRaf kinase, vascular endothelial growth factor receptor-2, platelet-derived growth factor receptor-beta or T-LAK cell-derived protein kinase.

9. The use of a compound according to any one of claims 1 to 4, and pharmaceutically acceptable salts or pharmaceutical compositions for the manufacture of a medicament for the treatment of a disease associated with dysregulation of BRaf kinase, vascular endothelial growth factor receptor-2, platelet derived growth factor receptor-beta or T-LAK cell derived protein kinase.

10. The use according to claim 9, wherein the diseases associated with the dysregulation of BRaf kinase, vegf receptor-2, vegf receptor-beta or T-LAK cell-derived protein kinase are lung cancer, liver cancer, melanoma, colon cancer, rectal cancer, breast cancer, ovarian cancer, renal cancer.