CN102558144A - Aryl urea derivatives - Google Patents

Abstract

The invention discloses a group of aryl urea derivatives represented by a general structure, belonging to the field of medicinal chemistry. The invention also discloses a preparation method of the aryl urea derivative represented by the general structure. The compound of the invention has the function of raf kinase inhibitor and can be used for preparing medicine for treating tumor diseases.

Description

A kind of aryl urea derivative

technical field

The invention relates to the field of medicinal chemistry, in particular to a group of aryl urea compounds represented by the general formula, their preparation method and use as raf kinase inhibitor.

Background technique

The p21ras oncogene is one of the main reasons for the occurrence and development of human parenchymal cancer, and this gene is mutated in 30% of cancer patients (Bolton et al., Ann. Rep. Med. Chem, 1994, 29, 165-74; Bos. Cancer. Res. 1989, 49, 4682-9). The non-mutated normal form ras protein is a key element in the signaling cascade directed by growth factor receptors in almost all tissues (Avruch et al., Trends Biochem. Sci. 1994, 19, 279-83). Biochemically, ras is a protein that binds guanine nucleotides, and the cycle between the GTP-bound active state and the GDP-bound resting state is tightly controlled by ras endogenous GTPase activity and other regulatory proteins. Mutant ras in cancer cells has increased endogenous GTPase activity and, consequently, downstream effectors of this protein, such as raf kinase, signal constitutive growth. Cancerous growth of cells bearing these mutants is thus caused (Magnuson et al., Semin. Cancer Biol. 1994, 5, 247-53). Inhibition of the effects of active ras by inhibition of raf kinase signaling, for example by administering an inactivating antibody to raf kinase or co-expression of dominant negative raf kinase or dominant negative MEK as a substrate for raf kinase, is known to allow conversion The cells revert to a normal growth phenotype (see; Daum et al., Trends Biochem. Sci. 1994, 19, 474-80; Fridman et al., J. Biol. Chem. 1994, 269, 30105-8). Kolch et al. (Nature, 1991, 349, 426-28) further pointed out that in membrane-associated oncogenes, inhibition of raf expression by antisense RNA can inhibit cell proliferation. Similarly, raf kinase inhibition (with antisense oligodeoxynucleotides) was found to correlate with inhibition of growth of various human tumors both in vitro and in vivo (Monia et al., Nat. Med. 1996, 2, 668-75).

Recent research focuses on finding potent raf kinase inhibitors. Clinical data show that inhibitors of signal transduction pathways are less toxic than traditional chemotherapy drugs. Some experts predict that such drugs will become the standard drug for cancer treatment in the next two decades and be widely used in clinical practice. Arylureas often have inhibitory activity against other kinases in tumor signaling pathways, including vascular endothelial growth factor receptor 2/3 (VEGFR-2, VEGFR-3), platelet-derived growth factor receptor beta (PDGFR -β), KIT, FLT-3, RET. Such drugs can not only inhibit the proliferation of tumor cells, but also prevent the formation of tumor neovascularization. This further enhances the clinical effect and research value of these compounds in inhibiting tumors.

Contents of the invention

The invention establishes a structure-activity relationship model and a drug screening model of aryl urea compounds by means of pharmacophore model and other computer-aided drug design methods, and designs a series of aryl urea compounds with new structures on the basis of these.

The aryl urea compounds of the present invention are used as raf kinase inhibitors for the inhibition of human or animal raf kinase pathways, for example, for the treatment of cancerous growth of tumors or cells mediated by raf kinases. In particular, these compounds are useful in the treatment of human or animal cancers such as melanoma, liver cancer, renal cancer, acute leukemia, non-small cell lung cancer, prostate cancer, thyroid cancer, skin cancer, colorectal cancer, pancreatic cancer , ovarian cancer, breast cancer, myelodysplastic syndrome, esophageal cancer, gastrointestinal cancer or mesothelioma, etc.

Therefore, the present invention relates to a compound of general formula I or a pharmaceutically acceptable salt thereof:

Wherein, R1 is a substituted or unsubstituted five-membered aromatic heterocyclic ring represented by formula a, wherein R4, R5, and R6 are each independently selected from a carbon atom, a nitrogen atom, an oxygen atom, or a sulfur atom, and the substituents R8, R9, Each R10 is independently selected from hydrogen, halogen, C1-4 alkyl, or C1-4 alkoxy;

R2 is selected from fluorine, chlorine, bromine, iodine, nitrile, C1-4 alkoxy, or mercapto.

R1 is further a substituted or unsubstituted five-membered aromatic heterocyclic ring represented by formula a, wherein R4, R5, and R6 are each independently selected from a carbon atom, a nitrogen atom, or a sulfur atom, and the substituents R8, R9, and R10 are each independently is selected from hydrogen or methyl.

R2 is further selected from fluorine, chlorine, bromine, iodine, nitrile, methoxy, or mercapto.

R2 is further selected from fluorine.

R1 is further selected from:

R1 is further selected from:

The inventors introduced new substituents or functional groups into the active molecules through the study of the structure-activity relationship of the lead compounds, and synthesized hundreds of new compounds, and then carried out preliminary screening on all new compounds according to the experimental design of "determination of biological activity" According to the results of the primary screening, the structure is optimized, and the new compounds obtained from the primary screening are re-screened. The overall animal model is used for evaluation, pharmacology, pharmacodynamics, and toxicology studies are carried out, and the most preferred new compound disclosed in the present invention is finally determined.

The compound of general formula of the present invention and pharmaceutically acceptable salt thereof can be:

1-(4-chloro-3-(trifluoromethyl)phenyl)-3-(2-fluoro-4-(2-(1-methyl-4-pyrazolyl)-4-pyridyloxy) Phenyl) urea;

1-(4-Chloro-3-(trifluoromethyl)phenyl)-3-(2-fluoro-4-(2-(4-pyrazolyl)-4-pyridyloxy)phenyl)urea;

1-(4-Chloro-3-(trifluoromethyl)phenyl)-3-(2-fluoro-4-(2-(4-imidazolyl)-4-pyridyloxy)phenyl)urea;

1-(4-Chloro-3-(trifluoromethyl)phenyl)-3-(2-fluoro-4-(2-(3-pyrrolyl)-4-pyridyloxy)phenyl)urea;

1-(4-Chloro-3-(trifluoromethyl)phenyl)-3-(2-fluoro-4-(2-(3-thienyl)-4-pyridyloxy)phenyl)urea;

1-(4-Chloro-3-(trifluoromethyl)phenyl)-3-(2-fluoro-4-(2-(1-methyl-4-imidazolyl)-4-pyridyloxy)benzene base) urea;

1-(4-chloro-3-(trifluoromethyl)phenyl)-3-(2-fluoro-4-(2-(1-methyl-3-pyrrolyl)-4-pyridyloxy)benzene base) urea;

1-(4-chloro-3-(trifluoromethyl)phenyl)-3-(2-chloro-4-(2-(1-methyl-4-pyrazolyl)-4-pyridyloxy) Phenyl) urea;

1-(4-chloro-3-(trifluoromethyl)phenyl)-3-(2-bromo-4-(2-(1-methyl-4-pyrazolyl)-4-pyridyloxy) Phenyl) urea;

1-(4-Chloro-3-(trifluoromethyl)phenyl)-3-(2-iodo-4-(2-(1-methyl-4-pyrazolyl)-4-pyridyloxy) Phenyl) urea;

1-(4-Chloro-3-(trifluoromethyl)phenyl)-3-(2-methoxy-4-(2-(1-methyl-4-pyrazolyl)-4-pyridineoxy base) phenyl) urea;

1-(4-Chloro-3-(trifluoromethyl)phenyl)-3-(2-cyano-4-(2-(1-methyl-4-pyrazolyl)-4-pyridyloxy ) phenyl) urea;

1-(4-chloro-3-(trifluoromethyl)phenyl)-3-(2-mercapto-4-(2-(1-methyl-4-pyrazolyl)-4-pyridyloxy) phenyl) urea.

Particularly preferred compounds of the invention are:

According to IUPAC nomenclature, the formula IV compound of the present invention is named: 1-(4-chloro-3-(trifluoromethyl)phenyl)-3-(2-fluoro-4-(2-(1-methyl) -4-pyrazolyl)-4-pyridyloxy)phenyl)urea.

The pharmaceutically acceptable salts described in the present invention include the acid addition salts formed by the compound of general formula I and the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, citric acid, tartaric acid, phosphoric acid, lactic acid, pyruvic acid, acetic acid, malic acid, acid or benzoic acid.

The present invention also includes the use of the compound of general formula I or a pharmaceutically acceptable salt thereof in the preparation of medicaments for preventing or treating diseases related to raf kinase inhibitors. Among them, diseases related to Raf kinase inhibitors are melanoma, liver cancer, kidney cancer, acute leukemia, non-small cell lung cancer, prostate cancer, thyroid cancer, skin cancer, colorectal cancer, pancreatic cancer, ovarian cancer, breast cancer, bone marrow dysplasia syndrome, cancer of the esophagus, gastrointestinal tract, or mesothelioma.

The preparation method of the compound of general formula I of the present invention is as follows, and all compounds of the present invention can be obtained by using this preparation method: first, the aminophenol compound is subjected to nucleophilic substitution with 2,4-dihalopyridine under the action of a strong base Reaction, the obtained product undergoes Suzuki reaction with five-membered aromatic heterocyclic boronic acid pinacol ester under the catalysis of palladium catalyst, and the obtained product reacts with 3-trifluoromethyl-4-chloro-phenylisocyanate to obtain the compound of general formula I.

The preparation method of preferred formula IV compound of the present invention is as follows:

step 1:

Step 2:

Step 3:

As two intermediates in the preparation process of the above-mentioned preferred compounds of the present invention, the compound of formula II and the compound of formula III are also new compounds.

New intermediate compound of formula II: according to IUPAC nomenclature, the compound of formula II of the present invention is named: 2-chloro-4-(3-fluoro-4-aminophenoxy)pyridine.

New intermediate compound of formula III: according to IUPAC nomenclature, the compound of formula III of the present invention is named: 2-fluoro-4-(2-(1-methyl-4-pyrazolyl)-4-pyridyloxy)aniline.

The preparation method of formula II, formula III, formula IV compound of the present invention is specifically as follows:

Step 1: Dissolve 1.34g of 3-fluoro-4-aminophenol in anhydrous dimethyl sulfoxide, after 10min of nitrogen gas, add 1.24g of potassium tert-butoxide, stir at room temperature for 30min, then add 1.32g of 2-chloro -4-fluoropyridine, then slowly raise the temperature of the reaction system to 80°C and keep it warm for 2 hours. TLC detection shows that the reaction is complete. After cooling to room temperature, add water and ethyl acetate, and extract the aqueous phase twice with ethyl acetate , the ethyl acetate layers were combined, washed twice with water, then washed once with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and the obtained crude product was purified by column chromatography to obtain the compound of formula II;

Step 2: Under nitrogen protection, dissolve 1.6g 2-chloro-4-(3-fluoro-4-aminophenoxy)pyridine and 1.68g 1-methyl-4-pyrazoleboronic acid pinacol ester in tetrahydrofuran (THF), under stirring, add 2.77g of potassium carbonate and 4.5mL of water, then add 0.39g of tetrakistriphenylphosphine palladium catalyst under the dark, keep stirring at 70°C for 24h, TLC detects that the reaction is complete; cool to room temperature, and the reaction system Concentrate, then add ethyl acetate and water, after separation, the aqueous phase is extracted twice with ethyl acetate, the ethyl acetate layer is combined, washed twice with water, washed once with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated; the crude product After purification by column chromatography, the compound of formula III is obtained;

Step 3: Dissolve 10.2g 2-fluoro-4-(2-(1-methyl-4-pyrazolyl)-4-pyridyloxy)aniline in dichloromethane, add 7.95g 3- Trifluoromethyl-4-chloro-phenylisocyanate, after stirring at room temperature for 12 hours, a large amount of solids precipitated out, the system was concentrated, filtered with suction, washed with ethyl acetate, and dried to obtain the compound of formula IV.

Methods for enantiomeric and diastereomeric mixtures are familiar to those skilled in the art. The present invention includes any compound of formula I in isolated racemic or optically active form having raf kinase inhibitory activity.

The present invention also includes a pharmaceutical composition comprising a compound of general formula I and a physiologically acceptable carrier. The compounds of the present invention may be administered by injection, inhalation or spray or rectally, orally, dermally, parenterally, or in unit dosage form. "Administration by injection" includes intravenous, intramuscular, subcutaneous and parenteral injections, as well as the use of infusion techniques. Skin administration includes topical or transdermal administration. One or more compounds can coexist with one or more pharmaceutically acceptable non-toxic carriers, and other active ingredients as needed. Oral compositions may be prepared according to any suitable method known in the art for the manufacture of pharmaceutical compositions. To improve the mouthfeel of the preparation, the composition may contain one or more of the following agents: diluents, sweeteners, flavours, colorants and preservatives. Tablets contain the active ingredients in admixture with pharmaceutically acceptable non-toxic excipients suitable for the manufacture of tablets. Such excipients are, for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, such as cornstarch or alginic acid. Binders, such as magnesium stearate, stearic acid or talc. Tablets may be uncoated or coated by known techniques to delay their disintegration and absorption in the gastrointestinal tract and provide long-term sustained action. For example, a time delay material such as glyceryl monostearate or glyceryl distearate may be employed. The compounds can also be prepared in solid, immediate release form. Oral formulations may also be hard gelatin capsules in which the active ingredient is mixed with an inert solid diluent, such as calcium carbonate, calcium phosphate, or kaolin, or soft gelatin capsules in which the active ingredient is mixed with water or, for example, peanut oil, liquid paraffin, or olive oil. Oil and other oils mixed.

Aqueous suspensions may also be used which contain the active materials in admixture with excipients suitable for the manufacture of aqueous suspensions. The excipients are suspending agents such as sodium carboxymethylcellulose, methylcellulose, hydroxypropyl-methylcellulose, sodium alginate, polyvinylpyrrolidone, tragacanth and acacia; dispersing agents Or the wetting agent may be a natural phospholipid, such as lecithin, or a condensation product of ethylene oxide with a fatty acid, such as polyoxyethylene stearate, or a condensation product of ethylene oxide with a long chain fatty alcohol, such as seventeen Oxyethylene cetyl alcohol, or condensation products of ethylene oxide with partial esters of fatty acids and hexitols, such as polyoxyethylene sorbitan monooleate. Aqueous suspensions may also contain one or more preservatives, such as ethyl or n-propyl p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents, and one or more sweetening agents , such as sucrose or saccharin. In dispersible powders or granules suitable for addition to water for an aqueous suspension, the active ingredient is in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified by those already mentioned above. Other excipients, for example sweetening, flavoring and coloring agents, may also be present.

The pharmaceutical composition of the present invention may also be in the form of a non-aqueous liquid preparation, such as an oily suspension, which can be formulated by suspending the active ingredient in a vegetable oil such as peanut oil, olive oil, sesame oil or peanut oil, or a mineral oil such as liquid paraffin. The oily suspensions may contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol. In order to improve the taste, the above-mentioned sweeteners and spices can be added. The composition can be preserved by the addition of antioxidants such as ascorbic acid.

The pharmaceutical compositions of the present invention may also be in the form of oil-in-water emulsions. The oily phase may be a vegetable oil such as olive oil or arachis oil or a mineral oil such as liquid beeswax, or a mixture thereof. Suitable emulsifiers may be natural gums such as tragacanth and acacia, or natural phospholipids, such as soybean lecithin or lecithin: partial esters of fatty acids with anhydrohexitols, such as sorbitan oleate; Condensation products of the above-mentioned partial esters and ethylene oxide, such as polyoxyethylene sorbitan monooleate. The emulsions may also contain sweetening and flavoring agents.

Syrups may also be formulated with sweetening agents, for example glycerol, polypropylene glycol, sorbitol or sucrose. Such preparations may also contain a demulcent, a preservative and flavoring and coloring agents.

The compounds may also be administered rectally or vaginally in the form of suppositories. Such compositions can be prepared by mixing the drug with a suitable non-irritating excipient which is solid at ordinary temperatures but liquid at rectal or vaginal temperature and, therefore, will melt in the rectum or vagina to release the drug. Such materials include cocoa butter and polyethylene glycols.

In the description and claims of the present invention, the naming of compounds is based on the chemical structural formula. If the naming of the compound is inconsistent with the chemical structural formula when representing the same compound, the chemical structural formula or chemical reaction formula shall prevail.

Now in conjunction with embodiment, the present invention will be further described:

Detailed ways

Example 1:

1a: Synthesis of 2-chloro-4-(3-fluoro-4-aminophenoxy)pyridine:

Dissolve 1.34g (10.5mmol) of 3-fluoro-4-aminophenol in 10mL of anhydrous dimethyl sulfoxide, pass nitrogen gas for 10min, add 1.24g (11.0mmol) of potassium tert-butoxide, and stir at room temperature for 30min, 1.32 g (10.0 mmol) of 2-chloro-4-fluoropyridine was added, and then the reaction system was slowly heated to 80° C. and kept for 2 h. TLC detection showed that the reaction was complete. After cooling to room temperature, 100 mL of water and 100 mL of ethyl acetate were added, and after separation, the aqueous phase was extracted twice with 100 mL of ethyl acetate, and the ethyl acetate layers were combined, washed twice with water (100 mL/time), and washed once with saturated brine ( 100mL/time), dried over anhydrous sodium sulfate, filtered, concentrated, and the resulting crude product was purified by column chromatography to obtain 1.60g of a light yellow solid with a yield of 67%. 1H NMR (300MHz, CDCl3): δ3.76(br s, 2H), 6.70-6.84(m, 5H), 8.21(d, J=5.7Hz, 1H). MS(ESI+): 239.1[M+H] ⁺

1b: Synthesis of 2-fluoro-4-(2-(1-methyl-4-pyrazolyl)-4-pyridyloxy)aniline:

Under nitrogen protection, 1.6g (6.7mmol) 2-chloro-4-(3-fluoro-4-aminophenoxy)pyridine and 1.68g (8.1mmol) 1-methyl-4-pyrazole boronic acid pinacol Dissolve the ester in 13mL of tetrahydrofuran (THF), add 2.77g (20.0mmol) of potassium carbonate and 4.5mL of water under stirring, then add 0.39g (0.335mmol) of tetrakistriphenylphosphorous palladium catalyst in the dark, and keep stirring at 70°C After 24 hours, TLC detected that the reaction was complete.

Cool to room temperature, concentrate the reaction system, then add 50 mL each of ethyl acetate and water, extract the aqueous phase twice with 50 mL ethyl acetate after separation, combine the ethyl acetate layers, wash twice with water (50 mL/time), and wash with saturated salt Wash once with water (50 mL/time), dry over anhydrous sodium sulfate, filter, and concentrate. The crude product was purified by column chromatography to obtain 1.61 g of light yellow solid with a yield of 84%. 1H NMR (300MHz, CDCl3): δ3.75(br s, 2H), 3.93(s, 3H), 6.61(d, J=5.7Hz, 1H), 6.72-6.84(m, 3H), 6.95(s, 1H), 7.86(s, 2H), 8.36(d, J=5.7Hz, 1H). MS(ESI+): 285.1[M+H] ⁺

1c: 1-(4-Chloro-3-(trifluoromethyl)phenyl)-3-(2-fluoro-4-(2-(1-methyl-4-pyrazolyl)-4-pyridineoxy base) phenyl) urea synthesis:

Dissolve 10.2g (35.8mmol) of 2-fluoro-4-(2-(1-methyl-4-pyrazolyl)-4-pyridyloxy)aniline in 100mL of dichloromethane, and add 7.95g of (35.8mmol) 3-trifluoromethyl-4-chloro-phenylisocyanate, after stirring at room temperature for 12h, a large amount of solids precipitated, the system was concentrated to 30mL of solvent remaining, suction filtered, washed with ethyl acetate, dried to obtain a white solid 14.25g, yield 79%. 1H NMR (300MHz, CDCl3): δ3.87(s, 3H), 6.69(d, J=5.7Hz, 1H), 7.03(d, J=8.7Hz, 1H), 7.24-7.27(m, 2H), 7.63(s, 2H), 7.97(s, 1H), 8.12(s, 2H), 8.26(s, 1H), 8.39(d, J=5.7Hz, 1H), 8.69(s, 1H), 9.51(s , 1H).MS(ESI+): 506.0[M+H] ⁺

Example 2: 1-(4-chloro-3-(trifluoromethyl)phenyl)-3-(2-fluoro-4-(2-(4-pyrazolyl)-4-pyridyloxy)benzene base) urea

The preparation method is the same as in Example 1, wherein 1-methyl-4-pyrazoleboronic acid pinacol ester in 1b is replaced by 4-pyrazoleboronic acid pinacol ester, and dichloromethane in 1c is replaced by ethyl acetate.

¹ H NMR (300MHz, DMSO-d ₆ ): δ6.42 (br s, 1H), 6.39 (d, J=3.9Hz, 1H), 7.45 (d, J=8.4Hz, 2H), 7.57-7.69( m, 4H), 7.96(s, 1H), 8.12(s, 1H), 8.24(s, 1H), 8.37(d, J=5.4Hz, 1H), 8.93(s, 1H), 9.17(s, 1H ).MS(ESI+): 494.8[M+H] ⁺

Example 3: 1-(4-chloro-3-(trifluoromethyl)phenyl)-3-(2-fluoro-4-(2-(4-imidazolyl)-4-pyridyloxy)phenyl ) urea

The preparation method is the same as in Example 1, wherein the 1-methyl-4-pyrazoleboronic acid pinacol ester in 1b is replaced by 4-imidazoleboronic acid pinacol ester.

¹ H NMR (300MHz, DMSO-d ₆ ): δ6.42 (br s, 1H), 6.39 (d, J=3.9Hz, 1H), 7.45 (d, J=8.4Hz, 2H), 7.57-7.69( m, 4H), 7.96(s, 1H), 8.12(s, 1H), 8.24(s, 1H), 8.37(d, J=5.4Hz, 1H), 8.93(s, 1H), 9.17(s, 1H ).MS(ESI+): 494.8[M+H] ⁺

Example 4: 1-(4-chloro-3-(trifluoromethyl)phenyl)-3-(2-fluoro-4-(2-(3-pyrrolyl)-4-pyridyloxy)phenyl ) urea

The preparation method is the same as in Example 1, wherein 1-methyl-4-pyrazole borate pinacol ester in 1b is replaced by 3-pyrrole borate pinacol ester.

¹ H NMR (300MHz, DMSO-d ₆ ): δ6.41 (br s, 1H), 6.65 (d, J=3.9Hz, 1H), 7.15 (d, J=8.4Hz, 2H), 7.57-7.69( m, 5H), 7.96(s, 1H), 8.17(s, 1H), 8.24(s, 1H), 8.37(d, J=5.4Hz, 1H), 8.93(s, 1H), 9.17(s, 1H ).MS(ESI+): 491.8[M+H] ⁺

Example 5: 1-(4-chloro-3-(trifluoromethyl)phenyl)-3-(2-fluoro-4-(2-(3-thienyl)-4-pyridyloxy)phenyl ) urea

The preparation method is the same as in Example 1, wherein the 1-methyl-4-pyrazoleboronic acid pinacol ester in 1b is replaced by 3-thiopheneboronic acid pinacol ester.

¹ H NMR (300MHz, DMSO-d ₆ ): δ6.52(s, 1H), 6.65(d, J=3.9Hz, 1H), 7.15(d, J=8.4Hz, 2H), 7.57-7.69(m , 4H), 7.96(s, 1H), 8.17(s, 1H), 8.26(s, 1H), 8.37(d, J=5.4Hz, 1H), 8.93(s, 1H), 9.17(s, 1H) .MS(ESI+): 508.8[M+H] ⁺

Example 6: 1-(4-chloro-3-(trifluoromethyl)phenyl)-3-(2-fluoro-4-(2-(1-methyl-4-imidazolyl)-4-pyridine Oxy)phenyl)urea

The preparation method is the same as in Example 1, wherein the 1-methyl-4-pyrazoleboronic acid pinacol ester in 1b is replaced by 1-methyl-4-imidazoleboronic acid pinacol ester.

¹ H NMR (300MHz, DMSO-d ₆ ): δ3.75(s, 3H), 6.65(d, J=3.9Hz, 1H), 7.15(d, J=8.4Hz, 2H), 7.57-7.69(m , 4H), 7.91(s, 1H), 8.15(s, 1H), 8.24(s, 1H), 8.32(d, J=5.4Hz, 1H), 8.99(s, 1H), 9.19(s, 1H) .MS(ESI+): 506.8[M+H] ⁺

Example 7: 1-(4-chloro-3-(trifluoromethyl)phenyl)-3-(2-fluoro-4-(2-(1-methyl-3-pyrrolyl)-4-pyridine Oxy)phenyl)urea

The preparation method is the same as in Example 1, wherein 1-methyl-4-pyrazole borate pinacol ester in 1b is replaced by 1-methyl-3-pyrrole borate pinacol ester.

¹ H NMR (300MHz, DMSO-d ₆ ): δ3.65(s, 3H), 6.63(d, J=3.9Hz, 1H), 6.82(s, 1H), 7.15(d, J=8.4Hz, 2H ), 7.57-7.69(m, 4H), 7.91(s, 1H), 8.15(s, 1H), 8.24(s, 1H), 8.32(d, J=5.4Hz, 1H), 8.99(s, 1H) , 9.17(s, 1H).MS(ESI+): 505.8[M+H] ⁺

Example 8: 1-(4-chloro-3-(trifluoromethyl)phenyl)-3-(2-chloro-4-(2-(1-methyl-4-pyrazolyl)-4- Pyridyloxy)phenyl)urea

The preparation method is the same as in Example 1, wherein 4-aminophenol in 1a is replaced by 3-chloro-4-aminophenol.

¹ H NMR (300MHz, DMSO-d ₆ ): δ3.89(s, 3H), 6.34(br s, 1H), 6.63(d, J=5.4Hz, 1H), 7.15(d, J=8.4Hz, 2H), 7.57-7.69(m, 4H), 8.12(s, 1H), 8.24(d, J=2.4Hz, 1H), 8.37(d, J=5.4Hz, 1H), 8.93(s, 1H), 9.17(s, 1H).MS(ESI+): 523.3[M+H] ⁺

Example 9: 1-(4-chloro-3-(trifluoromethyl)phenyl)-3-(2-bromo-4-(2-(1-methyl-4-pyrazolyl)-4- Pyridyloxy)phenyl)urea

The preparation method is the same as in Example 1, wherein 4-aminophenol in 1a is replaced by 3-bromo-4-aminophenol.

¹ H NMR (300MHz, DMSO-d ₆ ): δ3.81(s, 3H), 6.58(dd, J=2.1, 5.4Hz, 1H), 6.82(d, J=8.4Hz, 1H), 7.05(dd , J=2.4, 8.4Hz, 1H), 7.25(d, J=2.4Hz, 1H), 7.51-7.64(m, 3H), 7.96(s, 1H), 8.16(s, 1H), 8.29(d, J=2.4Hz, 1H), 8.37(d, J=5.4Hz, 1H), 8.93(s, 1H), 9.17(s, 1H). MS(ESI+): 567.7[M+H] ⁺

Example 10: 1-(4-chloro-3-(trifluoromethyl)phenyl)-3-(2-iodo-4-(2-(1-methyl-4-pyrazolyl)-4- Pyridyloxy)phenyl)urea

The preparation method is the same as in Example 1, wherein 4-aminophenol in 1a is replaced by 3-iodo-4-aminophenol.

¹ H NMR (300MHz, DMSO-d ₆ ): 3.81(s, 3H), 6.38(d, J=5.4Hz, 1H), 6.72(d, J=2.4Hz, 1H), 6.87(dd, J=2.4 , 5.4Hz, 1H), 6.95(d, J=8.4Hz, 1H), 7.51-7.64(m, 3H), 7.96(s, 1H), 8.16(s, 1H), 8.29(d, J=8.1Hz , 1H), 8.37(d, J=5.4Hz, 1H), 8.93(s, 1H), 9.17(s, 1H). MS(ESI+): 614.7[M+H] ⁺

Example 11: 1-(4-chloro-3-(trifluoromethyl)phenyl)-3-(2-methoxy-4-(2-(1-methyl-4-pyrazolyl)- 4-pyridyloxy)phenyl)urea

The preparation method is the same as in Example 1, wherein 4-aminophenol in 1a is replaced by 3-methoxy-4-aminophenol.

¹ H NMR (300MHz, DMSO-d ₆ ): δ3.57(s, 3H), 3.81(s, 3H), 6.59(d, J=5.4Hz, 1H), 6.72(d, J=2.4Hz, 1H ), 6.87(dd, J=2.4, 5.4Hz, 1H), 6.95(d, J=8.4Hz, 1H), 7.51-7.64(m, 3H), 7.96(s, 1H), 8.16(s, 1H) , 8.29(d, J=8.1Hz, 1H), 8.37(d, J=5.4Hz, 1H), 8.93(s, 1H), 9.17(s, 1H). MS(ESI+): 518.8[M+H] ⁺

Example 12: 1-(4-chloro-3-(trifluoromethyl)phenyl)-3-(2-cyano-4-(2-(1-methyl-4-pyrazolyl)-4 -pyridyloxy)phenyl)urea

The preparation method is the same as in Example 1, wherein 4-aminophenol in 1a is replaced by 3-cyano-4-aminophenol.

¹ H NMR (300MHz, DMSO-d ₆ ): 3.81(s, 3H), 6.43(d, J=5.4Hz, 1H), 6.72(d, J=2.4Hz, 1H), 6.88(dd, J=2.4 , 5.4Hz, 1H), 6.95(d, J=8.4Hz, 1H), 7.51-7.64(m, 3H), 7.96(s, 1H), 8.16(s, 1H), 8.29(d, J=8.1Hz , 1H), 8.37(d, J=5.4Hz, 1H), 8.93(s, 1H), 9.17(s, 1H). MS(ESI+): 513.8[M+H] ⁺

Example 13: 1-(4-chloro-3-(trifluoromethyl)phenyl)-3-(2-mercapto-4-(2-(1-methyl-4-pyrazolyl)-4- Pyridyloxy)phenyl)urea

The preparation method is the same as in Example 1, wherein 4-aminophenol in 1a is replaced by 3-mercapto-4-aminophenol.

¹ H NMR (300MHz, DMSO-d ₆ ): 3.81(s, 3H), 6.65(d, J=5.4Hz, 1H), 6.72(d, J=2.4Hz, 1H), 6.89(dd, J=2.4 , 5.4Hz, 1H), 6.94(d, J=8.4Hz, 1H), 7.51-7.64(m, 3H), 7.96(s, 1H), 8.20(s, 1H), 8.29(d, J=8.1Hz , 1H), 8.37(d, J=5.4Hz, 1H), 8.93(s, 1H), 9.17(s, 1H). MS(ESI+): 520.1[M+H] ⁺

Experimental example 1: Raf screening in vitro

The activity of the various isoforms of raf serine/threonine kinase can be assayed by providing ATP, the MEK substrate, and measuring the transfer of the phosphate moiety to the MEK residue. Raf recombinant isoforms were obtained by purification from sf9 insect cells infected with a human raf recombinant baculovirus expression vector. Recombinant kinase-inactive MEK was expressed in E. coli, purified and labeled with biotin. For each assay, test compounds were serially diluted in DMSO and then mixed with raf (0.50 nM) and kinase-inactive biotin-MEK (50 nM) in reaction buffer with ATP (1 uM). Reactions were incubated for an additional 2 hours at room temperature and stopped by the addition of 0.5M EDTA. The stopped reaction mixture was transferred to neutradavin coated plates (pierce) and incubated for 1 hour. Phosphorylation products were measured by DELFIA time-resolved fluorescence system (Wallac) using rabbit anti-p-MEK (Cell Signaling) as primary antibody and europium-labeled anti-rabbit as secondary antibody. Time-resolved fluorescence was read on a Wallac 1232 DELFIA fluorometer. Concentration of 50% inhibition (IC50) of each compound was calculated by non-linear regression using XL fit data analysis software.

Using the above procedure, the compound of Example 1 was shown to have raf kinase inhibitory activity with an IC50 of less than 5 [mu]M.

Experimental Example 2: The Compound of the Invention Inhibits the Growth of ACHN Renal Cancer

1 Materials and methods

1.1 Experimental materials

Female BALB/c-nu/nu nude mice, 4 weeks old, with an average body weight of 14.5g (13.4-15.6)g. ACHN renal carcinoma cell line was purchased from American Type Species Collection (ATCC).

1.2 Experimental method

2.0×106/0.2ml of ACHN renal cancer cells were inoculated subcutaneously on the right back of each nude mouse, and 16 nude mice were randomly divided into a treatment group and a control group according to body weight numbers. There was no significant difference in body weight between the two groups. From the 3rd day after the inoculation, administration began every other day, and the medication group was given the compound of Example 1 (60mg/kg; solvent: 3% dehydrated alcohol+97% normal saline), and the matched group was given the solvent (3% dehydrated alcohol+97% normal saline). % normal saline). Inject subcutaneously away from the tumor, 0.2ml each time. During the administration period, the general condition of the mice was observed, and the body weight and tumor size of the mice were measured every other day, using the formula: V=1/2×a×b2, (a is the long diameter, b is the short diameter). On the 31st day after inoculation, the mice were killed by neck dissection, and the tumor volume was measured and the mice were weighed before dissection. Subcutaneous tumors were dissected and weighed, and mouse lungs were excised. Subcutaneous tumors and mouse lungs were fixed with FAA (glacial acetic acid + formalin + ethanol), and embedded in paraffin. Calculate the mouse weight again (mouse weight=tumor-bearing mouse weight-tumor weight). HE staining of the largest section of the coronal plane of both lungs, counting pulmonary metastatic nodules under a microscope (100 times field of view).

1.3 Statistical processing

Tumor weight, volume, and rats between the two groups were tested by t-test, the growth curve of tumor volume was analyzed by SAS covariance, and the number of lung metastatic nodules was tested by exact probability method.

2 results

2.1 Subcutaneous tumor weight and tumor volume changes

On the 31st day of inoculation, the subcutaneous tumor weights of the two groups were 646.17±131.28 mg and 243.18±57.63 mg respectively, and the tumor weights of the drug group were significantly lower than those of the control group (p<0.01). At 31 days, the volumes of subcutaneous tumors in the two groups were 338.21±44.39mm3 and 113.92±57.19mm3 respectively (p<0.01). The tumor volume-time changes of the two groups are shown in Table 1:

Table 1: Tumor volume-time changes

The results in Table 1 show that the growth of tumor volume in the medication group was significantly lower than that in the control group.

2.2 Pulmonary metastatic nodules

Of the 8 mice in the control group, 6 had lung metastases, 3 had 1 metastatic nodule, and the rest had 3-5 metastatic nodules. No lung metastatic nodules were found in the treatment group. The difference between the two groups was tested by the exact probability method and had significant significance (p<0.05). The control group not only had a higher metastasis rate, but also had more metastatic nodules.

2.3 Observation of side effects during medication

During the medication period, the mice moved well, and no adverse reactions such as diarrhea were seen.

3 Experimental conclusions

The tumor weight of the compound medication group of the present invention is significantly lower than that of the control group, which is statistically significant (p<0.01), and the tumor volume growth of the medication group is significantly lower than that of the control group. The incidence of tumor metastasis, and the compound of the present invention has lower toxic and side effects.

Claims (19)

1. the compound of formula I or its pharmacy acceptable salt:

Wherein, Replacement or unsubstituted five yuan of fragrant heterocycles that R1 representes for formula a; Wherein, R4, R5, R6 are selected from carbon atom, nitrogen-atoms, Sauerstoffatom or sulphur atom independently of one another, and substituent R 8, R9, R10 are selected from hydrogen, halogen, C1-4 alkyl or C1-4 alkoxyl group independently of one another;

R2 is selected from fluorine, chlorine, bromine, iodine, itrile group, C1-4 alkoxyl group or sulfydryl.

2. replacement or unsubstituted five yuan of fragrant heterocycles that compound according to claim 1 or its pharmacy acceptable salt: R1 represent for formula a; Wherein, R4, R5, R6 are selected from carbon atom, nitrogen-atoms or sulphur atom independently of one another, and substituent R 8, R9, R10 are selected from hydrogen or methyl independently of one another.

3. compound according to claim 1 or its pharmacy acceptable salt: R2 are selected from fluorine, chlorine, bromine, iodine, itrile group, methoxyl group or sulfydryl.

4. compound according to claim 3 or its pharmacy acceptable salt: wherein, R2 is selected from fluorine.

5. according to any described compound or its pharmacy acceptable salt among the claim 1-4: wherein, R1 is selected from:

6. compound according to claim 5 or its pharmacy acceptable salt: wherein, R1 is selected from:

7. compound according to claim 1 or its pharmacy acceptable salt are selected from:

1-(4-chloro-3-(trifluoromethyl) phenyl)-3-(2-fluoro-4-(2-(1-methyl-4-pyrazolyl)-4-pyridyloxy) phenyl) urea;

1-(4-chloro-3-(trifluoromethyl) phenyl)-3-(2-fluoro-4-(2-(4-pyrazolyl)-4-pyridyloxy) phenyl) urea;

1-(4-chloro-3-(trifluoromethyl) phenyl)-3-(2-fluoro-4-(2-(4-imidazolyl)-4-pyridyloxy) phenyl) urea;

1-(4-chloro-3-(trifluoromethyl) phenyl)-3-(2-fluoro-4-(2-(3-pyrryl)-4-pyridyloxy) phenyl) urea;

1-(4-chloro-3-(trifluoromethyl) phenyl)-3-(2-fluoro-4-(2-(3-thienyl)-4-pyridyloxy) phenyl) urea;

1-(4-chloro-3-(trifluoromethyl) phenyl)-3-(2-fluoro-4-(2-(1-methyl-4-imidazolyl)-4-pyridyloxy) phenyl) urea;

1-(4-chloro-3-(trifluoromethyl) phenyl)-3-(2-fluoro-4-(2-(1-methyl-3-pyrryl)-4-pyridyloxy) phenyl) urea;

1-(4-chloro-3-(trifluoromethyl) phenyl)-3-(2-chloro-4-(2-(1-methyl-4-pyrazolyl)-4-pyridyloxy) phenyl) urea;

1-(4-chloro-3-(trifluoromethyl) phenyl)-3-(2-bromo-4-(2-(1-methyl-4-pyrazolyl)-4-pyridyloxy) phenyl) urea;

1-(4-chloro-3-(trifluoromethyl) phenyl)-3-(2-iodo-4-(2-(1-methyl-4-pyrazolyl)-4-pyridyloxy) phenyl) urea;

1-(4-chloro-3-(trifluoromethyl) phenyl)-3-(2-methoxyl group-4-(2-(1-methyl-4-pyrazolyl)-4-pyridyloxy) phenyl) urea;

1-(4-chloro-3-(trifluoromethyl) phenyl)-3-(2-cyanic acid-4-(2-(1-methyl-4-pyrazolyl)-4-pyridyloxy) phenyl) urea;

1-(4-chloro-3-(trifluoromethyl) phenyl)-3-(2-sulfydryl-4-(2-(1-methyl-4-pyrazolyl)-4-pyridyloxy) phenyl) urea.

8. compound according to claim 1 or its pharmacy acceptable salt, it is:

9. the preparation method of formula IV compound or its pharmacy acceptable salt:

10. preparation method according to claim 9 is characterized in that: 10.2g 2-fluoro-4-(2-(1-methyl-4-pyrazolyl)-4-pyridyloxy) aniline is dissolved in the methylene dichloride, and nitrogen protection adds 7.95g 3-trifluoromethyl-4-chloro-benzene isocyanic ester down; There are a large amount of solids to separate out after stirring 12h under the room temperature; System is concentrated, suction filtration, ETHYLE ACETATE is washed; Oven dry gets formula IV compound.

11. the compound of formula I or the preparation method of its pharmacy acceptable salt are: the aminophenols thing under the highly basic effect with 2; 4-dihalo pyridine generation nucleophilic substitution reaction; The Suzuki reaction takes place in products therefrom and five yuan of aromatic heterocyclic boric acid pinacol esters under the catalysis of palladium catalyst, products therefrom and 3-trifluoromethyl-4-chloro-benzene isocyanate reaction obtains the compound of formula I.

12. formula II compound or its pharmacy acceptable salt:

13. the preparation method of formula II compound or its pharmacy acceptable salt:

14. preparation method according to claim 13 is characterized in that: 1.34g 3-fluoro-4-amino-phenol is dissolved in the anhydrous dimethyl sulphoxide, behind the logical nitrogen 10min, adds potassium tert.-butoxide 1.24g; After stirring 30min under the room temperature, add 1.32g 2-chloro-4-fluorine pyridine, again reaction system slowly is warming up to 80 ℃ and insulation reaction 2h, TLC detects demonstration and reacts completely; After being cooled to room temperature, add entry and ETHYLE ACETATE, behind the separatory water with ethyl acetate extraction twice, the combined ethyl acetate layer; Wash 2 times, again with saturated common salt washing 1 time, anhydrous sodium sulfate drying; Filter, concentrate, get formula II compound behind the gained bullion column chromatography purification.

15. formula III compound or its pharmacy acceptable salt:

16. the preparation method of formula III compound or its pharmacy acceptable salt:

17. preparation method according to claim 16; It is characterized in that: under the nitrogen protection; 1.6g 2-chloro-4-(3-fluoro-4-amino-benzene oxygen) pyridine and 1.68g 1-methyl-4-pyrazoles boric acid pinacol ester are dissolved in (THF) in the THF, stir adding 2.77g salt of wormwood and 4.5mL water down, add 0.39g four triphenyl phosphorus palladium catalysts then under the lucifuge; In 70 ℃ of insulated and stirred 24h, the TLC detection reaction is complete; Be cooled to room temperature, reaction system is concentrated, add ETHYLE ACETATE and water then, water is with ethyl acetate extraction twice behind the separatory, and the combined ethyl acetate layer washes twice, and saturated common salt is washed once, and anhydrous sodium sulfate drying filters, and concentrates; Get formula III compound behind the thick product column chromatography purification.

18. the said compound of claim 1 or its pharmacy acceptable salt are used for preventing or the purposes of the medicine of treatment and raf SU11752 diseases associated in preparation.

19. purposes according to claim 18, wherein raf SU11752 diseases associated is melanoma, liver cancer, kidney, acute leukemia, nonsmall-cell lung cancer, prostate cancer, thyroid carcinoma, skin carcinoma, colorectal carcinoma, carcinoma of the pancreas, ovarian cancer, mammary cancer, myelodysplastisches syndromes, the esophageal carcinoma, gastrointestinal cancer or mesothelioma.