CN102532113B - Aryl urea derivative - Google Patents

Abstract

The invention discloses aryl urea derivative indicated through a group of general formula structures, which belongs to the field of medicinal chemistry. The invention further discloses a preparation method for the aryl urea derivative indicated by the general formula structures. The compound of the aryl urea derivative has an effect of raf kinase inhibitor and can be used for preparing medicine for curing tumor diseases.

Description

Arylurea Derivatives

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:

Formula I

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;

formula a

R2 is selected from hydrogen, hydroxyl, nitro, amino, C1-4 alkyl.

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 hydrogen, hydroxyl, nitro, amino, or methyl.

R2 is further selected from hydrogen.

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

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

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

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

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

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

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

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

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

1-(4-Chloro-3-(trifluoromethyl)phenyl)-3-(2-nitro-4-(2-(1-methyl-4-pyrazolyl)-4-pyridyloxy ) phenyl) urea.

Particularly preferred compounds of the invention are:

Formula IV

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

The pharmaceutically acceptable salts of 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 reacts with 2,4-dihalopyridine under the action of a strong base for nucleophilic substitution reaction , the obtained product undergoes a Suzuki reaction with the five-membered aromatic heterocyclic boronic acid pinacol ester under the catalysis of a palladium catalyst, and the obtained product reacts with 3-trifluoromethyl-4-chloro-phenylisocyanate to obtain a compound of the general formula I.

As a further example, the preparation method of the 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-(4-aminophenoxy)pyridine.

Formula II

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

Formula III

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

Step 1: Dissolve 4.35g of 4-aminophenol in anhydrous dimethyl sulfoxide, pass nitrogen gas for 10min, add 4.7g of potassium tert-butoxide, stir at room temperature for 30min, then add 5g 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, water and ethyl acetate are added. After liquid separation, the aqueous phase is extracted twice with ethyl acetate, and the ethyl acetate is combined layer, washed twice with water, 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 the protection of nitrogen, dissolve 5.7g 2-chloro-4-(4-aminophenoxy)pyridine and 6.47g 1-methyl-4-pyrazoleboronic acid pinacol ester in tetrahydrofuran, and add them under stirring 10.7g of potassium carbonate and 17.1mL of water, then add 1.5g of tetrakistriphenylphosphine palladium catalyst in the dark, keep stirring at 70°C for 24h, and TLC detects that the reaction is complete. Cool to room temperature, concentrate the reaction system, then add ethyl acetate and water, separate the aqueous phase and extract it twice with ethyl acetate, combine the ethyl acetate layer, wash twice with water, once with saturated brine, and dry over anhydrous sodium sulfate , filtered and concentrated. The crude product is purified by column chromatography to obtain the compound of formula III;

Step 3: Dissolve 6.7g 4-(2-(1-methyl-4-pyrazolyl)-4-pyridyloxy)aniline in ethyl acetate, add 5.6g 3-trifluoromethyl under nitrogen protection -4-Chloro-phenylisocyanate, after stirring at room temperature for 12 hours, a large amount of solids precipitated, 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: 1-(4-chloro-3-(trifluoromethyl)phenyl)-3-(4-(2-(1-methyl-4-pyrazolyl)-4-pyridyloxy) Phenyl)urea

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

Dissolve 4.35g (39.8mmol) of 4-aminophenol in 40mL of anhydrous dimethyl sulfoxide, and after 10min of nitrogen gas, add 4.7g (41.8mmol) of potassium tert-butoxide, stir at room temperature for 30min, then add 5g (38.0 mmol) of 2-chloro-4-fluoropyridine, 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, and concentrated, and the obtained crude product was purified by column chromatography to obtain 7.26g of light yellow solid with a yield of 86.8%. ¹ H NMR (300MHz, CDCl ₃ ): δ4.07(br s, 2H), 6.72(d, J=8.7Hz, 2H), 6.75-6.77(m, 2H), 6.88(d, J=8.7Hz, 2H), 8.19 (d, J = 5.4Hz, 1H). MS (ESI+): 221.1 [M+H] ⁺

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

Under nitrogen protection, 5.7g (25.9mmol) of 2-chloro-4-(4-aminophenoxy)pyridine and 6.47g (31.1mmol) of 1-methyl-4-pyrazoleboronic acid pinacol ester were dissolved in 70mL In tetrahydrofuran (THF), add 10.7g (77.5mmol) potassium carbonate and 17.1mL water under stirring, then add 1.5g (1.29mmol) tetrakistriphenylphosphine palladium catalyst under the dark, keep stirring at 70°C for 24h, TLC detection The response is 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 5.85 g of light yellow solid with a yield of 85%. ¹ H NMR (300MHz, CDCl ₃ ): δ3.84(br s, 2H), 3.92(s, 3H), 6.60(dd, J=2.4, 5.7Hz, 1H), 6.71(d, J=8.7Hz, 2H), 6.91(d, J=8.7Hz, 2H), 6.94(d, J=2.1Hz, 1H), 7.86(s, 2H,), 8.34(d, J=5.7Hz, 1H).MS(ESI+ ): 221.1[M+H] ⁺

1c: 1-(4-chloro-3-(trifluoromethyl)phenyl)-3-(4-(2-(1-methyl-4-pyrazolyl)-4-pyridyloxy)phenyl ) Synthesis of urea:

Dissolve 6.7g (25.1mmol) of 4-(2-(1-methyl-4-pyrazolyl)-4-pyridyloxy)aniline in 80mL of ethyl acetate, and add 5.6g (25.1mmol) of 3-Trifluoromethyl-4-chloro-phenylisocyanate, after stirring at room temperature for 12 hours, a large amount of solids precipitated out, the system was concentrated to 40mL of solvent remaining, filtered with suction, washed with ethyl acetate, and dried to obtain 7.5g of white solid, collected The rate is 60.9%. ¹ H NMR (300MHz, DMSO-d ₆ ): δ3.88(s, 3H), 6.63(d, J=3.9Hz, 1H), 7.15(d, J=8.4Hz, 2H), 7.21(s, 1H ), 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+): 488.1[M+H] ⁺

Example 2: 1-(4-Chloro-3-(trifluoromethyl)phenyl)-3-(4-(2-(4-pyrazolyl)-4-pyridyloxy)phenyl)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 ): δ6.40(br s, 1H), 6.63(d, J=3.9Hz, 1H), 7.15(d, J=8.4Hz, 2H), 7.21(s, 1H), 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+): 474.1[M+H] ⁺

Example 3: 1-(4-Chloro-3-(trifluoromethyl)phenyl)-3-(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 ): δ6.30(br s, 1H), 6.63(d, J=3.9Hz, 1H), 7.15(d, J=8.4Hz, 2H), 7.21(s, 1H), 7.57-7.69(m, 4H), 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+): 474.1[M+H] ⁺

Example 4: 1-(4-chloro-3-(trifluoromethyl)phenyl)-3-(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 ): δ6.35(br s, 1H), 6.63(d, J=3.9Hz, 1H), 7.15(d, J=8.4Hz, 2H), 7.21(s, 1H), 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+): 473.1[M+H] ⁺

Example 5: 1-(4-Chloro-3-(trifluoromethyl)phenyl)-3-(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), 6.63(d, J=3.9Hz, 1H), 7.15(d, J=8.4Hz, 2H), 7.21(s, 1H ), 7.57-7.69(m, 4H), 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+): 490.1[M+H] ⁺

Example 6: 1-(4-chloro-3-(trifluoromethyl)phenyl)-3-(4-(2-(1-methyl-4-imidazolyl)-4-pyridyloxy)benzene base) 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), 6.63(d, J=3.9Hz, 1H), 7.15(d, J=8.4Hz, 2H), 7.28(s, 1H ), 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+): 488.1[M+H] ⁺

Example 7: 1-(4-chloro-3-(trifluoromethyl)phenyl)-3-(4-(2-(1-methyl-3-pyrrolyl)-4-pyridyloxy)benzene base) 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 ): δ3.67(s, 3H), 6.63(d, J=3.9Hz, 1H), 6.82(s, 1H), 7.15(d, J=8.4Hz, 2H ), 7.28(s, 1H), 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+): 487.1[M+H] ⁺

Example 8: 1-(4-chloro-3-(trifluoromethyl)phenyl)-3-(2-hydroxyl-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 4-amino-1,3-benzenediol.

¹ H NMR (300MHz, DMSO-d ₆ ): δ3.88(s, 3H), 6.21(br s, 1H), 6.63(d, J=5.4Hz, 1H), 7.15(d, J=8.4Hz, 2H), 7.57-7.69(m, 4H), 7.96(s, 1H), 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+): 504.1[M+H] ⁺

Example 9: 1-(4-chloro-3-(trifluoromethyl)phenyl)-3-(2-nitro-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-nitro-4-aminophenol.

¹ H NMR (300MHz, DMSO-d ₆ ): δ3.81(s, 3H), 6.61(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+): 533.1[M+H] ⁺

Example 10: 1-(4-chloro-3-(trifluoromethyl)phenyl)-3-(2-methyl-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-methyl-4-aminophenol.

¹ H NMR (300MHz, DMSO-d ₆ ): δ2.23(s, 3H), 3.81(s, 3H), 6.61(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+): 502.1[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 14.2g (12.6-15.8)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 628.42±149.87mg and 262.25±61.82mg respectively, and the tumor weights of the medication 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 326.85±58.21mm3 and 110.85±47.66mm3 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 Lung metastatic nodules

Of the 8 rats in the control group, 7 had lung metastases, 3 had 1 metastatic nodule, and the rest had 3-5 metastatic nodules. No lung metastatic nodule was 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.

Experimental example 3: Evaluation of the curative effect of the compound of the present invention on 786-O nude mouse xenograft tumor

1. Purpose of the test

The inhibitory effect of the compound of the present invention on the growth of transplanted tumor in human 786-O nude mice was investigated, and the toxicity of the compound was preliminarily observed.

2. Test material

Test compound: Compound of Example 1 of the present invention (No. A1); production provider: Founder Pharmaceutical Research Institute Co., Ltd.

Positive control drug: Sorafenib; production provider: Founder Pharmaceutical Research Institute Co., Ltd.

Comparative compound:

Comparative Compound 1 (No. B1): Chinese Patent Publication No. CN101801383A, publication date: 2010-08-11, the compound "1-(4-chloro-3-(trifluoromethane Base) phenyl) -3-(2-fluoro-4-(2-(1-methyl-1H-pyrazol-4-yl) pyridin-4-yloxy) phenyl) urea", preparation method: Same as Example 1 of the present invention, wherein the raw material 4-aminophenol is replaced by 3-fluoro-4-aminophenol.

Comparative compound 2 (No. B2): Chinese Patent Publication No. CN101801383A, publication date: 2010-08-11, the compound "1-(4-(2- (1-Methyl-1H-pyrazol-4-yl)pyridin-4-yloxy)phenyl)-3-(3-(trifluoromethyl)phenyl)urea", the preparation method is implemented with the present invention Example 1, wherein the starting material 3-trifluoromethyl-4-chloro-phenylisocyanate was replaced by 3-trifluoromethyl-phenylisocyanate.

Solvent material:

Absolute ethanol

Shanghai Runjie Chemical Reagent Co., Ltd.

Product batch number: 20100525

Cremophor EL: polyoxyethylene castor oil

Beijing Fengli Jingqiu Trading Co., Ltd.

Product batch number: 57219156PO

HPMC-K4M: Hypromellose

Production Unit: Shanghai Colorcon Coating Technology Co., Ltd.

Batch number: PD224587

Appearance: white powder

SLS: Sodium Lauryl Sulfate

Production unit: Anhui Shanhe Pharmaceutical Excipients Co., Ltd.

Batch number: 080602

Appearance: white powder

Test animals:

Strain: BALB/C nude mice (SPF grade)

Source: Shanghai Slack Experimental Animal Co., Ltd.

Production license number: SCXK (Shanghai) 2007-0005

License number: SYXK (Shanghai) 2009-0075

Gender: Male.

Age: 6 weeks old

Transplanted Tumor Strains

Name: Human renal carcinoma 786-O

Source: Breeds kept in our laboratory, BALB/C nude mice subcutaneously transplanted and maintained

3. Test method

Dosing preparations and configuration methods:

Vehicle 1: 0.5% HPMC-K4M & 0.2% SLS in water.

Preparation method: Weigh an appropriate amount of HPMC-K4M and SLS into a 250mL blue cap bottle, add an appropriate amount of ultrapure water, and stir with a magnetic stirrer for more than 4hrs; after all the solid substances are evenly dispersed, let the liquid stand still for 2-3hrs before use.

Vehicle 2: 50% Ethanol & 50% Polyoxyethylene Castor Oil

Preparation method: Weigh an appropriate amount of polyoxyethylene castor oil, put it into a 250ml bottle with a blue cap, add the same volume of absolute ethanol, oscillate and shake, and mix the two evenly.

Sorafenib preparation method: Weigh an appropriate amount of Sorafenib into a 50ml centrifuge tube, add an appropriate weight of solvent 2 to the centrifuge tube; shake and mix on a vortex mixer for about 1 min, put it in an ice bath for ultrasonic treatment for more than 20 minutes; wait until the compound is completely After dissolving (uniformly dispersed), add an appropriate volume of ultrapure water, shake the centrifuge tube to make the suspension evenly mixed, the concentrations of alcohol and polyoxyethylene castor oil in the suspension are both 12.5%; store at 4°C for later use.

Compound preparation method of Example 1: Weigh an appropriate amount of sample into a 50ml centrifuge tube; add an appropriate weight of solvent 2 into the centrifuge tube; shake and mix on a vortex mixer for about 1 minute, then put it in an ice bath for ultrasonic treatment for more than 20 minutes; After the compound is completely dissolved (uniformly dispersed), add an appropriate volume of ultrapure water, shake the centrifuge tube, and mix the suspension evenly. The concentration of alcohol and polyoxyethylene castor oil in the suspension is both 12.5%; store at 4°C for later use .

Vehicle control: 12.5% ethanol & 12.5% polyoxyethylene castor oil aqueous solution

Preparation method: Weigh an appropriate amount of polyoxyethylene castor oil, put it into a 250ml bottle with a blue cap, add the same volume of absolute ethanol, oscillate and shake, and mix the two evenly. Add an appropriate volume of ultrapure water so that the final concentrations of both ethanol and polyoxyethylene castor oil are 12.5%.

Animal model preparation:

The well-grown 786-O solid tumor was taken, cut into small pieces of uniform size about 1 mm ³ under aseptic conditions, and inoculated subcutaneously in the right forelimb armpit of nude mice with a trocar. The tumor growth was observed regularly until the tumor volume grew to 250-550 mm ³ .

Grouping and administration:

Animals with too large or too small tumor volume and irregular tumor shape were eliminated, and a total of 72 tumor-bearing mice with a tumor volume of ^250-550mm3 in good condition were selected, and the animals were divided into 9 groups, which were respectively used as vehicle control group and 2 mice. Positive control group, 2 test product groups and 4 control compound groups. Positive control, test product and control compound group were all administered by intragastric administration, once a day; the vehicle control group was given 12.5% ethanol & 12.5% polyoxyethylene castor oil ultrapure aqueous solution, once a day; 10ml/kg.

During the administration period, the tumor diameter was measured twice a week, and the tumor volume was calculated, and the body weight of the animal was recorded at the same time. The state of the animals was observed each time the drug was administered, and the abnormal state was recorded.

To collect plasma:

Animals were fasted from 5:30 p.m. the day before the end of the experiment (water was not allowed). Each dosing group was divided into 4 subgroups. Blood was collected at 0hr, 2hr, 4hr, and 8hr time points (anticoagulated with sodium heparin) after intragastric administration, and plasma was centrifuged at 2000g for 10min. Protected from light and stored in a -80°C refrigerator for further testing.

To sacrifice an animal:

The animals were sacrificed with CO ₂ , the tumors were removed, weighed, and photographed. The animals were dissected grossly, and the viscera were observed with naked eyes for abnormalities.

Observation indicators:

1) Tumor volume (TV), the calculation formula is:

TV=1/2×a×b ²

Where a is the long diameter of the tumor, b is the short diameter,

2) Relative tumor volume (relative tumor volume, RTV), the calculation formula is:

RTV=Vt/V ₀ ,

V ₀ is the tumor volume measured at the time of grouping (ie d0), and Vt is the tumor volume at each measurement.

3) Relative tumor proliferation rate T/C (%):

T/C(%)＝(T _RTV /C _RTV )×100%

T _RTV : relative tumor volume of the treatment group;

C _RTV : vehicle control group relative tumor volume.

4) Tumor growth inhibition rate (GI):

GI＝100×{1-[(T _Vt -T _V0 )/(C _Vt -C _V0 )]}

T _Vt : Tumor volume measured each time in the treatment group;

T _V0 : initial tumor volume in the treatment group;

C _Vt : Tumor volume measured each time in the vehicle control group;

C _V0 : the initial tumor volume of the vehicle control group;

5) Tumor Inhibition Rate (IR)

IR＝(W _C -W _T )/WC

Among them, W _C and W _T represent the average tumor weight of the vehicle control group and the average tumor weight of the drug-administered group, respectively.

statistical methods:

The experimental data were calculated and related statistical processing with Microsoft Office Excel 2003 software. Unless otherwise specified, the data are expressed as mean ± standard error (Mean ± S.E), and t-test was used for comparison between two groups.

4. Test results

4.1 Effects of each group of compounds on the growth of 786-O nude mice xenografts

Thirty-one days after the 786-O tumor mass transplantation, the tumor mass grew to about 380mm ³ , and the drugs were divided into groups and administered orally once a day for a total of 14 days.

The specific results are shown in Table 2:

4.2 Effects of each group of compounds on the tumor weight of 786-O nude mice xenografts

The specific results are shown in Table 3:

Table 3 Effects of each group of compounds on human kidney cancer 786-O nude mice xenograft tumor weight

Note: 1. Compared with Vehicle Control group, *P<0.05, **P<0.01;

2./ All animals died, no corresponding data.

Experimental results:

Oral administration of the compound of Example 1 showed obvious anti-tumor activity in vivo in human 786-O nude mouse xenograft tumor model, and its drug effect was significantly better than that of Bayer's first-line drug Sorafenib for the treatment of advanced renal cancer.

In addition, the compound of Example 1 is obviously better than the reference compounds B1 and B2, which are highly toxic, have low drug efficacy, and basically have no druggability.

Experimental Example 4: 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 compounds of Examples 2-10 showed raf kinase inhibitory activity with IC50 all less than 5 μM.

Claims (6)

1. The compound shown in formula IV or its pharmaceutically acceptable salt, it is: 2. according to the preparation method of formula IV compound shown in claim 1 or its pharmaceutically acceptable salt: 3. preparation method according to claim 2 is characterized in that: 6.7 parts by weight 4-(2-(1-methyl-4-pyrazolyl)-4-pyridyloxy) aniline is dissolved in ethyl acetate , under nitrogen protection, add 5.6 parts by weight of 3-trifluoromethyl-4-chloro-phenylisocyanate, stir at room temperature for 12 hours, a large amount of solids precipitate out, concentrate, filter with suction, wash with ethyl acetate, and dry to obtain the compound of formula IV . the 4. The preparation method of the compound shown in formula IV according to claim 1 or a pharmaceutically acceptable salt thereof, is characterized in that: the aminophenol compound reacts with 2,4-dihalopyridine under the action of a strong base Nucleophilic substitution reaction, the resulting product reacts with the five-membered aromatic heterocyclic base boronic acid pinacol ester under the catalysis of a palladium catalyst, and the resulting product reacts with 3-trifluoromethyl-4-chloro-phenylisocyanate to obtain the formula IV the indicated compounds. the 5. Use of the compound of claim 1 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for preventing or treating diseases related to raf kinase inhibitors. the 6. The use according to claim 5, wherein the 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 cancer, ovarian cancer, breast cancer, myelodysplastic syndrome, esophageal cancer, gastrointestinal cancer, or mesothelioma. the