CN102153558B

CN102153558B - Derivative of multi-target antitumor inhibitor 2-aminopyrrole-triazine and synthesis method thereof

Info

Publication number: CN102153558B
Application number: CN2011100432181A
Authority: CN
Inventors: 孟佳伦
Original assignee: YANGZHOU YONGJI MEDICAL NEW TECHNOLOGY CO LTD
Current assignee: Qingdao Jia Jia Novartis Medical Technology Co Ltd
Priority date: 2011-02-23
Filing date: 2011-02-23
Publication date: 2012-11-21
Anticipated expiration: 2031-02-23
Also published as: CN102153558A

Abstract

The invention discloses a derivative of a multi-target antitumor inhibitor, namely 2-aminopyrrole-triazine and a synthesis method thereof, and relates to the technical field of the treatment of abnormal cells in mammals. The derivative is synthesized by taking ethyl isocyanoacetate as a starting point through a chemical synthesis method, and has the advantages of mature process, short production period, environment friendliness, low cost and the like. The product can be used as a heat shock protein (Hsp) 90 multi-target antitumor inhibitor, can inhibit Hsp90, and promote the degradation of Hsp90 effect proteins which play an important role in tumor growth signal pathways through ubiquitination, so that multiple targets of the tumor growth signal pathways are blocked and the growth of tumor is effectively prevented. The derivative has higher affinity with tumor cells, can selectively kill the tumor cells, and promote the degradation of primer proteins thereof through ubiquitin-proteasome pathways, so that the effects of tumor treatment and resistance of the targets are achieved, and the derivative has strong action and small side effect.

Description

Multi-target antitumor inhibitor 2-aminopyrrole-triazine derivative and synthesis method thereof

Technical Field

The present invention relates to the field of treating abnormal cells, particularly cancer cells, in a mammal.

Background

Heat shock proteins (Hsps) were discovered by geneticist Ritossa in 1962. This is a class of proteins that are highly conserved during biological evolution and widely present in prokaryotes and eukaryotes. Hsp is a multigene family in which the products of different genes differ in their expression, function and localization in cells. It is common in the art to distinguish between members of this family by differences in molecular weight, such as Hsp90, Hsp70, and Hsp 27. In addition, a portion of heat shock proteins are classified as related glycoproteins, such as GRP94 and GRP 75. Hsps act as molecular chaperones in the body, participate in maintaining the normal folding of proteins to form the normal structure of proteins, and play an important role in regulating the balance of protein synthesis and degradation and protein localization. When the cells are stimulated by various environments, the expression of heat shock protein in the cells is increased, the cells can be rapidly and transiently protected against the attack of endogenous stress, the repair function of the cells is enhanced, and the tolerance degree of the cells to the stress is improved. Many diseases occur in humans and are associated with misfolding of proteins. Therefore, the development of the medicine for regulating the molecular chaperone becomes a new idea. A large number of documents show that Hsps are closely related to the occurrence and development of tumors. There is evidence that the expression of Hsps in tumors changes at different stages of tumor progression. Moreover, tumor cells adapt to the harsh environment of the surroundings more readily than normal cells, which is clearly closely related to the role played by molecular chaperones. Recent studies suggest that Hsp90 plays a very important role in the survival of tumor cells, and is an important component of many oncogene pathways; heat shock protein 90(Hsp90) has become a new target for anti-tumor, and the anti-tumor activity of some natural products is directed against Hsp90 as a molecular target. Hsp90 and its inhibitors are the hot spot and frontier of current antitumor studies.

Biological properties of Hsp 90: hsp90 is one of the most active chaperones in the cell, and Hsp90 is relied upon for the normal functioning of many signal transduction proteins. When the cell is in stress reaction, it can interact with the protein whose self conformation is changed by environmental stimulation, ensure the proper folding of the protein and prevent the non-specific aggregation of the protein, thus maintaining the normal activity of the cell. Hsp90 is also an important buffer factor in cell mutation, and presumably it can also correct the misfolding of the mutant protein. Hsp90 is also an important regulator of cell function under normal physiological conditions. It is known at present to regulate the spatial structure and mutations of about 40 proteins, which can be mainly divided into three classes: steroid hormone receptors; serine/threonine or tyrosine kinases; other proteins such as mutant p53, telomerase hTERT subunit, etc. All these proteins are important regulatory proteins in the course of physiological and biochemical changes of cells. Two types, Hsp90ct and Hsp9013, exist in the cytoplasm, GRP94 is present in the endoplasmic reticulum, and Hsp 75/tumor necrosis factor receptor-related protein1 (tumor necrosis factor receptor associated protein1, TRAP1) is present in the mitochondrial matrix. It is now believed that the mode of action of these 4 members is almost identical, but because of their different localization within the cell, the proteins to which they bind differ. For example, the ErbB2 receptor, a tyrosine kinase, is a downstream protein (client protein) specific to GRP94, the type I tumor necrosis factor receptor (tnfr1), and the retinal glioma protein r (retinoblastoma protein) are downstream proteins of TRAP 1. The Hsp90 monomer is formed by a conserved 25 kDa N-terminus and a 55 kDa c-terminus joined by a linking region (TRAP1 does not contain this region). Both ends can be combined with downstream protein or other substrate polypeptide such as coactive molecular chaperone. In addition, the N-terminal has a special ATP binding site, which has a structure similar to that of Topo II 13 and is a novel nucleotide binding domain.

Hsp90 functions in dependence on the presence of ATP, and the ATP/ADP binding site assumes the role of a conformational transition region, regulating the assembly of the multiple chaperone complex it participates in. Hsp90 in an ADP-bound conformation, Hsp90 forms a complex with p60HOP to bind Hsp 70. Since the effect of Hsp90 is usually that proteins bind to the Hsp 70/Hsp 40 complex first and then to Hsp 90. When ATP is substituted for ADP, Hsp90 undergoes a conformational change and the p60HOP and Hsp 70/Hsp 4O complexes are released, while additional co-chaperones, including p23, to which certain immunophilins or p50cdc37 bind, stabilize the actor protein and are in a conformational state that is capable of binding to a ligand (if the actor protein is a steroid hormone receptor) or of responding to a stimulus (e.g., the actor protein is a protein kinase). This conversion is of course a dynamic, bi-directional process. When ATP is hydrolysed to ADP, the conformation of Hsp90 is changed again, and the co-chaperone to which it is bound undergoes a corresponding switch again, and the bound actor protein is released and degraded by the action of proteasomes ".

In the early 9O's of the 2O century, Srivastava demonstrated that heat shock proteins extracted from tumor cells could lead to strong immune responses in the host, probably because they could serve as molecular chaperones for some tumor antigens. Ciocca et al, in 1992, have found that breast cancer cells that highly express Hsps are resistant to some chemotherapeutic drugs. These results all suggest that Hsps may be chaperones for some proteins essential in tumor cell growth. Since Hsp90 has many downstream proteins involved in the regulation of different signal transduction pathways, the mechanism of action in the development of tumor development is complicated. Studies on the effect of Hsp90 were found more by using inhibitors thereof. A large number of experiments also demonstrated that Hsp90 has different effects after inhibition in different cell lines, which is related to different genetic backgrounds and biological characteristics of different tumor cells. However, in general, the spectrum of antitumor effects of Hsp90 inhibitors is quite broad.

In 1992, Whitesell et al found that benzoquinone ansamycins (benzoquinone ansamycins) antibiotics exhibited potent antitumor activity both in vitro and in vivo, an effect that is difficult to explain with the known inhibitory effects on some tyrosine kinases. Intensive research shows that the antibiotics, especially GA, can be specifically combined with Hsp90 to block the combination of Hsp90 and v-Src protein, thereby causing the protein to be unstable and inhibiting the activity of v.src kinase. The discovery of this class of inhibitors makes Hsp90 an exciting new target for tumor therapy. The current Hsp90 inhibitors have entered clinical trials.

Geldanamycin (ga), the earliest discovered Hsp90 inhibitor, has made it possible to study the biological function of Hsp90 and to study the role of Hsp 90-dependent proteins in cancer development. GA belongs to the benzoquinone ansamycin class of antibiotics. These antibiotics are structurally linked by a benzoquinone moiety and a planar macrocyclic ansa bridge. It is a natural product isolated from actinomycetes broth in the first 70 th 20 th century, but it was not until 80 th 20 th century that the compound had been found to have anti-tumor activity. It can reverse malignant change of fibroblast transfected with v-Src proto-oncogene, and its antineoplastic activity is successively verified in vitro experiment and animal body. Originally, such compounds were considered inhibitors of tyrosine kinases. However, as the research goes into, it is found that the anti-tumor effect is mainly dependent on the inactivation of the oncogene protein and protein kinase through the proteolysis route, rather than the direct inhibition of the catalytic activity of the kinase. Immunoprecipitation and affinity experiments revealed that the main mechanism of action of GA was achieved by binding to Hsp 90. Although GA shows good antitumor activity, its serious hepatotoxicity limits its clinical application, yet its unique target of action and biological activity still draws attention from pharmacologists and biologists. At present, the compound is more used as a tool medicine for researching the in-vivo biological function of Hsp90 and is researched as a lead compound for developing a medicine taking Hsp90 as an action target.

Because of its important role in maintaining the normal structure, function and stability of various tumor proteins, Hsp90 has been considered as a new target for tumor therapy. Moreover, the wide distribution of Hsp90 in normal tissues also provides a barrier to its targeting for tumor therapy. Recent in vitro and in vivo experiments found that the affinity of tumor tissue-derived Hsp90 with 17-AAG was 100 times higher than that of normal tissue-derived Hsp 90. Kamal et al believe that this is due to the fact that Hsp90 in tumor tissues usually constitutes an active multi-chaperone complex with other proteins, and that ATPase is also more active than Hsp90 in normal tissues, where Hsp90 is present in an inactive form. Therefore, the discovery of the therapeutic target can provide a novel method with specificity, effectiveness and less side effects for treating the tumor. The search for Hsp90 inhibitors is becoming an ongoing focus of research. Benzoquinone ansamycins have been clinically studied as a first generation Hsp9O inhibitor, and in addition, several natural products of different structures have been shown to inhibit Hsp 90. However, the existing inhibitors have strong toxic and side effects, and limit the clinical application of the inhibitors. In addition, whether a proper administration scheme can be established is also a restrictive factor for the compounds to exert the anti-tumor effect. Therefore, the search for small molecule compounds with good biological activity and small toxic and side effects for resisting Hsp90 has become the current research direction. Examples of such heat shock protein 90 inhibitory compounds include those described in published patent applications including WO2006/071789, WO2006/008503, WO2006/090094, WO 2005/021552.

Disclosure of Invention

The invention aims to provide a main molecular structure of a small molecular compound, a derivative thereof, a heat shock protein 90(Hsp90) inhibitor, namely a multi-target anti-tumor inhibitor 2-aminopyrrole-triazine derivative with strong activity, low toxicity and small side effect.

The chemical molecular formula of the invention is as follows:

wherein,

r1a is hydrogen radical, C₁-C₆Alkyl of (C)₂ -C₈Alkenyl of, C₂-C₈Alkynyl of (A), C₆-C₁₄Aryl of (C)₂-C₉Heteroaryl of (A), C₂-C₉Is iso-cycloalkyl or C₃-C₈Optionally one functional group in the cycloalkyl group of (a);

r1b is hydrogen radical, C₁-C₆Alkyl of (C)₂ -C₈Alkenyl of, C₂-C₈Alkynyl of (A), C₆-C₁₄Aryl of (C)₂-C₉Heteroaryl of (A), C₂-C₉Is iso-cycloalkyl or C₃-C₈Optionally one functional group in the cycloalkyl group of (a);

r2 is Cl, F, C₁-C₃Alkyl or fluoro substituted C1 to 6₁-C₃An alkyl group;

r3 is Cl, F, C₁-C₃Alkyl or fluoro substituted C1 to 6₁-C₃An alkyl group;

r4 is- (C)₁-C₆Alkenyl) -OH, -O- (C)₁-C₆Alkyl) - (C)₁-C₆Alkenyl) -O- (C₁-C₆Alkyl) - (C)₁-C₆Alkenyl)_p-O-(C₁-C₆Alkenyl)_p-(C₆-C₁₀-aryl) - (C)₁-C₆Alkenyl)_p-O-(C₁-C₆Alkenyl)_p-(C₃-C₁₀-cycloalkyl) - (C)₁-C₆Alkenyl)_p-O-(C₁-C₆Alkenyl)_p- (5-10 heterocycle) - (C)₁-C₆Alkenyl)_p-O-(C₁-C₆Alkenyl)_p- (3-10 heterocycle) - (C)₁-C₆Alkenyl)_p-O-(C₂-C₆Alkenyl), -O- (C)₂-C₆Alkenyl) - (3-10 heterocycle), -O- (C)₂-C₆Alkyl) - (3-10 heterocycle) or C₁-C₈Any one functional group of an alkoxy group.

The derivative of the 2-aminophenylpyrrolo [2,1-f ] -1,2, 4-triazine carboxamide is a derivative of a multi-target antitumor inhibitor 2-aminopyrrole-triazine (according to the regulation of sFDA, the multi-target antitumor inhibitor belongs to a class of innovative drugs). The protein can be used as a heat shock protein 90 multi-target anti-tumor inhibitor, can inhibit Hsp90, and promotes the degradation of Hsp90 effector protein playing an important role in a tumor growth signal pathway through ubiquitination, thereby blocking multiple targets of the tumor proliferation growth signal pathway and effectively preventing the growth of tumors. The compound and the salt thereof can promote the degradation of substrate protein thereof through a ubiquitin-proteasome pathway, thereby exerting the anti-tumor effect of the compound and the salt thereof. The invention has definite anti-tumor activity as heat shock protein 90 inhibitor, has higher affinity with tumor cells, and can selectively kill the tumor cells. The specificity of target treatment determines the strong therapeutic effect and the small side effect of the target medicine.

Another object of the present invention is to provide a method for synthesizing the compound having the above molecular structural characteristics.

The synthesis steps are as follows:

1) synthesis of diethyl ester 1H-pyrrole-2, 4 dicarboxylic acid:

dissolving ethyl isocyanoacetate and 1, 8-diazabicyclo [5.4.0] undec-7-ene in tetrahydrofuran to form a solution, adding formaldehyde into the solution, and stirring the reaction mixture to react at the temperature of 50 +/-0.5 ℃; after the reaction is finished, cooling the reaction mixture to normal temperature, respectively washing the reaction mixture with a saturated sodium bicarbonate aqueous solution and a saturated sodium chloride aqueous solution by using ethyl acetate, combining the ethyl acetate organic layers after washing, and obtaining diethyl ester 1H-pyrrole-2, 4 dicarboxylic acid through dehydration, drying and vacuum concentration;

2) synthesis of 1-aminodiethyl-1H-pyrrole-2, 4-dicarboxylic acid:

mixing diethyl 1H-pyrrole-2, 4-dicarboxylic acid, ammonium chloride, methyl trioctyl ammonium chloride, sodium hydroxide aqueous solution and methyl tert-butyl ether to form a solvent, adding concentrated ammonium into the solvent, then adding bleaching water, and stirring the formed reaction mixture to react at room temperature; after the reaction is finished, filtering the reaction mixture, taking filtrate, respectively washing the filtrate with ethyl acetate, saturated sodium bicarbonate water solution and saturated sodium chloride water solution, combining the ethyl acetate organic layers after washing, and obtaining 1-aminodiethyl-1H-pyrrole-2, 4 dicarboxylic acid through dehydration, drying and vacuum concentration;

3) [1,2,4] triazine-6-carboxylic acid for synthesizing diethylamino-4-4-hydroxypyrrolo [2,1-f ]:

mixing and stirring 1-aminodiethyl-1H-pyrrole-2, 4-dicarboxylic acid and formamidine hydrochloride and dimethyl sulfoxide to react at the temperature of 150 +/-0.5 ℃; cooling the reaction mixture to normal temperature, respectively washing the reaction mixture with ethyl acetate, saturated aqueous sodium bicarbonate solution and saturated aqueous sodium chloride solution, combining the organic ethyl acetate layers after washing, dehydrating, drying and vacuum concentrating to obtain a crude product of [1,2,4] triazine-6-carboxylic acid of diethylamino-4-4-hydroxypyrrolo [2,1-f ], and finally purifying by a recrystallization method to obtain purified [1,2,4] triazine-6-carboxylic acid of diethylamino-4-hydroxypyrrolo [2,1-f ];

4) [1,2,4] triazine-6-carboxylic acid for the synthesis of ethyl-2- { (penta) - [ (dimethylamino) methylene ] amino } -4-hydroxypyrrolo [2,1-f ]:

mixing [1,2,4] triazine-6-carboxylic acid of diethylamino-4-hydroxypyrrolo [2,1-f ] with dimethylformamide, thionyl chloride and dichloromethane for reflux reaction; cooling the reaction mixture to normal temperature, respectively washing the reaction mixture with saturated aqueous sodium bicarbonate solution and saturated aqueous sodium chloride solution by using ethyl acetate, combining organic ethyl acetate layers after washing, dehydrating, drying and concentrating in vacuum to obtain a crude product of [1,2,4] triazine-6-carboxylic acid of ethyl-2- { (penta) - [ (dimethylamino) methylene ] amino } -4-hydroxypyrrolo [2,1-f ]; purifying by recrystallization to obtain purified [1,2,4] triazine-6-carboxylic acid of ethyl-2- { (penta) - [ (dimethylamino) methylene ] amino } -4-hydroxypyrrolo [2,1-f ];

5) synthesis of ethyl 4-chloro-2- { (penta) - [ (dimethylamino) methylene ] amino } pyrrolo [2,1-f ] [1,2,4] triazine-6-carboxylic acid:

stirring and reacting [1,2,4] triazine-6-carboxylic acid of ethyl-2- { (penta) - [ (dimethylamino) methylene ] amino } -4-hydroxypyrrolo [2,1-f ] and phosphorus oxychloride at 100 +/-0.5 ℃ to ensure that the color of the reaction mixture becomes uniform brown, and then preserving the heat for 30 minutes; then the reaction mixture was rapidly cooled to 0 ℃, then the pH of the reaction mixture was adjusted to 7.2 with NaOH solution, the white suspension was collected after filtration, washed with ethyl acetate, and the filtered filtrate was scooped up with brine to obtain an organic layer, which was then dehydrated, dried and concentrated in vacuo to obtain a crude product of ethyl 4-chloro-2- { (penta) - [ (dimethylamino) methylene ] amino } pyrrolo [2,1-f ] [1,2,4] triazine-6-carboxylic acid; finally purifying by silica gel column to obtain purified ethyl 4-chloro-2- { (penta) - [ (dimethylamino) methylene ] amino } pyrrolo [2,1-f ] [1,2,4] triazine-6-carboxylic acid;

6) [1,2,4] triazine-6-carboxylic acid to synthesize ethyl 4- (2, 4-dichloro-6-hydroxyphenyl) -2- { (penta) - [ (dimethylamino) methylene ] amino } pyrrolo [2,1-f ]:

mixing an aqueous sodium carbonate solution with ethyl 4-chloro-2- { (penta) - [ (dimethylamino) methylene ] amino } pyrrolo [2,1-f ] [1,2,4] triazine-6-carboxylic acid, (3, 5-dichloro-2- (hydroxyboron) phenol) (7) and 1,4 dioxane, purging with nitrogen, adding tetrakis (triphenylphosphine) palladium, and stirring at 80 + -0.5 deg.C to react; cooling to room temperature after the reaction is finished, then adding water to form a water mixture, extracting the water mixture by using ethyl acetate, and washing an extracted organic layer by using a saturated sodium chloride aqueous solution; then dehydrated, dried and concentrated in vacuum to obtain a crude product of [1,2,4] triazine-6-carboxylic acid of ethyl 4- (2, 4-dichloro-6-hydroxyphenyl) -2- { (penta) - [ (dimethylamino) methylene ] amino } pyrrolo [2,1-f ]; finally, the crude [1,2,4] triazine-6-carboxylic acid of ethyl 4- (2, 4-dichloro-6-hydroxyphenyl) -2- { (penta) - [ (dimethylamino) methylene ] amino } pyrrolo [2,1-f ] is purified by a silica gel column to obtain purified [1,2,4] triazine-6-carboxylic acid of ethyl 4- (2, 4-dichloro-6-hydroxyphenyl) -2- { (penta) - [ (dimethylamino) methylene ] amino } pyrrolo [2,1-f ];

7) synthesis of [1,2,4] triazine-6-carboxylic acid of diethylamino-4- (2, 4-dichloro-6-hydroxyphenyl) pyrrolo [2,1-f ]:

A4M dioxane solution of hydrochloric acid was reacted with [1,2,4] triazine-6-carboxylic acid of ethyl 4- (2, 4-dichloro-6-hydroxyphenyl) -2- { (penta) - [ (dimethylamino) methylene ] amino } pyrrolo [2,1-f ] and methanol with stirring at room temperature; vacuum evaporating the reaction liquid, neutralizing with saturated sodium carbonate solution, and extracting with ethyl acetate; washing the extracted organic layer with saturated sodium chloride aqueous solution, dehydrating, drying and vacuum concentrating to obtain crude [1,2,4] triazine-6-carboxylic acid product of diethylamino-4- (2, 4-dichloro-6-hydroxyphenyl) pyrrolo [2,1-f ]; finally, the crude product of [1,2,4] triazine-6-carboxylic acid of diethylamino-4- (2, 4-dichloro-6-hydroxyphenyl) pyrrolo [2,1-f ] is purified by a silica gel column to obtain purified [1,2,4] triazine-6-carboxylic acid of diethylamino-4- (2, 4-dichloro-6-hydroxyphenyl) pyrrolo [2,1-f ];

8) synthesis of [1,2,4] triazine-6-carboxylic acid of diethylamino-4- {2, 4-dichloro-6- [2- (hydro-pyrazol-1-yl) ethoxy ] phenyl } pyrrolo [2,1-f ]:

potassium carbonate and 1- (2-chloroethyl) -1H-pyrazole (10), diethylamino-4- (2, 4-dichloro-6-hydroxyphenyl) pyrrolo [2, 1-f)]Is [1,2,4] of]Triazine-6-carboxylic acid, dimethyl formamide 120 plus or minus 0.5^oStirring and reacting under the condition of C; cooling to room temperature after the reaction is finished, adding water and ethyl acetate into the reaction mixture, and stirring for 30 minutes to obtain a water mixture; extracting the aqueous mixture with ethyl acetate, washing the extracted organic layer with saturated aqueous sodium chloride solution, dehydrating, drying, and vacuum concentrating to obtain diethylamino-4- {2, 4-dichloro-6- [2- (hydro-pyrazol-1-yl) ethoxy ] ethyl]Phenyl } pyrrolo [2,1-f]Is [1,2,4] of]Crude triazine-6-carboxylic acid; finally, diethylamino-4- {2, 4-dichloro-6- [2- (hydro-pyrazol-1-yl) ethoxy]Phenyl } pyrrolo [2,1-f]Is [1,2,4] of]Purifying the crude triazine-6-carboxylic acid product with silica gel column to obtain purified diethylamino-4- {2, 4-dichloro-6- [2- (hydro-pyrazol-1-yl) ethoxy group]Phenyl } pyrrolo [2,1-f]Is [1,2,4] of]Triazine-6-carboxylic acid;

9) synthesis of [1,2,4] triazine-6-carboxylic acid of 2-amino-4- {2, 4-dichloro-6- [2- (1H-pyrazol-1-yl) ethoxy ] phenyl } pyrrolo [2,1-f ]:

stirring sodium hydroxide aqueous solution, diethylamino-4- {2, 4-dichloro-6- [2- (hydrogen-pyrazol-1-yl) ethoxy ] phenyl } pyrrolo [2,1-f ], [1,2,4] triazine-6-carboxylic acid and ethanol for reaction at room temperature; after the reaction is finished, adding a hydrochloric acid aqueous solution to obtain a mixture with the pH value of 7.0 +/-1; extracting the mixture with ethyl acetate, washing the extracted organic layer with saturated aqueous sodium chloride solution, dehydrating, drying, and vacuum concentrating to obtain [1,2,4] triazine-6-carboxylic acid of 2-amino-4- {2, 4-dichloro-6- [2- (1H-pyrazol-1-yl) ethoxy ] phenyl } pyrrolo [2,1-f ];

10) synthesis of [1,2,4] triazine-6-carboxamide of 2-amino-N-cyclopropyl-4- (2, 4-dichloro-6- [2- (1H-pyrazol-1-yl) ethoxyphenyl) pyrrolo [2,1-f ]:

1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride and [1,2,4] triazine-6-carboxylic acid, cyclopropylamine, triethylamine and dimethylformamide of 2-amino-4- {2, 4-dichloro-6- [2- (1H-pyrazol-1-yl) ethoxy ] phenyl } pyrrolo [2,1-f ] are stirred to react at room temperature; after the reaction is finished, extracting by using ethyl acetate, washing the extracted organic layer by using a saturated sodium bicarbonate aqueous solution and a saturated sodium chloride aqueous solution, and then dehydrating, drying and vacuum concentrating to obtain a crude product of the [1,2,4] triazine-6-formamide of the 2-amino-N-cyclopropyl-4- (2, 4-dichloro-6- [2- (1H-pyrazol-1-yl) ethoxyphenyl) pyrrolo [2,1-f ]; finally, the crude product of [1,2,4] triazine-6-carboxamide of 2-amino-N-cyclopropyl-4- (2, 4-dichloro-6- [2- (1H-pyrazol-1-yl) ethoxyphenyl) pyrrolo [2,1-f ] was purified by a silica gel column to obtain purified [1,2,4] triazine-6-carboxamide of 2-amino-N-cyclopropyl-4- (2, 4-dichloro-6- [2- (1H-pyrazol-1-yl) ethoxyphenyl) pyrrolo [2,1-f ].

In addition, the synthesis steps of the 3, 5-dichloro-2- (hydroxyboron) phenol are as follows:

1) synthesizing diiodo-3, 5-dichlorophenol:

mixing p-3, 5-dichlorophenol and anhydrous toluene, and adding a sodium hydride aqueous solution at 0 ℃ under a nitrogen atmosphere for reaction; the reaction mixture was warmed to room temperature and stirred for 30 minutes, the resulting suspension was cooled back to 0 ℃ and then iodine was added for further reaction; stirring the final reaction mixture, placing the reaction mixture in an environment of between 0 and room temperature for at least 24 hours, sequentially quenching the reaction mixture by using a hydrochloric acid aqueous solution, extracting the reaction mixture by using ethyl acetate, separating by using an ethyl acetate layer, quenching and washing by using a saturated sodium chloride aqueous solution, and dehydrating, drying and vacuum-concentrating a quenched organic layer to obtain a crude diiodo-3, 5-dichlorophenol product; purifying the crude product of diiodo-3, 5-dichlorophenol by using a silica gel column to obtain purified diiodo-3, 5-dichlorophenol;

2) synthesis of 1, 5-dichloro-3- (methoxyethoxy) -2-iodobenzene:

stirring diiodo-3, 5-dichlorophenol, chloromethoxyethane, cesium carbonate and dimethylformamide for reaction at room temperature; quenching and washing the reacted mixture by water and salt water in sequence, and dehydrating, drying and vacuum-concentrating the quenched organic layer to obtain a crude product of 1, 5-dichloro-3- (methoxyethoxy) -2-iodobenzene; purifying the crude product of the 1, 5-dichloro-3- (methoxyethoxy) -2-iodobenzene by using a silica gel column to obtain purified 1, 5-dichloro-3- (methoxyethoxy) -2-iodobenzene;

3) synthesis of 2- [2, 4-dichloro-6- (methoxyethoxy) phenyl ] -4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolane:

purging 1, 5-dichloro-3- (methoxyethoxy) -2-iodobenzene, 4,5, 5-methyl- [1,3,2] dioxaborone and triethylamine in dioxane solution by nitrogen for 30 minutes, adding palladium acetate and biphenyl-2-yl-dicyclohexylphosphine into a reaction mixture, and stirring the reaction mixture at 80 ℃ until the reaction is finished; after the reaction mixture is cooled, sequentially quenching and washing the reaction mixture by using a saturated ammonium chloride solution, water and a saturated sodium chloride aqueous solution; dehydrating, drying and vacuum concentrating the quenched organic layer to obtain a 2- [2, 4-dichloro-6- (methoxyethoxy) phenyl ] -4,4,5, 5-tetramethyl-1, 3, 2-dioxyboron lane crude product; purifying the crude product of the 2- [2, 4-dichloro-6- (methoxyethoxy) phenyl ] -4,4,5, 5-tetramethyl-1, 3, 2-dioxyboron lane by using a silica gel column to obtain purified 2- [2, 4-dichloro-6- (methoxyethoxy) phenyl ] -4,4,5, 5-tetramethyl-1, 3, 2-dioxyboron lane;

4) synthesis of 3, 5-dichloro-2- (hydroxyboron) phenol:

mixing 2- [2, 4-dichloro-6- (methoxyethoxy) phenyl ] -4,4,5, 5-tetramethyl-1, 3, 2-dioxyboron lane and anhydrous dichloromethane, and adding boron tribromide to react at 0 ℃ in a nitrogen atmosphere; after the reaction is finished, pouring the reaction mixture into water, adding a sodium hydroxide aqueous solution, and adjusting the pH value of the reaction mixture to 10 +/-1; separating to remove the organic layer; adjusting pH value of the separated water layer solution to 3 with 1N HCl solution, extracting with ethyl acetate, washing the extracted organic layer with saturated sodium chloride aqueous solution, dehydrating, drying, and vacuum concentrating to obtain 3, 5-dichloro-2- (hydroxyboron) phenol.

The synthetic method successfully applies a computer-aided drug small molecule design technology, designs and researches anti-cancer subjects, successfully synthesizes brand-new small molecules with high activity, and has the advantages of mature synthetic process, short production period, environmental friendliness, low cost and the like.

Detailed Description

The synthesis method comprises the following steps:

the raw materials used in the invention are all from market.

Hereinafter "DMF" is dimethylformamide, "DMAP" is dimethylaminopyridine, "TBME" is methyl tert-butyl ether, "Me" is methyl, "TEA" is triethylamine, "i-PrOH" is isopropanol, "DMSO" is dimethyl sulfone, "DCM" is dichloromethane, "TLC" is thin layer chromatography, and "MTBE" is methyl tert-butyl ether.

The following reaction steps illustrate one synthetic route of the present invention:

description of the specific operation of each reaction step:

1. the synthesis method of 1H-pyrrole-2, 4 dicarboxylic acid (2) of compound diethyl ester comprises the following steps:

ethyl isocyanoacetate (1) (4.56 g, 40mmol) and 1, 8-diazabicyclo [5.4.0 (9.2 g, 60 mmol) were combined]Dissolving undec-7-ene (DBU) in 200ml of tetrahydrofuran to form a solution, adding 80mmol of p-type formaldehyde into the solution, and stirring the reaction mixture at the temperature of 50 +/-0.5 ℃ for reaction; after 5 hours, the reaction mixture was cooled to normal temperature after the reaction was completed, and the reaction mixture was washed twice with 500ml of ethyl acetate and 100ml of saturated aqueous sodium bicarbonate solution and 100ml of saturated aqueous sodium chloride solution, and then the combined ethyl acetate organic layers were dehydrated and dried with sodium sulfate, and then concentrated in vacuo to collect 3.0 g of crude 1H-pyrrole-2, 4-dicarboxylic acid (2) as diethyl ester with a yield of 36%, which was a pale yellow solid and used directly in the next reaction without further purification.¹H NMR (400MHz, DMSO-D₆).ppm 1.52-1.58 (t,6H)， 1.891.98 (m, 6H)， 2.45-2.55 (m, 4H) ，4.32 (br,s,1H)， 5.23(s, 1H) ，7.09(s, 1H) ，8.23 (s, 1H)。

2. Synthesis of Compound 1-aminodiethyl-1H-pyrrole-2, 4-dicarboxylic acid (3):

diethyl (3.0 g, 14.3mmol) 1H-pyrrole-2, 4-dicarboxylic acid (2), (2.3 g, 42.9 mmol) ammonium chloride, (6.9 g, 17.16 mmol) methyltrioctylammonium chloride and (14.3 ml, 28.6 mmol) 2N aqueous sodium hydroxide were mixed with 200ml methyl tert-butyl ether to form a solvent, 40ml concentrated ammonium was added to the solvent, then 30ml bleaching water was added, and the resulting reaction mixture was stirred at room temperature for 6 hours. After completion of the reaction, the reaction mixture was filtered to remove solid salts, and the filtrate was taken to be saturated with 500ml of ethyl acetate and 100ml of water, respectivelyAqueous sodium bicarbonate and 100ml of saturated aqueous sodium chloride were washed twice, then the combined ethyl acetate organic layers were dried over sodium sulfate and concentrated in vacuo to yield 2.4 g of crude 1-aminodiethyl-1H-pyrrole-2, 4-dicarboxylic acid (3) as a pale yellow solid in 75% yield and used directly in the next reaction without further purification.¹H NMR (400MHz, DMSO-D₆).ppm 1.52-1.58 (t,6H)， 2.45-2.55 (m, 4H)， 4.45 (br,s,2H)， 7.24 (s, 1H)， 8.34 (s, 1H)。

3. Synthesis of [1,2,4] triazine-6-carboxylic acid (4) of the Compound diethylamino-4-hydroxypyrrolo [2,1-f ]:

1-Aminodiethyl-1H-pyrrole-2, 4-dicarboxylic acid (3) (2.4 g, 10.7 mmol) and chloroformamidine hydrochloride (19.7 g, 171.2 mmol) were charged into 200ml of dimethyl sulfoxide (DMSO) solvent and stirred at 150 ℃ for 1 hour. After the reaction is finished, cooling to normal temperature, respectively extracting and washing the reaction mixture twice with 500ml ethyl acetate and 100ml saturated sodium bicarbonate water solution and 100ml saturated sodium chloride water solution, combining the ethyl acetate organic layers after extraction and washing, dehydrating and drying with sodium sulfate, and then carrying out vacuum concentration to obtain the diethylamino-4-4-hydroxypyrrolo [2,1-f ] pyrrole]Is [1,2,4] of]The crude triazine-6-carboxylic acid (4) is purified by recrystallization to yield 1.45 g of diethylamino-4-hydroxypyrrolo [2,1-f ] as an off-white powder]Is [1,2,4] of]Triazine-6-carboxylic acid (4) in a yield of 61%.¹H NMR (400MHz, DMSO-D₆). ppm 1.52-1.58 (t,3H)， 2.45-2.55 (m, 2H)， 6.53 (br,s,2H)， 7.12(s, 1H)， 8.35(s, 1H)， 10.23(s, 1H)。

4. Synthesis of [1,2,4] triazine-6-carboxylic acid (5) of the compound ethyl-2- { (penta) - [ (dimethylamino) methylene ] amino } -4-hydroxypyrrolo [2,1-f ]:

the reaction solution was stirred (1.45 g, 6.6 mmol) with diethylamino-4-hydroxypyrrolo [2,1-f ]]Is [1,2,4] of]Triazine-6-carboxylic acid (4), 6ml of dimethylformamide, (1.01 ml,3.2 mmol) of thionyl chloride and dichloromethane solution were refluxed for 3 hours. The reaction mixture was cooled, and the reaction mixture was extracted twice with 500ml of ethyl acetate and 100ml of saturated aqueous sodium bicarbonate solution and 100ml of saturated aqueous sodium chloride solution, and the combined organic layers of ethyl acetate after extraction were dried over sodium sulfate and concentrated in vacuo to give ethyl-2- { (penta) - [ (dimethylamino) methylene]Amino } -4-hydroxypyrrolo [2, 1-f)]Is [1,2,4] of]Crude triazine-6-carboxylic acid (5). The crude product (5) was purified by recrystallization, and 1.8 g of ethyl-2- { (penta) - [ (dimethylamino) methylene ] are collected as an off-white powder]Amino } -4-hydroxypyrrolo [2, 1-f)]Is [1,2,4] of]Triazine-6-carboxylic acid (5) in a yield of 91%.¹H NMR (400MHz, DMSO-D₆).ppm 1.28（d, J=5.81Hz, 6H），1.52-1.58 (t,3H)， 2.45-2.55 (m, 2H)， 4.56-4.89(m, 1H)， 6.53 (br,s,2H) 7.12(s, 1H)， 8.35(s, 1H)， 10.23(s, 1H)。

5. Synthesis of the compound ethyl 4-chloro-2- { (penta) - [ (dimethylamino) methylene ] amino } pyrrolo [2,1-f ] [1,2,4] triazine-6-carboxylic acid (6):

the reaction mixture was washed with water (1.8 g, 6.5 mmol) ethyl-2- { (penta) - [ (dimethylamino) methylene]Amino } -4-hydroxypyrrolo [2, 1-f)]Is [1,2,4] of]Triazine-6-carboxylic acid (5) and (31 ml, 332 mmol) phosphorus oxychloride were vigorously stirred at 100 ℃ for 30 minutes until the color of the reaction mixture became a uniform brown color and then heating was continued for an additional 30 minutes. The reaction mixture is then rapidly cooled to 0 ℃ in ice or a water bath and then titrated with 2N aqueous NaOH to adjust the pH of the reaction mixture to 7.2. The white suspension thus produced was then filtered, washed with 600ml of ethyl acetate, the filtrate was scooped up with brine, the organic layer was dried over sodium sulfate and concentrated in vacuo to give ethyl 4-chloro-2- { (penta) - [ (dimethylamino) methylene]Amino } pyrrolo [2,1-f][1,2,4]A crude triazine-6-carboxylic acid product (6); purifying the crude product (6) with silica gel column (with ethyl acetate/n-hexane, 0-30% solvent), and collecting the purified product1.2 g (4.23 mmol) ethyl 4-chloro-2- { (penta) - [ (dimethylamino) methylene as a light yellow solid]Amino } pyrrolo [2,1-f][1,2,4]Triazine-6-carboxylic acid (6) in a yield of 65%.¹H NMR (400MHz, DMSO-D₆).ppm 1.28（d, J=5.81Hz, 6H），1.52-1.58 (t,3H)， 2.45-2.55 (m, 2H)， 4.56-4.89(m, 1H)， 6.53 (br,s,2H)， 7.12(s, 1H) 8.35(s, 1H)。

6. Synthetic route to the compound 3, 5-dichloro-2- (hydroxyboron) phenol (7 i.e., F):

6.1 Synthesis of Compound diiodo-3, 5-dichlorophenol (2):

p- (70 g, 0.43 mol) p-3, 5-dichlorophenol was mixed with 1 l of anhydrous toluene, and sodium hydride (51.5 g, 1.29 mol) was added thereto at 0 ℃ under a nitrogen atmosphere. The reaction mixture was allowed to warm to room temperature and stirred for 30 minutes, the resulting suspension was cooled back to 0 ℃ and then iodine (253.8 g, 91.5 mol) was added slowly. And (3) stirring the final reaction mixture, placing the reaction mixture in an environment of between 0 and room temperature, sequentially quenching the reaction mixture by using 1L of 1N hydrochloric acid solution after at least 24 hours, extracting the reaction mixture by using 1L of ethyl acetate, separating an ethyl acetate layer, then quenching and washing the reaction mixture by using 100ml of saturated sodium chloride aqueous solution, dehydrating and drying a quenched organic layer by using sodium sulfate, and then carrying out vacuum concentration to obtain a crude diiodo-3, 5-dichlorophenol product. The crude diiodo-3, 5-dichlorophenol product was purified by silica gel column (ethyl acetate/n-hexane, 0-20% solvent) to collect diiodo-3, 5-dichlorophenol (2) in 85 g yield of 68% as a white solid.

6.2 Synthesis of the Compound 1, 5-dichloro-3- (methoxyethoxy) -2-iodobenzene (3):

(67 g, 0.23 mol) diiodo-3, 5-dichlorophenol (2), (31.8 g, 0.29 mol) chloromethoxyethane and (63.7 g, 0.2 mol) cesium carbonate were mixed with 600ml dimethylformamide and stirred at room temperature for 2 hours. After the reaction is finished, the mixture is quenched and washed by 500ml of ethyl acetate, 100ml of x3 times of water and 100ml of saturated sodium chloride aqueous solution, the quenched organic layer is dehydrated and dried by sodium sulfate, and then the organic layer is concentrated in vacuum, so that the crude product of the 1, 5-dichloro-3- (methoxyethoxy) -2-iodobenzene is obtained. The crude product was purified over silica gel column (ethyl acetate/n-hexane, 0-50% solvent) and 80 g of 1, 5-dichloro-3- (methoxyethoxy) -2-iodobenzene (3) were collected as a yellow solid in 99% yield.

6.3 Synthesis of the Compound 2- [2, 4-dichloro-6- (methoxyethoxy) phenyl ] -4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolane (4):

(77 g, 0.22 mol) 1, 5-dichloro-3- (methoxyethoxy) -2-iodobenzene (3), (57 g, 0.44 mol) 4,4,5, 5-methyl- [1,3,2] dioxaborolane and (92 ml, 0.66 mol) triethylamine were put into 500ml of a dioxane solution, purged with nitrogen for 30 minutes, and then (2.7 g, 0.011 mol) palladium acetate and (8.5 g, 0.22 mol) biphenyl-2-yl-dicyclohexylphosphine were added to the reaction mixture, and the reaction mixture was stirred at 80 ℃ for 1.5 hours. After cooling down, the reaction mixture was quenched with 500ml of ethyl acetate followed by 100ml of saturated ammonium chloride solution, 100ml of water and 100ml of saturated aqueous sodium chloride solution. The quenched organic layer was dried over sodium sulfate and concentrated in vacuo to give a crude 2- [2, 4-dichloro-6- (methoxyethoxy) phenyl ] -4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolane. The crude product was purified again on silica gel column (ethyl acetate/n-hexane, 0-50% solvent) and collected as a brown solid in 35 g of 2- [2, 4-dichloro-6- (methoxyethoxy) phenyl ] -4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolane (4) with a yield of 45%.

6.4 Synthesis of the Compound 3, 5-dichloro-2- (hydroxyboron) phenol (F):

2- [2, 4-dichloro-6- (methoxyethoxy) phenyl (35 g, 0.1 mol)]-4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolane (4) and 200ml of anhydrous dichloromethane are mixed and stirred, boron tribromide (125 g, 0.5 mol) is slowly added under an ambient condition of 0 ℃ and nitrogen atmosphere, after stirring for a further 20 minutes, the reaction mixture is poured into water, then 100ml of 3N sodium hydroxide solution is added to adjust the pH of the reaction mixture to about 10, and then the organic layer is removed by separation. 500ml of 1N HCl solution was added to the separated aqueous layer to make the pH of the separated aqueous layer mixture about 3, and the separated aqueous layer mixture was extracted 3 times with 500ml of ethyl acetate, respectively. The organic layers after extraction were combined, washed twice with 500ml and saturated aqueous sodium chloride solution, respectively, then dehydrated over sodium sulfate and dried, and concentrated in vacuo to yield 40 g of 3, 5-dichloro-2- (hydroxyboron) phenol (F) as a white solid in 80% yield and used directly in the next reaction without further purification.¹H NMR (400MHz, MeOD).ppm 6.875-6.878(d, 1H), 6.727-6.737 (d, 1H)。

7. Synthesis of [1,2,4] triazine-6-carboxylic acid (8) of the compound ethyl 4- (2, 4-dichloro-6-hydroxyphenyl) -2- { (penta) - [ (dimethylamino) methylene ] amino } pyrrolo [2,1-f ]:

sodium carbonate (1.84 g, 16.92 mmol), 33ml of water, (1.2 g, 4.23 mmol) ethyl 4-chloro-2- { (penta) - [ (dimethylamino) methylene]Amino } pyrrolo [2,1-f][1,2,4]Triazine-6-carboxylic acid (6) (1.2 g, 5.8 mmol) (3, 5-dichloro-2- (hydroxyboron) phenol) (F) and 70ml1,4 dioxane were mixed and reacted, and the reaction solution was purged with nitrogen several times and then tetrakis (triphenylphosphine) palladium (490 mg, 0.423 mmol) was added to the reaction mixture. The reaction mixture was stirred at 80 ℃ for 12 hours. After cooling to room temperature, 100ml of water were added and the aqueous mixture was extracted 2 times with 500ml of ethyl acetate. The organic layers after extraction were combined, washed with 100ml of saturated aqueous sodium chloride solution, dehydrated and dried over sodium sulfate, concentrated in vacuo to give ethyl 4- (2, 4-dichloro-6-hydroxyphenyl) -2- { (penta) - [ (dimethylamino) methylene]Amino } pyrrolo [2,1-f]Is [1,2,4] of]Crude triazine-6-carboxylic acid, which was purified by silica gel column (ethyl acetate/n-hexane, 0-50% solvent) to collect the purified product as a pale gray solid (1.34 g, 3.17 mmol) ethyl 4- (2, 4-dichloro-6-hydroxyphenyl) -2- { (penta) - [ (dimethylamino) methylene ether]Amino } pyrrolo [2,1-f]Is [1,2,4] of]Triazine-6-carboxylic acid (8) in a yield of 75%.¹H NMR (400MHz, DMSO-D₆).ppm 1.28（d, J=5.81Hz, 6H），1.52-1.58 (t,3H)， 2.45-2.55 (m,2H)， 4.56-4.89(m,1H)， 6.53 (br,s,2H)， 6.95(d,J=1.77Hz, 1H)， 7.08-7.21 (m,1H)， 7.32(s, 1H)， 8.35(s, 1H),10.67(s, 1H)。

8. Synthesis of [1,2,4] triazine-6-carboxylic acid (9) of the compound diethylamino-4- (2, 4-dichloro-6-hydroxyphenyl) pyrrolo [2,1-f ]:

a solution of (12 ml, 48 mmol) 4M hydrochloric acid in dioxane, (1.34 g, 3.17 mmol) ethyl 4- (2, 4-dichloro-6-hydroxyphenyl) -2- { (penta) - [ (dimethylamino) methylene]Amino } pyrrolo [2,1-f]Is [1,2,4] of]Triazine-6-carboxylic acid (8) was mixed with 10 ml of methanol and reacted with stirring at room temperature for 12 hours. The reaction solution was evaporated in vacuo and neutralized with saturated sodium carbonate solution to obtain a water mixture. The aqueous mixture was extracted 2 times with 500ml ethyl acetate, the combined organic layers were washed with 100ml and saturated aqueous sodium chloride, dried over sodium sulfate, and concentrated under vacuum to give diethylamino-4- (2, 4-dichloro-6-hydroxyphenyl) pyrrolo [2,1-f]Is [1,2,4] of]Crude triazine-6-carboxylic acid. The crude product was purified on a silica gel column (ethyl acetate/n-hexane, 0-50% solvent) and the purified product was collected as a pale grey solid, (1.01 g, 2.69 mmol) diethylamino-4- (2, 4-dichloro-6-hydroxyphenyl) pyrrolo [2,1-f ] p]Is [1,2,4] of]Triazine-6-carboxylic acid (9) in a yield of 85%.¹H NMR (400MHz, DMSO-D₆).ppm 1.52-1.58 (t,3H)， 2.45-2.55 (m,2H)，6.53 (br,s,2H)， 6.95(d,J=1.77Hz, 1H)， 7.08-7.21 (m,1H)， 7.32(s, 1H)， 8.35(s, 1H),10.67(s, 1H)。

9. Synthesis of [1,2,4] triazine-6-carboxylic acid (11) of the compound diethylamino-4- {2, 4-dichloro-6- [2- (hydro-pyrazol-1-yl) ethoxy ] phenyl } pyrrolo [2,1-f ]:

potassium carbonate (1.12 g, 8.07 mmol), (1.05 g, 8.07 mmol) 1- (2-chloroethyl) -1H-pyrazole (10), (1.01 g, 2.69 mmol) diethylamino-4- (2, 4-dichloro-6-hydroxyphenyl) pyrrolo [2,1-f ] was added]Is [1,2,4] of]Triazine-6-carboxylic acid (9) and 70ml dimethylformamide at 120^oThe reaction was stirred under C for 12 hours, and after cooling to room temperature, 100ml of water and 500ml of ethyl acetate were added to the reaction mixture and stirred for 30 minutes to form a water mixture. The aqueous mixture was extracted 2 times with 500ml of ethyl acetate, the organic layers after extraction were combined, washed with 100ml of saturated aqueous sodium chloride solution, dried over sodium sulfate and concentrated in vacuo to give diethylamino-4- {2, 4-dichloro-6- [2- (hydro-pyrazol-1-yl) ethoxy ] ethyl acetate]Phenyl } pyrrolo [2,1-f]Is [1,2,4] of]Crude triazine-6-carboxylic acid. The crude product was purified by silica gel column (ethyl acetate/n-hexane, 0-50% solvent) and the purified product was collected as diethylamino-4- {2, 4-dichloro-6- [2- (hydro-pyrazol-1-yl) ethoxy ] amino as a light yellow solid (0.741 g, 1.64 mmol)]Phenyl } pyrrolo [2,1-f]Is [1,2,4] of]Triazine-6-carboxylic acid (11) in a yield of 61%.¹H NMR (400MHz, DMSO-D₆).ppm 1.52-1.58 (t,3H)， 2.45-2.55 (m,2H),3.29-3.59（m,1H）, 3.77(d,J=22.8Hz,1H),4.20-4.28(m, 1H), 4.29-4.35(m, 2H), 6.53 (br,s,2H)， 6.95(d,J=1.77Hz, 1H)，7.01(s, 1H),7.04(s, 1H), 7.08-7.21 (m,1H)， 7.32(s, 1H)， 8.35(s, 1H)。

10. Synthesis of [1,2,4] triazine-6-carboxylic acid (12) of the compound 2-amino-4- {2, 4-dichloro-6- [2- (1H-pyrazol-1-yl) ethoxy ] phenyl } pyrrolo [2,1-f ]:

2N aqueous sodium hydroxide (4 ml, 8.2 mmol), (0.741 g, 1.64 mmol) diethylamino-4- {2, 4-dichloro-6- [2- (hydro-pyrazol-1-yl) ethoxy ] was added]Phenyl } pyrrolo [2,1-f]Is [1,2,4] of]Triazine-6-carboxylic acid (11) and 20ml of ethanol were mixed and reacted with stirring at room temperature for 12 hours. A2N aqueous hydrochloric acid solution was then added to bring the pH of the reaction mixture to about 7.0. The reaction mixture was extracted 2 times with 500ml of ethyl acetate, respectively. The organic layers after extraction were combined, washed with 100ml of saturated aqueous sodium chloride solution, dried over sodium sulfate and concentrated in vacuo to give a pale grey solid, (0.554 g, 1.28 mmol) 2-amino-4- {2, 4-dichloro-6- [2- (1H-pyrazol-1-yl) ethoxy ] ethoxy]Phenyl } pyrrolo [2,1-f]Is [1,2,4] of]Triazine-6-carboxylic acid (12) in 78% yield was obtained without further purification and was used in the next step.¹H NMR (400MHz, DMSO-D₆).ppm 3.29-3.59（m,1H）, 3.77(d,J=22.8Hz,1H),4.20-4.28(m, 1H), 4.29-4.35(m, 2H), 6.53 (br,s,2H)， 6.95(d,J=1.77Hz, 1H)，7.01(s, 1H),7.04(s, 1H), 7.08-7.21 (m,1H)， 7.32(s, 1H)， 8.35(s, 1H)，10.67（s，1H）。

11. Synthesis of [1,2,4] triazine-6-carboxamide (13) of the compound 2-amino-N-cyclopropyl-4- (2, 4-dichloro-6- [2- (1H-pyrazol-1-yl) ethoxyphenyl) pyrrolo [2,1-f ]:

1-Ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC HCl) (490 mg, 2.56 mmol), (0.554 g, 1.28 mmol) 2-amino-4- {2, 4-dichloro-6- [2- (1H-pyrazol-1-yl) ethoxy-1-yl)]Phenyl } pyrrolo [2,1-f]Is [1,2,4] of]Triazine-6-carboxylic acid (12), (146 mg, 2.56 mmol) cyclopropylamine, (0.53 ml, 3.84 mmol) triethylamine and 10 ml dimethylformamide were mixed and the reaction was stirred at room temperature for 12 hours. After the reaction was completed, the reaction mixture was extracted 2 times with 500ml of ethyl acetate, respectively. The organic layers after extraction were combined and washed with 100ml of saturated sodium bicarbonateThe aqueous solution and 100ml and saturated aqueous sodium chloride solution were washed, dehydrated and dried with sodium sulfate, and concentrated in vacuo to give 2-amino-N-cyclopropyl-4- (2, 4-dichloro-6- [2- (1H-pyrazol-1-yl) ethoxyphenyl) pyrrolo [2,1-f]Is [1,2,4] of]Crude triazine-6-carboxamide. The crude product was purified by silica gel column (ethyl acetate/N-hexane, 0-50% solvent) and the purified product was collected as a pale off-white solid, (490 mg, 1.04 mmol) 2-amino-N-cyclopropyl-4- (2, 4-dichloro-6- [2- (1H-pyrazol-1-yl) ethoxyphenyl) pyrrolo [2,1-f ] pyrrole [2]Is [1,2,4] of]Triazine-6-carboxamide (13) in 81% yield.¹H NMR (400MHz, DMSO-D₆).ppm 0.33-0.44(m, 2H),0.46-0.59(m,2H), 0.80-0.91(m,1H),3.29-3.59（m,1H）, 3.77(d,J=22.8Hz,1H),4.20-4.28(m, 1H), 4.29-4.35(m, 2H), 6.23(d, J=2.78Hz, 1H),6.53 (br,s,2H)， 6.95(d,J=1.77Hz, 1H)，7.01(s, 1H),7.04(s, 1H), 7.08-7.21 (m,1H)， 7.32(s, 1H)， 8.35(s, 1H)。

Secondly, compounds 14-17 can also be synthesized using similar synthetic methods as described above, with the following molecular structural formulas and analytical data:

and thirdly, biochemical analysis of the heat shock protein 90:

evaluation of biological Activity of Compounds of the invention Heat shock protein 90 biological activity was tested using Scintillation Proximity (SPA) competitive binding assays. Either full-length or N-terminal heat shock protein 90, loaded with its C-terminal binding 6 click tag versus yttrium silicate scintillant bead binding copper through its tag. Tritiated propyl geldanamycin, the structure of which is shown below, is a derivative of a natural inhibitor. Tritiated geldander (pGA), which contains tritiated propylamine group added at position 17, binds heat shock protein 90 and brings it to the small eye curtain isotope. 17-N-propylamine-gelder may be synthesized as described in U.S. Pat. No. 4261,989 and incorporated by reference. The second tritium-treated compound, shown below, can also be used in this experiment as one of the named compound a.

In the above formula, "T" indicates the position of the tritium-treated hydrogen atom label in the structure of compound a. The compound has K_d: 40nM and can be synthesized as follows. Compound a was synthesized from the parent compound. N-allyl-2- (5-chloro-2, 4-dihydroxybenzoyl) isoindoline-1-carboxamide is illustrated by the following synthesis:

allylamine (2.5 ml, 5.0mmol in 2M tetrahydrofuran) was added to the reaction mixture tert-butylcarbamic acid (R, S) 1, 3-dihydro-2H-isoindolecarboxylic acid (263 mg, 1.0 mmol), diisopropylethylamine (0.9 ml, 5.0 mmol) and 2- (7-aza-1H-benzotriazol-1-yl) -1,1,3, 3-tetramethyluronium hexafluorophosphate, 2- (7-aza-benzotriazol) -N, N, N ', N' -tetramethyluronium Hexafluorophosphate (HATU) (420 mg, 1.1 mmol) in 5.0 ml of dimethylformamide and stirring at room temperature under a nitrogen atmosphere for 12 hours. The mixture was extracted with ethyl acetate (500 ml x 2), the combined organic layers were extracted with saturated aqueous sodium bicarbonate (100 ml) and brine (100 ml) and then dried over sodium sulfate and the crude product was collected by vacuum concentration and purified on silica gel column (ethyl acetate/n-hexane, 0-50% solvent) to collect the purified product as tert-butyl-1- [ (allylamino) carbonyl ] -1, 3-dihydro-2H-isoindole-2-carboxylic acid (321 mg, quantitative yield).

4M hydrochloric acid solution in dioxane (3.0 ml, 12 mmol) was added to the reaction mixture tert-butyl-1- [ (allylamino) carbonyl]-1, 3-dihydro-2H-isoindole-2-carboxylic acid (1.0 mmol) in 5.0 ml of dichloromethane and in 40^oThis mixture was concentrated in vacuo to collect the product N-allylisoindoline-1-carboxamide as an oil which was used in the next reaction without further purification.

N-allylisoindoline-1-carboxamide (1.0 mmol) was added to the reaction mixture pentachloro-2, 4-bis (methoxymethoxy) benzoic acid (which may be synthesized as described in the writing of WO2006/1175669 and incorporated by reference) (340 mg, 1.2 mmol), 4-methylmorpholine (2.2 ml, 20.0 mmol), 1-ethyl- (3-dimethylaminopropyl) carbonyldiimidate hydrochloride (edchcl) (460 mg, 2.4 mmol) and 1-hydroxybenzotriazole (330 mg, 2.4 mmol) in 12 ml dimethylformamide under a nitrogen atmosphere and stirred at room temperature for 12 hours. Water (50 ml) was added to the reaction mixture to stop the reaction, the mixture was extracted with ethyl acetate (500 ml x 2), the combined organic layers were extracted with saturated aqueous sodium bicarbonate (100 ml) and brine (100 ml) and dried over sodium sulfate and the crude product was collected by vacuum concentration and purified on silica gel column (with ethyl acetate/N-hexane, 50-60% solvent) to collect the desired intermediate (423 mg, 92% yield) N-allyl-2- [ 5-chloro-2, 4-bis (methoxymethyl) benzoyl ] isoindoline-1-carboxamide.

4M hydrochloric acid solution in dioxane (3.0 ml, 12 mmol) was added to the reaction mixture N-allyl-2- [ 5-chloro-2, 4-bis (methoxymethyl) benzoyl ] isoindoline-1-carboxamide (392 mg, 0.85 mmol) in 5.0 ml dichloromethane and stirred at room temperature for 12 hours. Saturated aqueous sodium bicarbonate was then added to neutralize to a pH of about 7.0. The mixture was extracted with ethyl acetate (500 ml x 2) and the combined organic layers were washed with brine (100 ml) and dried over sodium sulfate and concentrated in vacuo to collect the desired end product as the parent compound (221 mg, 70% yield) N-allyl-2- (5-chloro-2, 4-dihydroxybenzoyl) isoindoline-1-carboxamide as a white solid.

¹H NMR (400MHz, DMSO-D₆).ppm 3.57（d,J=79.33Hz,2H）,4.65-4.93(m,1H),4.97-5.19(m,1H),5.42-5.60(m,1H),5.68-5.95(m,1H),6.40-6.71(m,1H),6.92(s,1H),7.15-7.67(m,4H),8.28(s,1H),10.06(s,1H),10.40(s,1H)。Anal.Calcd.forC₁₉H₁₇ClN₂O₄: C,61.21;H,4.60;N,7.51,Found:C,61.02;H,4.62;N,7.36 。

Once the parent compound was prepared, compound a was prepared using standard hydrogenation preparation methods and using tritium gas. Emitting isotopically excited scintillations at the test signal will create a measurable scintillating signal. When competitive compounds are added to the mixture tested, they compete to constrain tritium-treated geldand (pGA) or compound a to the N-terminal heat shock protein 90 to the ATP binding site. When the compound replaces gelder (pGA) or labels compound a, the signal decreases (the beta particle is no longer in proximity to the bead). This reduced signal is used to quantify the intense competition between the inhibitor/compound and geldand (pGA) or compound a to some extent.

For the³Detection of H-pGA (designated G1) and Compound A (designated G2) SPA binding of heat shock protein 90 was performed in 96-well flat-bottomed protein plates (Corning # 3604). For G1, the reaction typically contained 30nM of heat shock protein 90 and 200nM³Binding buffer for H-pGA (100 mM medium, pH7.5 and 150mM potassium chloride). For G2, a typical reaction solution contained 5nM of heat shock protein 90 and 50nM of Compound A. For the case of the G1, the case,³H-pGA was first diluted to 33% of tag and unlabeled pGA which was synthesized and purified to give a final concentration of 200 nM. For G2, dilution of labeled compound a with non-labeled compound a provided one label: the unlabeled ratio was 1:2 with a final concentration of 50 nM. The inhibitor is added into the heat shock protein 90-³H-pGA (or heat shock protein 90/Compound A) in 11 different concentrationsMeasurement of the activity (Ki) as an enzyme in solution. The concentration range of the inhibitor is 100uM or an appropriate concentration range may be used for solid samples, a concentration range of 10uM may be used for target compound libraries, and a concentration range of 10mM may be used for liquid samples. To determine the percentage of inhibitory activity, compounds will be tested at 1 and 10 uM. The concentration of the sample in the dimethylsulfoxide solution was 4%. copper-labeled-Ysi beads (Amersham # RPNQ 5096) were used to dilute in binding buffer and then added to each well of the plate to give a final concentration of 100 ug/well. After the panels were sealed, the lids were closed with aluminum foil lids and shaken for 30 minutes at room temperature. The panel beads were allowed to settle for 30 minutes and then counted using a Hewlett packard NXT instrument. This process also employs a mesoscale throughput and is tested using an instrument from beckmann Biomek FX. Samples were tested in duplicate and used on two independent days to ensure accurate determination of enzyme activity (Ki).

For the determination of enzyme activity (Ki), GraphPad Prim software was used and plotted with the correct cpm's (actual cpm's minus background) versus inhibitor concentration. These data are for general IC₅₀Equation, Y = YI/(1 + [ X ]]/IC₅₀) Wherein YI = Y-intercept and [ X ]]Are competing ligands/inhibitors. IC thereof₅₀Ki was then used for calculation and calculated by the Cheng-Prusoss equation:

where Cl = cold ligand concentration (variable), hI = hot ligand concentration (200 nM or 50 nM) and Kd { hI } =240nM (for cold ligand concentration), hI = hot ligand concentration (for hot ligand concentration) (for cold ligand concentration, hot ligand concentration, and cold ligand concentration, hI = hot ligand concentration (200 nM or 50 nM), and Kd { hI } =240nM (for hot ligand concentration³H-pGA) or 40nM (for Compound A). Error calculation method as follows and IC thereof₅₀error/IC₅₀Value = relative error, fractional error: ki values/Ki error.

In some cases, the inhibitor binds to heat shock protein 90 so tightly that the free inhibitor molecule is able to take up the enzyme inhibitor complex formed thereby, and thus the above equation does not applyAnd is valuable. This is usually when the IC is observed₅₀Is about the same concentration as heat shock protein 90. For tight binding inhibitors the following formula will apply:

EL and EL are complexes of radioligand-heat shock protein 90 presence and absence of inhibitors, EL/EL_oIndicating the presence of a fractional signal in the inhibitor, I_o，E_oAnd L_oThe concentrations of inhibitor, heat shock protein 90 and radioligand, respectively. Ki is the inhibition constant of the ligand, K_LIs the affinity constant between the enzyme (heat shock protein 90) and the ligand.

The following table is the Ki assay data for compounds 13-17:

compound #	(G2) Activity of the enzyme Ki (nM)	Half inhibition rate Akt Lum IC ₅₀（uM）
			13	50.65	0.55
14	100.75	2.44
			15	10.32	0.051
16	25.82	0.61
			17	210.75	4.55

The enzyme activity Ki and half inhibition ratio Akt Lum IC of the compounds 13-17 in the table above₅₀The detection data show that the compound has a certain anti-tumor biological activity, and when the compound and the salt thereof are tightly combined with the terminal of the heat shock protein 90 at the N-terminal of the ATP binding site, the compound can inhibit the heat shock protein 90(Hsp90), promote the degradation of Hsp90 effect protein playing an important role in a tumor growth signal pathway through ubiquitination, thereby blocking a plurality of targets of the tumor proliferation and growth signal pathway and effectively preventing the growth of tumors. Activity Ki and half inhibition ratio Akt Lum of enzyme and IC₅₀The smaller the value of (A), the stronger the biological activity of the compound is, the higher the affinity with tumor cells is, and the compound can selectively kill the tumor cells.

Heat shock protein 90(Hsp90) is used as molecular chaperone to regulate and maintain the conformation and function of various intracellular proteins and to help cells grow normally under the stimulation of stress environment. Many oncogene proteins are target of Hsp90, so inhibiting Hsp90 function would promote degradation of these oncogene proteins, contributing to cancer treatment.

The compounds and salts thereof of the present invention confirm the antitumor activity of Hsp90 inhibitor through the biochemical analysis and in vitro and in vivo experiments of heat shock protein 90, and the compounds 13 to 17 of the present invention are characterized in that the antitumor effect is mainly exerted by inactivating the oncoproteins and protein kinase through the proteolytic pathway, rather than directly inhibiting the catalytic activity of the kinase. Immunoprecipitation and affinity experiments revealed that the main mechanism of action of the compounds of the invention is through binding to Hsp 90. x-ray crystallography and biochemical experimental results show that the compound of the invention can compete for the ATP binding site of Hsp90 and inhibit the endogenous and atpase activities of Hsp 90. In vitro experiments prove that: the compound of the invention can make tumor cells enter a stationary phase and can also induce the cells to generate apoptosis. The compounds of the invention inhibit endogenous ATPase activity by binding to the N-terminus of Hsp 90. The compounds of the invention act on the N-terminal ATP/ADP binding site of Hsp 90. It is a small molecule compound designed according to the result of x-ray crystal diffraction. The compounds of the present invention can also degrade many of the actor proteins of Hsp 90.

Annotation of related terms:

"pharmaceutically acceptable preparation" refers to a combination of a compound or physiologically/pharmaceutically acceptable salt of the present invention with carriers and adjuvants. The preparation of the pharmaceutical formulation is carried out using known art skills and general procedures. For example, the compounds of the present invention may be formulated with a common adjuvant diluent or carrier to form tablets, capsules, and the like. Such adjuvant diluents or carriers include the following: fillers and extenders such as starches, sugars, mannitol, and silicic acid derivatives; such as carboxymethyl cellulose and other cellulose derivative binders, alginates, gelatin, polyvinyl pyrrolidone, humectants such as glycerol; disintegrating agents such as povidone, sodium starch glycolate, sodium carboxymethylcellulose, agar, calcium carbonate, sodium bicarbonate; retarders such as paraffin, absorption accelerators such as quaternary ammonium compounds; surfactants such as cetyl alcohol, glycerol monostearate; adsorption carriers such as kaolin, bentonite; lubricating oils such as talc, calcium stearate, magnesium and solid polyethylene glycols. The final formulation may be a pill, tablet, powder, lozenge, sachet, or sterile packaged powder, depending on the type of adjuvant used. Furthermore, it is a specific pharmaceutically acceptable formulation.

By "heat shock protein 90-inhibiting amount" is meant the amount or physiologically/pharmaceutically acceptable salt of a compound of the invention required to inhibit the activity of the heat shock protein 90 enzyme in vivo, e.g., in a mammal, or in vitro. The amount of such compound required to cause such inhibition can be determined without undue experimentation using the methods described and those of ordinary skill in the art.

By "heat shock protein 90 inhibits enzymatic activity" is meant the reduction of functional activity of heat shock protein 90 enzyme in vitro or in vivo such as in mammals and humans by contacting the enzyme with a compound of the invention.

By "therapeutically effective amount" is meant an amount of a compound or physiologically/pharmaceutically acceptable salt of the invention administered to a mammal sufficient to effect such treatment. By "therapeutically effective amount" is meant an amount sufficient to modulate or inhibit the activity of the heat shock protein 90 enzyme, which results in the reduction or amelioration of the disease condition through mediated activity of the heat shock protein 90 enzyme.

"treating" or "treatment" refers to any heat shock protein 90 mediated treatment of a disease in a mammal, particularly a human. Which comprises the following steps: 1) preventing the onset of the disease or condition that occurs, 2) modulating or inhibiting the disease or condition, i.e., arresting its development, 3) alleviating the disease or condition, i.e., causing a disease or condition that results from rehabilitation or 4) alleviating and/or alleviating the disease or condition, the disease or condition that causes these symptoms, e.g., reducing the inflammatory response. With respect to cancer, this simply means that the life span of a person will increase with the effect of cancer or that the symptoms of one or more diseases will decrease.

In reference to the treatment of cancer, a therapeutically effective amount means at least one of the following effects: 1) reducing the size of the tumor; 2) inhibit (i.e., slow to some extent, preferably stop) tumor metastasis; 3) inhibit (i.e., slow to some extent, preferably stop) tumor growth to some extent; 4) to some extent alleviate (or preferably eliminate) one or more symptoms associated with cancer.

"Compound" refers to any of the above compounds, and also includes those compounds described or presented generally. Also refers to pharmaceutically acceptable salts or solvates of such compounds.

"abnormal cell growth," unless otherwise indicated, refers to the growth of a cell, which is an independent normal regulatory mechanism (e.g., loss of inhibitory contact), including abnormal growth of normal cells and abnormal cell growth. This includes, but is not limited to, growth abnormalities: tumor cells this includes, but is not limited to, abnormal growth: tyrosine kinases that proliferate in tumor cells express mutated or overexpressed receptor tyrosine kinases; abnormal tyrosine kinase activation in other benign and malignant proliferative disease cells; receptor tyrosine kinases for any tumor cell proliferation; aberrant proliferative serine/threonine kinase activation of any tumor occurs; activation of other aberrant serine/threonine kinases occurs in benign and malignant cell proliferative diseases; benign and malignant one of activated RAS oncogene expression; tumor cells, benign and malignant, where RAS protein is mutated in another gene leading to activation of carcinogenesis; other diseases of benign and malignant cell proliferation in which RAS activation is aberrant. Examples of such benign proliferative diseases are psoriasis, benign prostatic hyperplasia, Human Papilloma Virus (HPV). "abnormal cell growth" is also meant to include abnormal growth, benign and malignant cells resulting from farnesyl protein transferase activity.

The use of "abnormal cell growth" and "hyperproliferation" are used interchangeably.

"steric structure" refers to spaces having the same chemical structure of a compound, but arranged for different atoms or functional groups.

"enantiomer" means that the two stereoisomers of the compound are not superimposable mirror images of each other.

"racemic" or "racemic mixture" refers to a mixture of enantiomers 1:1 of a particular compound.

"diastereomers", on the other hand, refer to two or more asymmetric centers of isomeric composition between, and do not reflect the relationship of the image pairs to each other.

"combination of agents" refers to a mixture of one or more of the compounds described, or physiologically/pharmaceutically acceptable salts, solvates, hydrates, or prodrugs and combinations thereof with other chemical ingredients, such as or physiologically/pharmaceutically acceptable carriers and excipients. The purpose of the pharmaceutical composition is to facilitate the delivery of a compound to an organism such as a mammal, including a human.

Cancer cells of the invention for treating abnormal cell growth include, but are not limited to: mesothelioma, hepatobiliary (liver and biliary tract), primary or secondary central nervous system tumor, primary or secondary brain tumor, lung cancer (non-small cell lung cancer and non-small cell lung cancer), bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular melanoma tumor, ovarian cancer, colon cancer, rectal cancer, cancer of the anal region, stomach cancer, cancer of the gastrointestinal tract (stomach, large intestine, duodenum), breast cancer, uterine cancer, cancer of the fallopian tubes, endometrial cancer, cervix, vagina, vulva cancer, hodgkin's disease, esophagus, small intestine tumor, cancer of the endocrine system, thyroid gland, parathyroid gland, adrenal gland cancer, soft tissue sarcoma cancer, urethra, penile cancer, prostate cancer, testicular cancer, chronic or acute leukemia, chronic myelogenous leukemia, lymphocytic lymphoma, bladder, kidney, ureter cancer, renal cell carcinoma, renal pelvis cancer, central nervous system tumor, primary central nervous system lymphoma, non-hodgkin's lymphoma, spinal axis tumor, brain stem glioma, pituitary adenoma, adrenal cortex, biliary bladder cancer, multiple myeloma, cholangiocarcinoma, fibrosarcoma, neuroblastoma, retinoblastoma, or a combination of one or more of the foregoing cancers.

The cancer cells of the invention for treating abnormal cell growth include: a benign proliferative disease but not limited to psoriasis, benign prostatic hyperplasia.

In the present embodiment the cancer is preferably selected from lung cancer (non-small cell lung cancer and non-small cell lung cancer), head or neck cancer, ovarian cancer, colon cancer, rectal cancer, anal region, gastric cancer, breast cancer, renal or ureteral cancer, renal cell carcinoma, renal pelvis cancer, tumors of the central nervous system, primary central nervous system lymphoma, non-hodgkin's lymphoma, spinal axis tumors or a combination of one or more of the above.

In another preferred embodiment of the invention the cancer is selected from lung cancer (non-small cell lung cancer and non-small cell lung cancer), head or neck cancer, ovarian cancer, colon cancer, rectal cancer, anal region, gastric cancer, breast cancer, kidney or ureteral cancer, renal cell carcinoma, renal pelvis cancer, central nervous system tumor, primary central nervous system lymphoma, non-hodgkin's lymphoma, spinal axis tumor or a combination of one or more of the foregoing cancers.

In a preferred embodiment, the cancer of the invention is selected from lung cancer (non-small cell lung cancer and non-small cell lung cancer), head or neck cancer, ovarian cancer, colon cancer, rectal cancer, anal region cancer or a combination of one or more of the foregoing.

Claims

1. The compound of the multi-target anti-tumor inhibitor 2-aminopyrrole-triazine has the following molecular formula:

in the above formula, the first and second carbon atoms are,

R_1ais hydrogen radical or C₃-C₈Cycloalkyl groups of (a);

R_1bis C₃-C₈Cycloalkyl groups of (a);

R₂is chlorine;

R₃is chlorine;

R₄is-O- (C)₂-C₆Alkyl) - (3-10 heterocycle).

2. A method for synthesizing 2-amino-N-cyclopropyl-4- {2, 4-dichloro-6- [2- (1H-pyrazol-1-yl) ethoxy ] phenyl } pyrrolo [2,1-f ] [1,2,4] triazine-6-carboxamide, characterized by comprising the steps of:

1) synthesis of diethyl pyrrole-2, 4-dicarboxylate:

dissolving ethyl isocyanoacetate and 1, 8-diazabicyclo [5.4.0] undec-7-ene in tetrahydrofuran to form a solution, adding formaldehyde into the solution, and stirring the reaction mixture to react at the temperature of 50 +/-0.5 ℃; after the reaction is finished, cooling the reaction mixture to normal temperature, respectively washing the reaction mixture with a saturated sodium bicarbonate aqueous solution and a saturated sodium chloride aqueous solution by using ethyl acetate, combining the ethyl acetate organic layers after washing, and obtaining pyrrole-2, 4-dicarboxylic acid diethyl ester through dehydration, drying and vacuum concentration;

2) synthesis of diethyl 1-aminopyrrole-2, 4-dicarboxylate:

mixing pyrrole-2, 4-dicarboxylic acid diethyl ester, ammonium chloride, methyl trioctyl ammonium chloride, sodium hydroxide aqueous solution and methyl tert-butyl ether to form a solvent, adding concentrated ammonium into the solvent, then adding bleaching water, and stirring the formed reaction mixture at room temperature for reaction; after the reaction is finished, filtering the reaction mixture, taking filtrate, respectively washing the filtrate with ethyl acetate, saturated sodium bicarbonate water solution and saturated sodium chloride water solution, combining the ethyl acetate organic layers after washing, and obtaining 1-aminopyrrole-2, 4-dicarboxylic acid diethyl ester through dehydration, drying and vacuum concentration;

3) synthesis of ethyl 2-amino-4-hydroxypyrrolo [2,1-f ] [1,2,4] triazine-6-carboxylate:

mixing and stirring 1-aminopyrrole-2, 4-dicarboxylic acid diethyl ester and chloroformamidine hydrochloride with dimethyl sulfoxide under the environmental condition of 150 +/-0.5 ℃ for reaction; cooling the reaction mixture to normal temperature, respectively washing the reaction mixture with ethyl acetate, saturated sodium bicarbonate water solution and saturated sodium chloride water solution, combining the organic ethyl acetate layers after washing, dehydrating, drying and vacuum concentrating to obtain a crude product of 2-amino-4-hydroxypyrrole [2,1-f ] [1,2,4] triazine-6-carboxylic acid ethyl ester, and finally purifying by a recrystallization method to obtain purified 2-amino-4-hydroxypyrrole [2,1-f ] [1,2,4] triazine-6-carboxylic acid ethyl ester;

4) synthesis of ethyl 2- [ (E) -dimethylaminomethyleneamino ] -4-hydroxypyrrolo [2,1-f ] [1,2,4] triazine-6-carboxylate:

mixing ethyl 2-amino-4-hydroxypyrrole [2,1-f ] [1,2,4] triazine-6-carboxylate with dimethylformamide, thionyl chloride and dichloromethane for reflux reaction; cooling the reaction mixture to normal temperature, respectively washing the reaction mixture with ethyl acetate, saturated aqueous sodium bicarbonate solution and saturated aqueous sodium chloride solution, combining the organic ethyl acetate layers after washing, dehydrating, drying and concentrating in vacuum to obtain a crude product of 2- [ (E) -dimethylamino methylene amino ] -4-hydroxypyrrolo [2,1-f ] [1,2,4] triazine-6-carboxylic acid ethyl ester; purifying by recrystallization to obtain purified ethyl 2- [ (E) -dimethylaminomethyleneamino ] -4-hydroxypyrrolo [2,1-f ] [1,2,4] triazine-6-carboxylate;

5) synthesis of ethyl 4-chloro-2- [ (E) -dimethylaminomethylamino ] pyrrolo [2,1-f ] [1,2,4] triazine-6-carboxylate:

stirring and reacting 2- [ (E) -dimethylamino methylene amino ] -4-hydroxypyrrolo [2,1-f ] [1,2,4] triazine-6-carboxylic acid ethyl ester and phosphorus oxychloride at the temperature of 100 +/-0.5 ℃ to ensure that the color of a reaction mixture is changed into uniform brown, and then preserving heat for 30 minutes; then the reaction mixture is rapidly cooled to 0 ℃, then NaOH solution is used for adjusting the pH value of the reaction mixture to 7.2, white suspended matters are taken after filtration and washed by ethyl acetate, filtered filtrate is scooped up by saline to obtain an organic layer, and then the organic layer is dehydrated, dried and concentrated in vacuum to obtain a crude product of 4-chloro-2- [ (E) -dimethylamino methylene amino ] pyrrolo [2,1-f ] [1,2,4] triazine-6-carboxylic acid ethyl ester; finally purifying by a silica gel column to obtain purified 4-chloro-2- [ (E) -dimethylamino methylene amino ] pyrrolo [2,1-f ] [1,2,4] triazine-6-carboxylic acid ethyl ester;

6) synthesis of ethyl 2- [ (E) -dimethylaminomethyleneamino ] -4- (2, 4-dichloro-6-hydroxyphenyl) pyrrolo [2,1-f ] [1,2,4] triazine-6-carboxylate:

mixing an aqueous sodium carbonate solution with 4-chloro-2- [ (E) -dimethylamino methyleneamino ] pyrrolo [2,1-f ] [1,2,4] triazine-6-carboxylic acid ethyl ester, 2, 4-dichloro-6-hydroxyphenylboronic acid and 1, 4-dioxane, adding tetrakis (triphenylphosphine) palladium after purging with nitrogen, and stirring for reaction at the temperature of 80 +/-0.5 ℃; cooling to room temperature after the reaction is finished, then adding water to form a water mixture, extracting the water mixture by using ethyl acetate, and washing an extracted organic layer by using a saturated sodium chloride aqueous solution; then dehydrating, drying and vacuum concentrating to obtain a crude product of 2- [ (E) -dimethylamino methylene amino ] -4- (2, 4-dichloro-6-hydroxyphenyl) pyrrolo [2,1-f ] [1,2,4] triazine-6-carboxylic acid ethyl ester; finally purifying the crude product of ethyl 2- [ (E) -dimethylaminomethyleneamino ] -4- (2, 4-dichloro-6-hydroxyphenyl) pyrrolo [2,1-f ] [1,2,4] triazine-6-carboxylate by using a silica gel column to obtain purified ethyl 2- [ (E) -dimethylaminomethyleneamino ] -4- (2, 4-dichloro-6-hydroxyphenyl) pyrrolo [2,1-f ] [1,2,4] triazine-6-carboxylate;

7) synthesis of ethyl 2-amino-4- (2, 4-dichloro-6-hydroxyphenyl) pyrrolo [2,1-f ] [1,2,4] triazine-6-carboxylate:

reacting 4M dioxane solution of hydrochloric acid with ethyl 2- [ (E) -dimethylaminomethyleneamino ] -4- (2, 4-dichloro-6-hydroxyphenyl) pyrrolo [2,1-f ] [1,2,4] triazine-6-carboxylate and methanol with stirring at room temperature; vacuum evaporating the reaction liquid, neutralizing with saturated sodium carbonate solution, and extracting with ethyl acetate; washing the extracted organic layer with saturated sodium chloride aqueous solution, dehydrating, drying and vacuum concentrating to obtain crude product of 2-amino-4- (2, 4-dichloro-6-hydroxyphenyl) pyrrolo [2,1-f ] [1,2,4] triazine-6-carboxylic acid ethyl ester; finally, purifying the crude product of the ethyl 2-amino-4- (2, 4-dichloro-6-hydroxyphenyl) pyrrolo [2,1-f ] [1,2,4] triazine-6-carboxylate by using a silica gel column to obtain purified ethyl 2-amino-4- (2, 4-dichloro-6-hydroxyphenyl) pyrrolo [2,1-f ] [1,2,4] triazine-6-carboxylate;

8) synthesis of ethyl 2-amino-4- {2, 4-dichloro-6- [2- (1H-pyrazol-1-yl) ethoxy ] phenyl } pyrrolo [2,1-f ] [1,2,4] triazine-6-carboxylate:

potassium carbonate and 1- (2-chloroethyl) -1H-pyrazole, 2-amino-4- (2, 4-dichloro-6-hydroxyphenyl) pyrrolo [2,1-f][1,2,4]Triazine-6-carboxylic acid ethyl ester and dimethyl formamide within 120 +/-0.5^oStirring and reacting under the condition of C; cooling to room temperature after the reaction is finished, adding water and ethyl acetate into the reaction mixture, and stirring for 30 minutes to obtain a water mixture; extracting the aqueous mixture with ethyl acetate, washing the extracted organic layer with saturated aqueous sodium chloride solution, dehydrating, drying, and vacuum concentrating to obtain 2-amino-4- {2, 4-dichloro-6- [2- (1H-pyrazol-1-yl) ethoxy]Phenyl } pyrrolo [2,1-f][1,2,4]Crude product of triazine-6-carboxylic acid ethyl ester; finally, 2-amino-4- {2, 4-dichloro-6- [2- (1H-pyrazol-1-yl) ethoxy]Phenyl } pyrrolo [2,1-f][1,2,4]Purifying the crude product of triazine-6-carboxylic acid ethyl ester by silica gel column to obtain purified 2-amino-4- {2, 4-dichloro-6- [2- (1H-pyrazol-1-yl) ethoxy]Phenyl } pyrrolo [2,1-f][1,2,4]Triazine-6-carboxylic acid ethyl ester;

9) synthesis of 2-amino-4- {2, 4-dichloro-6- [2- (1H-pyrazol-1-yl) ethoxy ] phenyl } pyrrolo [2,1-f ] [1,2,4] triazine-6-carboxylic acid:

stirring and reacting sodium hydroxide aqueous solution with 2-amino-4- {2, 4-dichloro-6- [2- (1H-pyrazol-1-yl) ethoxy ] phenyl } pyrrolo [2,1-f ] [1,2,4] triazine-6-carboxylic acid ethyl ester and ethanol at room temperature; after the reaction is finished, adding a hydrochloric acid aqueous solution to obtain a mixture with the pH value of 7.0 +/-1; extracting the mixture with ethyl acetate, washing the extracted organic layer with saturated aqueous sodium chloride solution, dehydrating, drying, and vacuum concentrating to obtain 2-amino-4- {2, 4-dichloro-6- [2- (1H-pyrazol-1-yl) ethoxy ] phenyl } pyrrolo [2,1-f ] [1,2,4] triazine-6-carboxylic acid;

10) synthesis of 2-amino-N-cyclopropyl-4- {2, 4-dichloro-6- [2- (1H-pyrazol-1-yl) ethoxy ] phenyl } pyrrolo [2,1-f ] [1,2,4] triazine-6-carboxamide:

reacting 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride with 2-amino-4- {2, 4-dichloro-6- [2- (1H-pyrazol-1-yl) ethoxy ] phenyl } pyrrolo [2,1-f ] [1,2,4] triazine-6-carboxylic acid, cyclopropylamine, triethylamine and dimethylformamide under stirring at room temperature; after the reaction is finished, extracting by using ethyl acetate, washing the extracted organic layer by using a saturated sodium bicarbonate aqueous solution and a saturated sodium chloride aqueous solution, and then dehydrating, drying and vacuum concentrating to obtain a crude product of 2-amino-N-cyclopropyl-4- {2, 4-dichloro-6- [2- (1H-pyrazol-1-yl) ethoxy ] phenyl } pyrrolo [2,1-f ] [1,2,4] triazine-6-formamide; finally, the crude product of 2-amino-N-cyclopropyl-4- {2, 4-dichloro-6- [2- (1H-pyrazol-1-yl) ethoxy ] phenyl } pyrrolo [2,1-f ] [1,2,4] triazine-6-carboxamide was purified by silica gel column to obtain purified 2-amino-N-cyclopropyl-4- {2, 4-dichloro-6- [2- (1H-pyrazol-1-yl) ethoxy ] phenyl } pyrrolo [2,1-f ] [1,2,4] triazine-6-carboxamide.

3. The method of synthesizing 2-amino-N-cyclopropyl-4- {2, 4-dichloro-6- [2- (1H-pyrazol-1-yl) ethoxy ] phenyl } pyrrolo [2,1-f ] [1,2,4] triazine-6-carboxamide according to claim 2, wherein the step of synthesizing 2, 4-dichloro-6-hydroxybenzeneboronic acid is:

1) synthesizing 2-iodine-3, 5-dichlorophenol:

mixing p-3, 5-dichlorophenol and anhydrous toluene, and adding a sodium hydride aqueous solution at 0 ℃ under a nitrogen atmosphere for reaction; the reaction mixture was warmed to room temperature and stirred for 30 minutes, the resulting suspension was cooled back to 0 ℃ and then iodine was added for further reaction; stirring the final reaction mixture, placing the reaction mixture in an environment of between 0 and room temperature for at least 24 hours, sequentially quenching the reaction mixture by using a hydrochloric acid aqueous solution, extracting the reaction mixture by using ethyl acetate, separating by using an ethyl acetate layer, quenching and washing by using a saturated sodium chloride aqueous solution, and dehydrating, drying and vacuum-concentrating a quenched organic layer to obtain a crude product of the 2-iodine-3, 5-dichlorophenol; purifying the crude product of the 2-iodine-3, 5-dichlorophenol by using a silica gel column to obtain purified 2-iodine-3, 5-dichlorophenol;

2) synthesis of 1, 5-dichloro-3- (ethoxymethoxy) -2-iodobenzene:

stirring 2 iodine-3, 5-dichlorophenol, chloromethoxy ethane, cesium carbonate and dimethylformamide for reaction at room temperature; quenching and washing the reacted mixture by water and salt water in sequence, and dehydrating, drying and vacuum-concentrating the quenched organic layer to obtain a crude product of 1, 5-dichloro-3- (ethoxymethoxy) -2-iodobenzene; purifying the crude product of the 1, 5-dichloro-3- (ethoxymethoxy) -2-iodobenzene by using a silica gel column to obtain purified 1, 5-dichloro-3- (ethoxymethoxy) -2-iodobenzene;

3) synthesis of 2- [2, 4-dichloro-6- (ethoxymethoxy) phenyl ] -4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan:

purging 1, 5-dichloro-3- (ethoxymethoxy) -2-iodobenzene, bis-pinacol borate and triethylamine in a dioxane solution for 30 minutes by using nitrogen, adding palladium acetate and biphenyl-2-yl-dicyclohexylphosphine into a reaction mixture, and stirring the reaction mixture at 80 ℃ until the reaction is finished; after the reaction mixture is cooled, quenching and washing the reaction mixture by saturated ammonium chloride solution, water and brine in sequence; dehydrating, drying and vacuum concentrating the quenched organic layer to obtain a crude product of 2- [2, 4-dichloro-6- (ethoxymethoxy) phenyl ] -4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan; purifying the crude product of 2- [2, 4-dichloro-6- (ethoxymethoxy) phenyl ] -4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan by using a silica gel column to obtain purified 2- [2, 4-dichloro-6- (ethoxymethoxy) phenyl ] -4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan;

4) synthesis of 2, 4-dichloro-6-hydroxyphenylboronic acid:

mixing 2- [2, 4-dichloro-6- (ethoxymethoxy) phenyl ] -4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan and anhydrous dichloromethane, and adding boron tribromide to react at 0 ℃ under the nitrogen atmosphere; after the reaction is finished, pouring the reaction mixture into water, adding a sodium hydroxide aqueous solution, and adjusting the pH value of the reaction mixture to 10 +/-1; separating to remove the organic layer; adjusting pH value of the separated water layer solution to 3 with 1N HCl solution, extracting with ethyl acetate, washing the extracted organic layer with saturated sodium chloride aqueous solution, dehydrating, drying, and vacuum concentrating to obtain 2, 4-dichloro-6-hydroxyphenylboronic acid.