CN104829613B

CN104829613B - Diaryl-substituted pyrazolo ring derivative, preparation method thereof and application thereof in medical field

Info

Publication number: CN104829613B
Application number: CN201510174036.6A
Authority: CN
Inventors: 安晓霞; 别平彦; 杨午立; 庄戈诗
Original assignee: Chifeng Mengguang Biotechnology Co ltd
Current assignee: Chifeng Mongolia Biotechnology Co., Ltd.
Priority date: 2015-04-13
Filing date: 2015-04-13
Publication date: 2020-06-09
Anticipated expiration: 2035-04-13
Also published as: CN104829613A

Abstract

The invention discloses a diaryl substituted pyrazolo ring derivative with a structure shown as a formula I, a preparation method thereof and application thereof in the field of medicines. The diaryl-substituted pyrazolo ring derivative has obvious SK-MEL28 cell inhibition activity, can be used as a BRAF inhibitor, is used for preparing medicaments for preventing or treating diseases including melanoma and diseases related to B-RAF V600E mutation, and has wide application prospect and value.

Description

Diaryl-substituted pyrazolo ring derivative, preparation method thereof and application thereof in medical field

Technical Field

The invention relates to diaryl substituted pyrazolo ring derivatives, a preparation method and application thereof, in particular to diaryl substituted pyrazolo ring derivatives with SK-MEL28 cell inhibition activity highly expressed by B-RAF V600E mutation, a preparation method and application thereof in medicine.

Background

Tumors are one of the most serious diseases threatening human health, and the chemotherapy of tumors has been changed greatly in recent years with the development of cell biology and tumor pharmacology. The traditional chemotherapy drugs are gradually abandoned because cell division is nonspecifically blocked, so that normal cell death is caused while tumor cells are killed, and meanwhile, by taking key node proteins in abnormally activated signal channels in the tumor cells as targets, the discovery of high-efficiency, low-toxicity and strong-specificity micromolecule inhibitors becomes an important direction for the research and development of current antitumor drugs. Receptor Tyrosine Kinases (RTKs) which are abnormally expressed and activated in tumors play a key role in various links such as tumor occurrence and development, invasion and metastasis, chemotherapy resistance and the like, and become hot spots for research on antitumor drugs.

Cytokinin-activated protein kinases (MAPKs) mediate intracellular signaling through a number of cell surface receptors. Among the MAPK-mediated pathways, the RAF/MEK/ERK signaling cascade is of great interest because it plays an important role in the development of a large number of human cancers, particularly those associated with RAF protein activation.

Studies have shown that activation of RAF involves mainly the release of inhibition of the RAF catalytic domain by the N-terminal regulatory region. Both RAS binding regions and cysteine rich regions are involved in kinase domain interactions and in the self-inhibition of RAF. This basic mode of action applies to all three Raf proteins (A-RAF, B-RAF and C-RAF). However, for both A-RAF and C-RAF, additional steps are required to achieve maximal activation, such as phosphorylation of activating amino acid residues and dephosphorylation of negatively regulated amino acid residues. Thus, B-RAF is the most readily activated protein in this family by RAS. In addition, the B-RAF kinase activity is much higher than that of C-RAF and A-RAF.

According to the results of the current research, B-RAF is an important isoform protein associated with cell proliferation and an important target of oncogenic RAS. Abnormal mutations in vivo have been shown to occur only in the case of B-RAF, which is thought to occur at a rate of 30-60% in cutaneous malignant melanoma (Nature,2002,417,949-954), 30-50% in thyroid cancer, 5-20% in colorectal cancer, and 30% or less in ovarian cancer (Nature Rev.mol.cell Biology,2004,5, 875-885). To date, 45 or more B-RAF mutations have been found. Mutant B-RAF proteins are converted in NIH3T3 cells (Nature,2002,417,949-954) and melanoma cells (Cancer Res.,2004,64, 2338-2342), and are essential for melanoma survival and conversion (Cancer Res.,2003,63, 5198-5202). Thus, B-Raf, which is at the core of tandem signaling in Raf/MEK/ERK, plays a crucial role in tumor survival.

RAF kinase inhibitors have been used to prevent tumor cell proliferation and thereby treat the following cancers: histiocytic lymphoma, small cell lung and pancreatic cancer, and breast cancer. Sorafenib is an oral, cytostatic, multi-target kinase inhibitor approved by the FDA for the treatment of advanced renal cell carcinoma. Sorafenib prevents tumor growth by inhibiting tumor cell proliferation and tumor angiogenesis (Clark et al, 2005, Clinical Cancer Res 11(15): 5472-.

However, the use of such targeted therapies is not ideal and problems such as drug resistance and lack of tumor response in the general population arise. Therefore, there is a great need in the art to develop new inhibitors to overcome many of the problems in tumor therapy.

Disclosure of Invention

The invention aims to provide a novel BRAF inhibitor with high efficiency and low toxicity.

The first aspect of the present invention provides a compound represented by formula I, a pharmaceutically acceptable salt thereof, a prodrug thereof, a hydrate thereof, or a solvate thereof:

in the formula:

n is 1 or 2;

x is N or CH;

y is halogen or NR₂R₃；

R₁Selected from the group consisting of: substituted or unsubstituted C₁-C₈Alkyl, substituted or unsubstituted C₃-C₈Cycloalkyl, substituted or unsubstituted C₆-C₁₀Aryl, heteroaryl containing 1-3 heteroatoms, wherein said substitution means having a substituent selected from the group consisting of: c₁-C₃Alkyl radical, C₁-C₃Haloalkyl, C₃-C₈Cycloalkyl radical, C₆-C₁₀Aryl radical, C₁-C₆Alkoxy, halogen;

R₂selected from the group consisting of: H. formyl, alkoxycarbonyl, -L₁NHC(O)OR_1a、-L₁NHC(O)NHR_1aSubstituted or unsubstituted C₃-C₆Cycloalkyl, substituted or unsubstituted C₁-C₃Alkyl, substituted or unsubstituted acyl;

wherein, the "substituted" means having a substituent selected from the group consisting of: cyano, -C (O) NH₂Hydroxy or C₁-C₆An alkyl group;

L₁is optionally substituted by 1-3 substituents each independently selected from halogen, C₁-C₄Alkyl or halogen substituted C₁-C₄Alkyl substituted C₁-C₄An alkylene group;

R_1ais H, C₁-C₄Alkyl or halogen substituted C₁-C₄An alkyl group;

R₃is H or methyl;

R₄is H, halogen or C₁-C₄An alkyl group;

R₅is H or halogen;

R₆is H, halogen, C₁-C₄Alkoxy radical, C₁-C₄An alkyl group;

R₇is halogen, H or C₁-C₄An alkyl group.

In another preferred embodiment, the aryl group is optionally substituted with 1-3 substituents each independently selected from halogen, CH₃Or CF₃Is substituted with the substituent(s).

In another preferred embodiment, the compound is selected from the group consisting of:

n- {3- [3- (2-amino-pyrimidin-4-yl) -4,5,6, 7-tetrahydro-pyrazolo [1,5-a ] pyridin-2-yl ] -phenyl } -2, 6-difluoro-benzenesulfonamide;

n- {3- [3- (2-amino-pyrimidin-4-yl) -4,5,6, 7-tetrahydro-pyrazolo [1,5-a ] pyridin-2-yl ] -phenyl } -methanesulfonamide;

n- {3- [3- (2-amino-pyrimidin-4-yl) -5, 6-dihydro-4H-pyrrolo [1,2-B ] pyrazol-2-yl ] -phenyl } -methanesulfonamide;

propane-1-sulfonic acid {3- [3- (2-amino-pyrimidin-4-yl) -4,5,6, 7-tetrahydro-pyrazolo [1,5-a ] pyridin-2-yl ] -phenyl } -amide;

propane-1-sulfonic acid {3- [3- (2-isobutylamino-pyrimidin-4-yl) -4,5,6, 7-tetrahydro-pyrazolo [1,5-a ] pyridin-2-yl ] -phenyl } -amide;

2, 6-difluoro-N- {3- [3- (2-isobutylamino-pyrimidin-4-yl) -4,5,6, 7-tetrahydro-pyrazolo [1,5-a ] pyridin-2-yl ] yl-phenyl } -benzenesulfonamide;

n- {3- [3- (2-amino-pyrimidin-4-yl) -5, 6-dihydro-4H-pyrrolo [1,2-b ] pyrazol-2-yl ] -phenyl } -2, 6-difluoro-benzenesulfonamide;

propane-1-sulfonic acid {3- [3- (2-amino-pyrimidin-4-yl) -5, 6-dihydro-4H-pyrrolo [1,2-B ] pyrazol-2-yl ] -phenyl } -amide;

n- {3- [3- (2-isobutylamino-pyrimidin-4-yl) -4,5,6, 7-tetrahydro-pyrazolo [1,5-a ] pyridin-2-yl ] -phenyl } -methanesulfonamide;

propane-1-sulfonic acid {3- [3- (2-isobutylamino-pyrimidin-4-yl) -5, 6-dihydro-4H-pyrrolo [1,2-b ] pyrazol-2-yl ] -phenyl } -amide;

propane-1-sulfonic acid (3- {3- [2- (2-cyanoethylamino) -pyrimidin-4-yl ] -5, 6-dihydro-4H-pyrrolo [1,2-b ] pyrazol-2-yl } -phenyl) -amide;

n- (3- {3- [2- (2-cyanoethylamino) -pyrimidin-4-yl ] -4,5,6, 7-tetrahydro-pyrazolo [1,5-a ] pyridin-2-yl } yl-phenyl) -2, 6-difluoro-benzenesulfonamide;

propane-1-sulfonic acid {3- [3- (2-cyano-ethylamino-pyrimidin-4-yl) -4,5,6, 7-tetrahydropyrazolo [1,5-a ] pyridin-2-yl ] -phenyl } -amide;

n- (3- {3- [2- (2-cyanoethylamino) -pyrimidin-4-yl ] -5, 6-dihydro-4H-pyrrolo [1,2-b ] pyrazol-2-yl } -phenyl) -2, 6-difluoro-benzenesulfonamide;

n- (3- {3- [ 2-aminopyridin-4-yl ] -4,5,6, 7-tetrahydropyrazolo [1,5-a ] pyridin-2-yl } -2-fluorophenyl) -2, 6-difluorobenzenesulfonamide;

n- (5-chloro 3- {3- [2- (2-cyanoethylamino) -pyrimidin-4-yl ] -5, 6-dihydro-4H-pyrrolo [1,2-b ] pyrazol-2-yl } -2-fluorophenyl) -2, 6-difluorobenzenesulfonamide;

n- (3- {3- [2- (2-cyanoethylamino) -pyrimidin-4-yl ] -5, 6-dihydro-4H-pyrrolo [1,2-b ] pyrazol-2-yl } -2, 4-difluorophenyl) -2, 6-difluorobenzenesulfonamide;

n- (3- {3- [2- (2-cyanoethylamino) -pyrimidin-4-yl ] -5, 6-dihydro-4H-pyrrolo [1,2-b ] pyrazol-2-yl } -2, 6-difluorophenyl) -2, 6-difluorobenzenesulfonamide;

methyl (S) - (1- ((4- (2- (3- ((2, 6-difluorophenyl) sulfonamido) -2-fluorophenyl) -5, 6-dihydro-4H-pyrrolo [1,2-b ] pyrazol-3-yl) pyrimidin-2-yl) amino) propan-2-yl) carbamate;

2, 6-difluoro-N- (2-fluoro-3- (3- (2- ((2- (methylsulfonyl) ethyl) amino) pyrimidin-4-yl) 4H-5, 6-dihydropyrrolo [3, 2-b ] pyrazol-2-yl) phenyl) benzenesulfonamide.

A second aspect of the present invention provides a process for the preparation of a compound of formula I, said process comprising the steps of:

and (3) performing cycloaddition reaction on the disubstituted alkyne intermediate V and the stednone III to obtain a pyrazolo ring derivative I:

in another preferred embodiment, the reaction temperature in this step is 170-180 ℃, preferably 175 ℃.

A third aspect of the invention provides a pharmaceutical composition comprising a therapeutically effective amount of a compound of formula I as described in the first aspect of the invention, or a pharmaceutically acceptable salt, tautomer, optical isomer, pharmaceutically acceptable solvate thereof.

In another preferred embodiment, the pharmaceutical composition is for the treatment of a disease associated with a mutation in B-RAF V600E or the pharmaceutical composition is for the treatment of a disease associated with a mutation in B-RAF V600E.

In another preferred embodiment, said disease associated with mutation of B-RAF V600E is selected from the group consisting of: abnormal cell proliferation, morphological changes, hyperkinesia, or a combination thereof.

In a fourth aspect, the present invention provides a B-RAF kinase inhibitor comprising an inhibitory effective amount of a compound of formula I as described in the first aspect of the invention, or a pharmaceutically acceptable salt, tautomer, optical isomer, pharmaceutically acceptable solvate thereof.

In another preferred embodiment, the pharmaceutically acceptable salt is a salt of a compound of formula I selected from the group consisting of: inorganic acid salts, organic acid salts, alkyl sulfonates, aryl sulfonates, or combinations thereof; preferably, the salt is selected from the group consisting of: hydrochloride, hydrobromide, nitrate, sulfate, phosphate, formate, acetate, propionate, benzoate, maleate, fumarate, succinate, tartrate, citrate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, or a combination thereof.

In another preferred embodiment, the pharmaceutically acceptable solvate refers to a solvate of the compound of formula I with a solvent selected from the group consisting of: water, ethanol, isopropanol, diethyl ether, acetone, or combinations thereof.

A fifth aspect of the invention provides the use of a compound of formula I:

(a) preparing a B-RAF inhibitor;

(b) for non-therapeutically inhibiting the activity of SK-MEL28 cells in vitro;

(c) for non-therapeutically inhibiting tumor cell growth in vitro;

(d) is used for preparing the medicine for treating the B-RAF V600E mutation inhibitory activity.

In a sixth aspect the present invention provides an in vitro non-therapeutic method of inhibiting B-RAF kinase, said method comprising the steps of:

culturing a cell in the presence of a compound of formula I, or a pharmaceutically acceptable salt thereof, thereby inhibiting B-RAF kinase activity in said cell.

A seventh aspect of the invention provides a method of treating a disease associated with a BRAF V600E mutation, the method comprising the steps of:

administering to a subject a therapeutically effective dose of a compound of formula I as described in the first aspect of the invention or a pharmaceutically acceptable salt, tautomer, optical isomer, pharmaceutically acceptable solvate thereof.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Detailed Description

The inventor of the invention has long and intensive research and unexpectedly prepares a compound with a structure shown as a formula I, and the compound is a type of effective B-RAF inhibitor and is particularly suitable to be used as an inhibitor of B-RAF V600E mutation inhibition activity. Based on the above findings, the inventors have completed the present invention.

Term(s) for

In the present invention, the alkyl group includes a linear or branched alkyl group, the alkenyl group includes a linear or branched alkenyl group, the alkynyl group includes a linear or branched alkynyl group, and the halogen is F, Cl, Br or I.

In the present invention, the term "substituted" means, unless otherwise specified, that one or more hydrogen atoms on a group are substituted with a substituent selected from the group consisting of: C1-C4 alkyl, C3-C7 cycloalkyl, C1-C4 alkoxy, halogen, hydroxyl, carboxyl (-COOH), C1-C4 aldehyde group, C2-C4 acyl, C2-C4 ester group, amino and phenyl; the phenyl group includes an unsubstituted phenyl group or a substituted phenyl group having 1 to 3 substituents selected from: halogen, C1-C4 alkyl, cyano, OH, nitro, C3-C7 cycloalkyl, C1-C4 alkoxy and amino.

In particular, herein, unless otherwise specified, reference to an atom includes all isotopic forms thereof, for example, when referring to a "hydrogen atom," it is intended to refer to a hydrogen atom, a deuterium atom, a tritium atom, or a combination thereof. In the present invention, the abundance of each isotopic atom of a certain element may be a state in which the element naturally occurs in nature, or a state in which the element is isotopically enriched.

The term "C1-C6 alkyl" refers to a straight or branched chain alkyl group having 1 to 6 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, or the like.

Specifically, in the present invention, when the number of carbon atoms of the group is not limited, it means a group having 1 to 10 carbon atoms, preferably 1 to 6 carbon atoms, unless otherwise specified.

In another preferred embodiment, the heterocyclic ring is a saturated ring or an unsaturated ring.

The term "C1-C4 alkoxy" refers to a straight or branched chain alkoxy group having 1 to 4 carbon atoms, such as methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, sec-butoxy, tert-butoxy, or the like.

The term "alkanoyl" or "alkylcarbonyl" refers to a group having the structure "-CO-alkyl", such as, for example, methylacyl, ethylacyl, propylacyl, isopropylacyl, butylacyl, isobutylacyl, sec-butylacyl, tert-butylacyl, or the like.

The term "pharmaceutically acceptable solvate" refers to a solvate of the corresponding compound with water, ethanol, isopropanol, diethyl ether, acetone.

A compound of formula I

The invention provides a compound shown as a formula I:

in the formula:

n is 1 or 2;

x is N or CH;

y is halogen or NR₂R₃；

R₁Selected from the group consisting of: substituted or unsubstituted C₁-C₈Alkyl, substituted or unsubstituted C₃-C₈Cycloalkyl, substituted or unsubstituted C₆-C₁₀Aryl, heteroaryl containing 1-3 heteroatoms, wherein said substitution is with a substituent selected from the group consisting ofSubstitution: c₁-C₃Alkyl radical, C₁-C₃Haloalkyl, C₃-C₈Cycloalkyl radical, C₆-C₁₀Aryl radical, C₁-C₆Alkoxy, halogen;

R_1ais H, C₁-C₄Alkyl or halogen substituted C₁-C₄An alkyl group;

R₃is H or methyl;

R₄is H, halogen or C₁-C₄An alkyl group;

R₅is H or halogen;

R₆is H, halogen, C₁-C₄Alkoxy radical, C₁-C₄An alkyl group;

R₇is halogen, H or C₁-C₄An alkyl group.

More preferably, the compounds of formula I according to the invention are as shown in table 1.

TABLE 1

Process for the preparation of compounds of formula I

The present invention also provides a process for the preparation of a compound of formula I, said process comprising the steps of:

Pharmaceutically acceptable salts

The pharmaceutical forms of the compounds of the invention may include the compounds themselves, as well as other pharmaceutically acceptable variations, such as optical isomers, cis-trans isomers, and the like, or pharmaceutically acceptable salts or solvates.

Preferably, the pharmaceutically acceptable salts include, but are not limited to: inorganic acid salts, organic acid salts, alkyl sulfonates, aryl sulfonates, or combinations thereof; preferably, the salt is selected from the group consisting of: hydrochloride, hydrobromide, nitrate, sulfate, phosphate, formate, acetate, propionate, benzoate, maleate, fumarate, succinate, tartrate, citrate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, or a combination thereof;

preferably, the pharmaceutically acceptable solvates include, but are not limited to: the compound is a solvate of water, ethanol, isopropanol, ether, acetone and the like.

Use of compounds of formula I

The compounds of formula I according to the invention are useful for:

(a) preparing a B-RAF inhibitor;

(b) for non-therapeutically inhibiting the activity of SK-MEL28 cells in vitro;

(c) for non-therapeutically inhibiting tumor cell growth in vitro;

The main advantages of the invention include:

compared with the prior art, the diaryl substituted pyridine provided by the inventionThe azolocyclic derivative has obvious inhibition effect on the human skin malignant melanoma SK-MEL28 cell strain with high mutation expression of B-RAF V600E; in particular compounds 17 and 18, the median inhibitory concentration IC₅₀The values are 137nM and 124nM respectively, which is better than positive control drug Verofenib (182nM), and has better development and application prospects.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out under conventional conditions or conditions recommended by the manufacturers. Unless otherwise indicated, percentages and parts are by weight.

The structures of the compounds prepared in the following examples are by nuclear magnetic resonance (¹HNMR) and Mass Spectrometry (MS), if necessary using NOE (nuclear over hauser Effect).

¹HNMR shift (δ) is given in units of parts per million (ppm).¹HNMR was measured using a Bruker AVANCE-400 NMR spectrometer using deuterated dimethyl sulfoxide (DMSO-d)₆) Deuterated chloroform (CDCl)₃) Internal standard is Tetramethylsilane (TMS), and chemical shift is 10^-6Given as a unit.

MS was determined using a FINNIGAN LCQAD (ESI) mass spectrometer (manufacturer: Therm, model: Finnigan LCQadvantage MAX).

IC₅₀The values were determined with a NovoStar microplate reader (BMG, Germany).

The thin silica gel layer is prepared from HSGF254 or GF254 silica gel plate.

The silica gel column chromatography uses 200-300 mesh silica gel of the Tibet Huanghai silica gel as a carrier.

In the following examples, unless otherwise specified, all reactions were carried out under a nitrogen atmosphere.

The nitrogen atmosphere means that the reaction flask is connected with a nitrogen balloon having a volume of about 1L.

In the following examples, the solutions in the reaction are all aqueous solutions unless otherwise specified.

Example 1

The first step is as follows:

bis (triphenylphosphine) palladium dichloride (47mg, 0.067mmol) and triphenylphosphine (35mg, 0.134mmol) were dissolved in THF (10ml) at room temperature, triethylamine (15ml) and 2, 4-dichloropyrimidine (2g, 13.4mmol) were added sequentially, stirring was carried out for 15 minutes under nitrogen protection, then cuprous iodide (26mg, 0.134mmol) and 3-ethynyl-aniline (1.77g, 14.77mmol) were added sequentially, stirring was carried out at an external temperature of 90 ℃ for 4 hours, TLC detection of the completion of the starting material, cooling, suction filtration, the filter cake was washed with ethyl acetate, the combined organic phases were dried over anhydrous sodium sulfate and concentrated under reduced pressure. Purification by silica gel column chromatography gave 3- (2-chloro-pyrimidin-4-ethynyl) -aniline (1.2g, light yellow solid) yield: 39 percent.¹MS m/z(ESI):230.6[M+1]

¹HNMR(400Hz,DMSO-d₆):8.82(m,1H),7.75(m,1H),7.13(t,1H),6.79(m,2H),6.74(m,1H),5.41(s,2H).

The second step is that:

3- (2-chloro-pyrimidin-4-ethynyl) -aniline (1g, 4.4mmol) and 2, 5-difluorobenzenesulfonyl chloride (1g, 4.84mol) were added to dichloromethane (20ml) at room temperature, followed by dropwise addition of pyridine (0.64ml) under ice bath, stirring at room temperature for 60h after completion of dropwise addition, TLC monitored for completion of the reaction of the starting materials, washing with 1N HCl solution (30ml), drying of the organic phase over anhydrous sodium sulfate, and concentration under reduced pressure. Purifying with silica gel column chromatography to obtain N- [3- (2-chloro-pyrimidine-4-ethynyl) -phenyl]-2, 6-difluoro-benzenesulfonamide (1g, white solid), yield: 56 percent.¹MS m/z(ESI):406.8[M+1]

¹HNMR(400Hz,DMSO-d₆):11.25(s,1H),8.85(m,1H),7.82-7.72(m,2H), 7.44(m,3H),7.32(m,3H).

The third step:

reacting N- [3- (2-chloro-pyrimidin-4-ethynyl) -phenyl at room temperature]-2, 6-difluoro-benzenesulfonamide (1.1g, 2.71mmol) was suspended in 80ml of mesitylene, and 4,5,6, 7-tetrahydro [1,2,3 ] was added]Oxadiazolo [3,4-a ]]Pyridin-8-ylium-3-phenoxide (0.42g, 3mmol), displaced three times with nitrogen and brought to an external temperature of 1 under nitrogenThe mixture was stirred for 24h at 75 ℃ and TLC monitored for complete reaction of the starting materials. The solvent was removed under reduced pressure and the crude product was purified by silica gel column chromatography to give N- {3- [3- (2-chloro-pyrimidin-4-yl) -4,5,6, 7-tetrahydro-pyrazolo [1,5-a]Pyridin-2-yl]-phenyl } -2, 6-difluoro-benzenesulfonamide (0.33g, yellow solid) yield: 24 percent.¹MS m/z(ESI):502.9[M+1]

¹HNMR(400Hz,DMSO-d₆):10.97(s,1H),8.42(d,1H),7.71(m,1H),7.32-7-13(m,6H),6.82(d,1H),4.15(m,2H),3.01(m,2H),2.00(m,2H),1.83(m,2H).

The fourth step:

n- {3- [3- (2-chloro-pyrimidin-4-yl) -4,5,6, 7-tetrahydro-pyrazolo [1,5-a ] is reacted at room temperature]Pyridin-2-yl]-phenyl } -2, 6-difluoro-benzenesulfonamide (0.1g, 0.2mmol) was added to 15ml of 7mol/L ammonia/methanol solution, the reaction was blocked at 90 ℃ for 60 hours, the reaction of the starting material was monitored by TLC to be complete, and after cooling to room temperature, 50ml of dichloromethane was added, and concentrated under reduced pressure. Purifying with silica gel column chromatography to obtain N- {3- [3- (2-amino-pyrimidin-4-yl) -4,5,6, 7-tetrahydro-pyrazolo [1,5-a]Pyridin-2-yl]-phenyl } -2, 6-difluoro-benzenesulfonamide (0.01g, white solid) yield: 10.4 percent.¹MS m/z(ESI):483.5[M+1]

¹HNMR(400Hz,DMSO-d₆):10.93(s,1H),7.92(d,1H),7.71(m,1H),7.28-7.09(m,6H),6.43(s,2H),5.97(d,1H),4.11(m,2H),2.97(m,2H),2.00(m,2H),1.80(m.2H).

Example 2

The first step is as follows:

3- (2-chloro-pyrimidin-4-ethynyl) -aniline (1.5g, 6.6mmol) and methanesulfonyl chloride (0.83g, 7.26mol) were added to dichloromethane (30ml) at room temperature, followed by dropwise addition of pyridine (0.6ml) under ice bath, stirring at room temperature for 60h after completion of dropwise addition, TLC monitored for completion of the reaction of the starting materials, washing with 1N HCl solution (30ml), drying of the organic phase over anhydrous sodium sulfate, and concentration under reduced pressure. Purifying with silica gel column chromatography to obtain N- [3- (2-chloro-pyrimidine-4-ethynyl) -phenyl]Methanesulfonamide (1.4g, yellow solid), yield: and 69 percent.¹MS m/z(ESI):308.7[M+1]

¹HNMR(400Hz,DMSO-d₆):10.06(s,1H),8.86(d,1H),7.83(d,1H),7.52-7.35(m,4H),3.06(s,3H).

The second step is that:

reacting N- [3- (2-chloro-pyrimidin-4-ethynyl) -phenyl at room temperature]-Methanesulfonamide (0.8g, 2.6mmol) was suspended in 25ml of mesitylene, and 4,5,6, 7-tetrahydro [1,2,3 ] was added]Oxadiazolo [3,4-a ]]Pyridin-8-ylium-3-phenoxide (0.37g,2.6mmol), displaced with nitrogen three times, warmed to 175 ℃ under nitrogen, stirred for 24h.TLC to monitor completion of the starting material reaction the solvent was removed under reduced pressure and the crude product was purified by silica gel column chromatography to give N- {3- [3- (2-chloro-pyrimidin-4-yl) -4,5,6, 7-tetrahydro-pyrazolo [1, 5-a-]Pyridin-2-yl]-phenyl } -methanesulfonamide (120mg, yellow solid) yield: 11.4 percent.¹MS m/z(ESI):404.8[M+1]

The third step:

n- {3- [3- (2-chloro-pyrimidin-4-yl) -4,5,6, 7-tetrahydro-pyrazolo [1,5-a ] is reacted at room temperature]Pyridin-2-yl]-phenyl } -methanesulfonamide (0.12g, 0.3mmol) was added to 15ml of 7mol/L ammonia/methanol solution, the reaction was blocked at 90 ℃ for 60 hours, the starting material was monitored by TLC for completion of the reaction, cooled to room temperature, added with 50ml of dichloromethane, and concentrated under reduced pressure. Purifying with silica gel column chromatography to obtain N- {3- [3- (2-amino-pyrimidin-4-yl) -4,5,6, 7-tetrahydro-pyrazolo [1,5-a]Pyridin-2-yl]-phenyl } -methanesulfonamide (20mg, white solid) yield: 17.4 percent.¹MS m/z(ESI):385.5[M+1]

¹HNMR(400Hz,DMSO-d₆):9.75(s,1H),8.03(d,1H),7.36(m,2H),7.21(m,2H),6.45(d,2H),6.20(d,1H),4.14(m,2H),2.98(m,5H),2.00(m,2H),1.80(m,2H).

Example 3

The first step is as follows:

reacting N- [3- (2-chloro-pyrimidine-4-ethynyl) -phenyl]-Methanesulfonamide (0.8g, 2.6mmol) was suspended in 25ml of mesitylene, and 5, 6-dihydro-4H-pyrrolo [1,2-C ] was added][1,2,3]Oxadiazole-7-onium-3-phenolate (0.33g, 6mmol), nitrogen replaced three times, warmed to 175 deg.C under nitrogen, stirred for 24h.TLC to monitor completion of the reaction of the starting materials, and then depressurizedRemoving solvent, and purifying the crude product by silica gel column chromatography to obtain N- {3- [3- (2-chloro-pyrimidin-4-yl) -5, 6-dihydro-4H-pyrrolo [1,2-b ]]Pyrazol-2-yl]-phenyl } -methanesulfonamide (80mg, yellow solid) yield: 7.9 percent.¹MS m/z(ESI):390.8[M+1]

The third step:

n- {3- [3- (2-chloro-pyrimidin-4-yl) -5, 6-dihydro-4H-pyrrolo [1,2-b ] at room temperature]Pyrazol-2-yl]-phenyl } -methanesulfonamide (80mg, 0.2mmol) was added to 15ml of 7mol/L ammonia/methanol solution, the reaction was blocked at 90 ℃ for 60h, the starting material was monitored by TLC for completion of the reaction, cooled to room temperature, 50ml of dichloromethane were added, and concentrated under reduced pressure. Purifying with silica gel column chromatography to obtain N- {3- [3- (2-amino-pyrimidin-4-yl) -5, 6-dihydro-4H-pyrrolo [1,2-b ]]Pyrazol-2-yl]-phenyl } -methanesulfonamide (15mg, white solid) yield: 20.2 percent.¹MS m/z(ESI):371.4[M+1]

¹HNMR(400Hz,DMSO-d₆):9.78(s,1H),8.03(d,1H),7.39-7.20(m,4H),6.39(m,2H),6.26(d,1H),4.14(m,2H),3.12(m,2H),2.96(s,3H),2.58(m,2H).

Example 4

The first step is as follows:

3- (2-chloro-pyrimidin-4-ethynyl) -aniline (1.5g, 6.6mmol) and propylsulfonyl chloride (1g, 7.26mol) were added to dichloromethane (30ml) at room temperature, followed by dropwise addition of pyridine (0.6ml) on an ice bath, stirring for 60h at room temperature after completion of the addition, TLC monitored for completion of the reaction of the starting materials, washed with 1N HCl solution (10ml), the organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure. Purifying with silica gel column chromatography to obtain propane-1-sulfonic acid [3- (2-chloropyrimidine-4-ethynyl) -phenyl]Amine (1.5g, yellow solid), yield: 68 percent.¹MS m/z(ESI):336.8[M+1]

¹HNMR(400Hz,DMSO-d₆):10.10(s,1H),8.85(d,1H),7.83(d,1H),7.49-7.35(m,4H),3.17(m,2H),1.74(m,2H),0.97(t,3H).

The second step is that:

propane-1-sulfonic acid [3- (2-chloropyrimidin-4-ethynyl) -phenyl ] at room temperature]Amine (0.8g, 1.85mmol) was suspended in 25ml of mesitylene, and 4,5,6, 7-tetrahydro [1,2,3 ] was added]Oxadiazolo [3,4-a ]]Pyridin-8-ylium-3-phenoxide (0.26g, 1.85mmol), displaced with nitrogen three times, warmed to 175 ℃ under nitrogen, stirred for 24h, TLC monitored the completion of the starting material reaction, removed the solvent under reduced pressure, and the crude product was purified by silica gel column chromatography to give propane-1-sulfonic acid {3- [3- (2-chloro-pyrimidin-4-yl) -4,5,6, 7-tetrahydro-pyrazolo [1,5-a ]]Pyridin-2-yl]-phenyl } -amine (0.1g, yellow solid) yield: 12.5 percent.¹MS m/z(ESI):432.9[M+1]

The third step:

propane-1-sulfonic acid {3- [3- (2-chloro-pyrimidin-4-yl) -4,5,6, 7-tetrahydro-pyrazolo [1,5-a ] at room temperature]Pyridin-2-yl]Phenyl } -amine (0.1g, 0.23mmol) was added to 15ml of 7mol/L ammonia/methanol solution, the reaction was blocked at 90 ℃ for 60 hours, the starting material was monitored by TLC for completion of the reaction, cooled to room temperature, 50ml of dichloromethane were added, and concentrated under reduced pressure. Purifying with silica gel column chromatography to obtain propane-1-sulfonic acid {3- [3- (2-amino-pyrimidin-4-yl) -4,5,6, 7-tetrahydro-pyrazolo [1,5-a]Pyridin-2-yl]-phenyl } -amide (60mg, white solid) yield: and (3.8).¹MS m/z(ESI):413.5[M+1]

¹HNMR(400Hz,DMSO-d₆):9.80(s,1H),8.02(d,1H),7.33-7.14(m,4H),6.46(s,2H),6.16(d,1H),4.13(m,2H),3.02(m,4H),2.00(m,2H),1.81(m,2H),1.68(m,2H),0.92(t,3H).

Example 5

Propane-1-sulfonic acid {3- [3- (2-chloro-pyrimidin-4-yl) -4,5,6, 7-tetrahydro-pyrazolo [1,5-a ] at room temperature]Pyridin-2-yl]Phenyl } -amine (0.1g, 0.23mmol), 5ml isobutylamine were added to a single-necked flask, stirred at room temperature overnight, TLC monitored for completion of the reaction of the starting materials, 50ml of dichloromethane were added, and washed with 10ml of 1N hydrochloric acid solution. Purifying with silica gel column chromatography to obtain propane-1-sulfonic acid {3- [3- (2-isobutylamino-pyrimidin-4-yl) -4,5,6, 7-tetrahydro-pyrazolo [1,5-a]Pyridin-2-yl]-phenyl } -amide (30mg, light yellow solid) yield: 28.0 percent.¹MS m/z(ESI):467.6[M+1]

¹HNMR(400Hz,DMSO-d₆):9.82(s,1H),8.03(d,1H),7.32-7.12(m,5H),6.16(d,1H),4.14(m,2H),3.00(m,5H),2.00(m,2H),1.75(m,2H),1.66(m,2H),1.25(m,2H),0.89(m,9H).

Example 6

N- {3- [3- (2-chloro-pyrimidin-4-yl) -4,5,6, 7-tetrahydro-pyrazolo [1,5-a ] is reacted at room temperature]Pyridin-2-yl]-phenyl } -2, 6-difluoro-benzenesulfonamide (0.1g, 0.2mmol), 5ml isobutylamine was added to a single-necked flask, stirred at room temperature overnight, TLC monitored for completion of the starting material reaction, added 50ml dichloromethane, and washed with 10ml 1N hydrochloric acid solution. Purifying with silica gel column chromatography to obtain 2, 6-difluoro-N- {3- [3- (2-isobutylamino-pyrimidin-4-yl) -4,5,6, 7-tetrahydro-pyrazolo [1,5-a]Pyridin-2-yl]Phenyl } -benzenesulfonamide (30mg, light yellow solid) yield: 28.0 percent.¹MS m/z(ESI):555.6[M+1]

¹HNMR(400Hz,DMSO-d₆):10.93(s,1H),7.95(d,1H),7.67(m,1H),7.28-7.07(m,6H),5.97(m,1H),4.15(m,2H),3.01(m,4H),2.00(m,2H),1.83(m,2H),1.25(m,1H),0.86(m,6H).

Example 7

The first step is as follows:

reacting N- [3- (2-chloro-pyrimidin-4-ethynyl) -phenyl at room temperature]-2, 6-difluoro-benzenesulfonamide (1.1g, 2.71mmol) was suspended in 25ml of mesitylene, and 5, 6-dihydro-4H-pyrrolo [1,2-C ] was added][1,2,3]Oxadiazole-7-onium-3-phenolate (0.34g, 2.71mmol) was replaced with nitrogen three times, warmed to an external temperature of 175 ℃ under nitrogen, stirred for 24h.TLC to monitor completion of the starting material reaction, the solvent was removed under reduced pressure, and the crude product was purified by silica gel column chromatography to give N- {3- [3- (2-chloro-pyrimidin-4-yl) -5, 6-dihydro-4H-pyrrolo [1,2-b ]]Pyrazol-2-yl]-phenyl } -2, 6-difluoro-benzenesulfonamide (0.22g, yellow solid) yield: 16.6 percent.¹MS m/z(ESI):488.9[M+1]

¹HNMR(400Hz,DMSO-d₆):10.99(s,1H),8.42(d,1H),7.74(m,1H),7.32-7.18(m,6H),6.88(d,1H),4.20(m,2H),3.14(m,2H),2.61(m,2H).

The second step is that:

n- {3- [3- (2-chloro-pyrimidin-4-yl) -5, 6-dihydro-4H-pyrrolo [1,2-b ] at room temperature]Pyrazol-2-yl]-phenyl } -2, 6-difluoro-benzenesulfonamide (0.1g, 0.2mmol) was added to 15ml of 7mol/L ammonia/methanol solution, the reaction was blocked at 90 ℃ for 60 hours, the reaction of the starting material was monitored by TLC to be complete, and after cooling to room temperature, 50ml of dichloromethane was added, and the mixture was concentrated under reduced pressure. Purifying with silica gel column chromatography to obtain N- {3- [3- (2-amino-pyrimidin-4-yl) -5, 6-dihydro-4H-pyrrolo [1,2-b ]]Pyrazol-2-yl]-phenyl } -2, 6-difluoro-benzenesulfonamide (0.04g, light yellow solid) yield: 43.0 percent.¹MS m/z(ESI):467.5[M+1]

¹HNMR(400Hz,DMSO-d₆):10.96(s,1H),7.91(d,1H),7.69(m,1H),7.32-7.13(m,6H),6.39(s,2H),6.04(d,1H),4.14(m,2H),3.09(m,2H),2.57(m,2H).

Example 8

The first step is as follows:

propane-1-sulfonic acid [3- (2-chloropyrimidin-4-ethynyl) -phenyl ] at room temperature]The amine (0.8g, 1.85mmol) was suspended in 25ml of mesitylene, and 5, 6-dihydro-4H-pyrrolo [1,2-C ] was added][1,2,3]Oxadiazole-7-onium-3-phenolate (0.24g, 1.85mmol) was replaced with nitrogen three times, the temperature was raised to 175 ℃ under nitrogen protection, and stirring was carried out for 24 hours. TLC to monitor the completion of the reaction, the solvent was removed under reduced pressure and the crude product was purified by silica gel column chromatography to give propane-1-sulfonic acid- {3- [3- (2-chloro-pyrimidin-4-yl) -5, 6-dihydro-4H-pyrrolo [1,2-b ]]Pyrazol-2-yl]-phenyl } -amine (0.08g, yellow solid) yield: 10.8 percent.¹MS m/z(ESI):418.9[M+1]

The second step is that:

propane-1-sulfonic acid- {3- [3- (2-chloro-pyrimidin-4-yl) -5, 6-dihydro-4H-pyrrolo [1,2-b ] at room temperature]Pyrazol-2-yl]Phenyl } -amine (0.08g, 0.19mmol) was added to 15ml of 7mol/L ammonia/methanol solution, the reaction was blocked at 90 ℃ for 60 hours, the starting material was monitored by TLC for completion of the reaction, cooled to room temperature, 50ml of dichloromethane were added, and concentrated under reduced pressure. Purifying by silica gel column chromatographyThe mixture is dissolved to obtain propane-1-sulfonic acid {3- [3- (2-amino-pyrimidine-4-yl) -5, 6-dihydro-4H-pyrrolo [1,2-B]Pyrazol-2-yl]-phenyl } -amide (0.06g, light yellow solid) yield: 79.6 percent.¹MS m/z(ESI):399.5[M+1]

¹HNMR(400Hz,DMSO-d₆):9.84(s,1H),8.01(d,1H),7.37(m,2H),7.22(m,2H),6.34(s,2H),6.22(d,1H),4.15(m,2H),3.17-3.01(m,4H),2.58(m,2H),1.70(m,2H),0.94(t,3H).

Example 9

N- {3- [3- (2-chloro-pyrimidin-4-yl) -4,5,6, 7-tetrahydro-pyrazolo [1,5-a ] is reacted at room temperature]Pyridin-2-yl]-phenyl } -methanesulfonamide (0.1g, 0.24mmol), 5ml isobutylamine were added to a single-necked flask, stirred at room temperature overnight, TLC monitored for completion of the reaction of the starting materials, added 50ml dichloromethane, and washed with 10ml 1N hydrochloric acid solution. Purifying with silica gel column chromatography to obtain N- {3- [3- (2-isobutylamino-pyrimidin-4-yl) -4,5,6, 7-tetrahydro-pyrazolo [1,5-a]Pyridin-2-yl]-phenyl } -methanesulfonamide (0.04g, light yellow solid) yield: 38.1 percent.¹MS m/z(ESI):441.5[M+1]

¹HNMR(400Hz,DMSO-d₆):9.75(s,1H),8.02(d,1H),7.32-7.14(m,5H),6.17(d,1H),4.12(m,2H),2.99-2.89(m,7H),2.01(m,2H),1.81(m,2H),1.22(m,1H),0.83(d,6H).

Example 10

Propane-1-sulfonic acid- {3- [3- (2-chloro-pyrimidin-4-yl) -5, 6-dihydro-4H-pyrrolo [1,2-b ] at room temperature]Pyrazol-2-yl]Phenyl } -amine (0.1g, 0.24mmol), 5ml isobutylamine were added to a single-necked flask, stirred at room temperature overnight, TLC monitored for completion of the reaction of the starting materials, 50ml of dichloromethane were added, and washed with 10ml of 1N hydrochloric acid solution. Purifying with silica gel column chromatography to obtain propane-1-sulfonic acid- {3- [3- (2-isobutylamino-pyrimidin-4-yl) -5, 6-dihydro-4H-pyrrolo [1,2-b ]]Pyrazol-2-yl]-phenyl } -amine (20mg, light yellow solid) yield: 18.3 percent.¹MS m/z(ESI):455.6[M+1]

¹HNMR(400Hz,DMSO-d₆):9.80(s,1H),8.03(d,1H),7.31-7.14(m,4H),6.16(d,1H),4.14(m,2H),3.02(m,5H),2.01(m,2H),1.82(m,2H),1.24(m,1H),0.92-0.83(m,9H).

Example 11

Propane-1-sulfonic acid- {3- [3- (2-chloro-pyrimidin-4-yl) -5, 6-dihydro-4H-pyrrolo [1,2-b ] at room temperature]Pyrazol-2-yl]Phenyl } -amine (0.1g, 0.24mmol), N, N-diisopropylethylamine (0.062g, 0.48mmol), 3-aminopropionitrile (0.05g, 0.72mmol), N-methylpyrrolidinone (2.5ml) were added to a single vial, sodium carbonate (0.051g, 0.48mmol) was added, the mixture was stirred overnight at 90 ℃ for external temperature, TLC monitored for completion of the reaction, 50ml ethyl acetate was added, and the mixture was washed successively with 10ml saturated sodium bicarbonate solution, 10ml water, and 10ml saturated brine. Dried over anhydrous sodium sulfate and the solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography to give propane-1-sulfonic acid (3- {3- [2- (2-cyanoethylamino) -pyrimidin-4-yl ester]-5, 6-dihydro-4H-pyrrolo [1,2-b]Pyrazol-2-yl } -phenyl) -amide (0.03g, light yellow solid) yield: 27.8 percent.¹MS m/z(ESI):452.5[M+1]

¹HNMR(400Hz,DMSO-d₆):9.81(s,1H),8.11(d,1H),7.53-7.11(m,4H),6.28(d,1H),4.14(m,2H),3.40(m,2H),3.03(m,3H),2.55(m,2H),2.02(m, 2H),1.83(m,2H),1.66(m,2H),0.92(t,3H).

Example 12

N- {3- [3- (2-chloro-pyrimidin-4-yl) -4,5,6, 7-tetrahydro-pyrazolo [1,5-a ] is reacted at room temperature]Pyridin-2-yl]-phenyl } -2, 6-difluoro-benzenesulfonamide (0.1g, 0.2mmol), N, N-diisopropylethylamine (0.052g, 0.4mmol), 3-aminopropionitrile (0.043g, 0.6mmol), N-methylpyrrolidone (2.5ml) were added to a one-necked flask, sodium carbonate (0.043g, 0.4mmol) was added, stirring was carried out overnight at an external temperature of 90 deg.C, the reaction of the starting materials was monitored by TLC to completion, 50ml of ethyl acetate was added, followed by 10ml of saturated hydrogen carbonateThe sodium solution was washed with 10ml of water and 10ml of saturated saline. Dried over anhydrous sodium sulfate and the solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography to give N- (3- {3- [2- (2-cyanoethylamino) -pyrimidin-4-yl group]-4,5,6, 7-tetrahydro-pyrazolo [1,5-a]Pyridin-2-yl } phenyl) -2, 6-difluoro-benzenesulfonamide (0.03g, light yellow solid) yield: 27.8 percent.¹MS m/z(ESI):536.6.[M+1]

¹HNMR(400Hz,DMSO-d₆):10.69(s,1H),8.01(d,1H),7.33-6.98(m,6H),6.67(m,1H),6.45(m,1H),6.04(m,1H),4.13(m,2H),3.53(m,2H),3.02(m,2H),2.67(m,2H),1.99(m,2H),1.82(m,2H).

Example 13

Propane-1-sulfonic acid {3- [3- (2-chloro-pyrimidin-4-yl) -4,5,6, 7-tetrahydro-pyrazolo [1,5-a ] at room temperature]Pyridin-2-yl]Phenyl } -amine (0.1g, 0.23mmol), N, N-diisopropylethylamine (0.059g, 0.46mmol), 3-aminopropionitrile (0.048g, 0.69mmol), N-methylpyrrolidone (2.5ml) were added to a single vial, sodium carbonate (0.049g, 0.46mmol) was added, stirring was carried out overnight at 90 ℃ for external temperature, TLC monitored for completion of the reaction of the starting material, 50ml of ethyl acetate was added, and washing was successively with 10ml of saturated sodium bicarbonate solution, 10ml of water, and 10ml of saturated saline. Dried over anhydrous sodium sulfate and the solvent was removed under reduced pressure. Purifying the residue with silica gel column chromatography to obtain propane-1-sulfonic acid {3- [3- (2-cyano-ethylamino-pyrimidin-4-yl) -4,5,6, 7-tetrahydropyrazolo [1,5-a]Pyridin-2-yl]-phenyl } -amide (0.03g, light yellow solid) yield: 28.0 percent.¹MS m/z(ESI):466.6.[M+1]

¹HNMR(400Hz,DMSO-d₆):9.81(s,1H),8.11(d,1H),7.35-7.13(m,4H),6.27(d,1H),4.16(m,2H),3.53(m,2H),3.02(m,4H),2.62(m,2H),2.02(m,2H),1.83(m,2H),1.64(m,2H),0.92(t,3H).

Example 14

N- {3- [3- (2-chloro-pyrimidin-4-yl) -5,6-dihydro-4H-pyrrolo [1,2-b]Pyrazol-2-yl]-phenyl } -2, 6-difluoro-benzenesulfonamide (0.1g, 0.2mmol), N, N-diisopropylethylamine (0.052g, 0.4mmol), 3-aminopropionitrile (0.043g, 0.6mmol), N-methylpyrrolidone (2.5ml) were added to a one-necked flask, sodium carbonate (0.043g, 0.4mmol) was added thereto, the mixture was stirred overnight at an external temperature of 90 ℃ and the reaction of the starting materials was monitored by TLC to be complete, 50ml of ethyl acetate was added, followed by washing with 10ml of saturated sodium bicarbonate solution, 10ml of water and 10ml of saturated saline. Dried over anhydrous sodium sulfate and the solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography to give N- (3- {3- [2- (2-cyanoethylamino) -pyrimidin-4-yl group]-5, 6-dihydro-4H-pyrrolo [1,2-b]Pyrazol-2-yl } -phenyl) -2, 6-difluoro-benzenesulfonamide (0.03g, light yellow solid) yield: 28.7 percent.¹MS m/z(ESI):522.5.[M+1]

¹HNMR(400Hz,DMSO-d₆):10.72(s,1H),8.02(d,1H),7.26-7.14(m,6H),6.66(d,1H),6.46(m,1H),6.10(m,1H),4.17(m,2H),3.52(m,2H),3.14(m,2H),2.69-2.57(m,4H).

Example 15

First step of

Methyl 3- (2, 6-difluoro-benzenesulfonamide) -2-fluoro-benzoate (2.95g,8.54mmol) was added to toluene (200ml) at room temperature, and a toluene solution of diisopropylaluminum hydride at a concentration of 1.5 mol per liter was added dropwise under ice bath, and the mixture was allowed to naturally warm to room temperature and stirred overnight. TLC monitoring the starting material reaction was complete, methanol (10ml) was added dropwise in ice bath, stirred for 0.5h, ethyl acetate (100ml) was added, the organic phase was washed successively with saturated aqueous tartaric acid (100ml), water (100ml), saturated brine (100ml), dried over anhydrous sodium sulphate and the solvent was removed under reduced pressure to give 2, 6-difluoro-N- (2-fluoro-3-hydroxymethyl-phenyl) -benzenesulfonamide (2.7g, pale yellow solid) yield: 99 percent.¹MS m/z(ESI):318.3.[M+1]

¹HNMR(400Hz,CDCl₃):7.59-7.46(m,2H),7.30(m,1H),7.22-6.97(m,4H),4.70(s,2H).

Second step of

Reacting 2, 6-difluoro-N- (2-fluoro-3-hydroxymethyl-phenyl) at room temperatureBenzenesulfonamide (3.1g, 10.00mmol) was added to dichloromethane (30ml), pyridinium chlorochromate (3.2g, 15.00mmol) was added under ice-bath, and stirred at room temperature overnight. TLC monitored complete reaction of starting material, suction filtered, filter cake dissolved in water (500ml), extracted with dichloromethane (100ml x 3), combined organic phases dried over anhydrous sodium sulfate, solvent removed under reduced pressure, residue TLC monitored complete reaction of starting material 2, 6-difluoro-N- (2-fluoro-3-formyl-phenyl) -benzenesulfonamide (2.74g, white solid) yield 86%.¹MS m/z(ESI):316.2.[M+1]

¹HNMR(400Hz,CDCl₃):10.25(s,1H),7.92(m,1H),7.65-7.52(m,2H),7.41(s,1H),7.27(m,1H),7.03(m,2H).

The third step

Adding iodomethyl triphenyl phosphorus iodide (6.9g, 13mmol) into new distilled tetrahydrofuran (90ml) at room temperature, adding 1 mol per liter of tetrahydrofuran solution (13ml) of lithium bis (trimethylsilyl) amide at ice bath, stirring for 1h at room temperature after the addition is finished, clarifying the solution, cooling to-60 ℃, adding hexamethyl phosphoric triamide (2.7ml,15mmol), cooling to-78 ℃, dropwise adding new distilled tetrahydrofuran (60ml) solution of 2, 6-difluoro-N- (2-fluoro-3-formyl-phenyl) -benzenesulfonamide, stirring for 0.5h at-78 ℃, then naturally heating to room temperature and stirring for 2 h. TLC monitoring the starting material reaction was complete, adding the system slowly to a saturated ammonium chloride solution (300ml) in ice bath, extracting with methyl tert-butyl ether (100ml _ 3), combining the organic phases drying over anhydrous sodium sulfate, removing the solvent under reduced pressure, adding the residue to tetrahydrofuran (5ml), adding a tetrahydrofuran solution of tetrabutylammonium fluoride at a concentration of 1 mol per liter, reacting at 85 ℃ in a glass-sealed tube for 18h, TLC monitoring the starting material reaction was complete, cooling to room temperature, adding the system to water (100ml), extracting with ethyl acetate (50ml _ 3), combining the organic phases drying over anhydrous sodium sulfate, removing the solvent under reduced pressure, and purifying the residue with silica gel chromatography to yield N- (3-ethynyl-2-fluoro-phenyl) -2, 6-difluoro-benzenesulfonamide (1g, light yellow solid): 37.0 percent.¹MS m/z(ESI):312.2.[M+1]。

¹HNMR(400Hz,CDCl₃):7.65(m,1H),7.55(m,1H),7.23(m,2H),7.08-6.98(m,3H),3.30(s,1H).

The fourth step

At room temperature, 4-bromopyridine-2-carbamic acid tert-butyl ester (0.055g, 0.2mmol), bis (triphenylphosphine) palladium dichloride (0.003g, 0.004mmol), cuprous iodide (0.0004g, 0.002mmol) were added in sequence into a three-necked flask, the system was replaced three times with a nitrogen balloon (nitrogen: hydrogen volume ratio about 4:1), and triethylamine (0.1ml) was added. The temperature was raised to an internal temperature of 80 ℃ and a solution of N- (3-ethynyl-2-fluoro-phenyl) -2, 6-difluoro-benzenesulfonamide (0.062g,0.2mmol) in acetonitrile (2.5ml) was added dropwise while hot, and the mixture was stirred under reflux for 1 h. TLC to monitor the reaction completion of the starting material, cooling to room temperature, adding water (10ml), extracting with ethyl acetate (5 ml. times.3), combining the organic phases, drying over anhydrous sodium sulfate, removing the solvent under reduced pressure, and purifying the residue by silica gel chromatography to give {4- [3- (2, 6-difluoro-benzenesulfonamide) -2-fluoro-phenylacetylene]-pyridin-2-yl } -carbamic acid tert-butyl ester (40mg, light yellow solid), yield: 39.7 percent.¹MS m/z(ESI):504.5.[M+1]。

¹HNMR(400Hz,DMSO-d₆):11.04(s,1H),10.03(s,1H),8.31(d,1H),7.91(s,1H),7.75(m,1H),7.55(m,1H),7.41-7.13(m,4H),7.15(d,1H),1.49(s,9H).

The fifth step

Reaction of {4- [3- (2, 6-difluoro-benzenesulfonamide) -2-fluoro-phenylacetylene at room temperature]-pyridin-2-yl } -carbamic acid tert-butyl ester (0.22g, 0.44mmol) was suspended in 25ml of mesitylene, and 4,5,6, 7-tetrahydro [1,2,3 ] was added]Oxadiazolo [3,4-a ]]Pyridin-8-ylium-3-phenoxide (0.062g,0.44mmol), displaced with nitrogen three times, warmed to an external temperature of 175 ℃ under nitrogen, stirred for 24h.TLC to monitor completion of the starting material reaction, the solvent was removed under reduced pressure and the crude product was purified by silica gel column chromatography to give N- (3- {3- [ 2-aminopyridin-4-yl)]-4,5,6, 7-tetrahydropyrazolo [1,5-a]Pyridin-2-yl } -2-fluorophenyl) -2, 6-difluorobenzenesulfonamide (0.014g, light yellow solid) yield: 13.2 percent.¹MS m/z(ESI):500.5.[M+1]

¹HNMR(400Hz,DMSO-d₆):7.65(m,2H),7.30-7.14(m,4H),6.26(s,1H),6.02-5.94(m,2H),4.14(m,2H),2.82(m,2H),2.01(m,2H),1.81(m,2H).

Example 16

In addition to the compounds disclosed in the above examples, compounds having the following test results were synthesized, and the synthesis methods and procedures of the compounds were referenced in example 15.

¹MS m/z(ESI):573.2[M+1]；

¹MS m/z(ESI):557.63[M+1]；

¹MS m/z(ESI):616.63[M+1]；

¹MS m/z(ESI):593.61[M+1]。

Example 17 biological experiments

Detection of proliferation half-inhibitory concentration IC of compound on human skin malignant melanoma cell line SK-MEL28 by CCK-8 detection kit₅₀

(1) Materials and methods

Cell lines:

SK-MEL28 human skin malignant melanoma cell line (institute of cell biology, Chinese academy of sciences)

Reagents and consumables:

Cell Counting Kit-8(Cat#CK04-13，Dojindo)

96-well culture plate (Cat #3599, Corning Costar)

Fetal bovine serum (Cat #10099-141, GIBCO)

Culture medium (Invitrogen)

Desktop Microplate reader SpectraMax M5Microplate reader (molecular devices)

(2) Reagent preparation

Preparation of a culture medium:

cell lines	Culture medium
		SK-MEL28	EMEM+10％FBS

Preparation of the compound:

the compound was diluted with DMSO to a final concentration of 1 mM.

(3) Cell culture

(i) Collecting cells in logarithmic growth phase, counting, and resuspending the cells with complete culture medium;

(ii) adjusting the cell concentration to a proper concentration, inoculating a 96-well plate, and inoculating 100 mu l of cell suspension in each well;

(iii) cells were incubated at 37 ℃ and 100% relative humidity, 5% CO₂Incubate in incubator for 24 hours.

(4)IC₅₀Experiment of

(i) Cells in the logarithmic growth phase were collected, counted, resuspended in complete medium, and the cell concentration adjusted to the appropriate concentration (SK-MEL28 cells, 6k cells/well) seeded into 96-well plates with 100 μ l cell suspension per well. Cells were incubated at 37 ℃ and 100% relative humidity, 5% CO₂After 24 hours incubation in the incubator.

(ii) After diluting the test compound 3-fold in gradient with DMSO 8 times, 5. mu.l of each well was added to 95. mu.l of complete medium to obtain a 20-fold dilution of the compound. The above compound was added to the cells at 25. mu.l/well. The final concentration of compound was from 10 μ M to 0 μ M, 3-fold dilution, for 10 concentration points.

(iii) Cells were incubated at 37 ℃ and 100% relative humidity, 5% CO₂Incubate in incubator for 72 hours. The medium was aspirated off, and a complete medium containing 10% CCK-8 was added and incubated in an incubator at 37 ℃ for 2-4 hours. After gentle shaking, absorbance at a wavelength of 450nm was measured on a SpectraMax M5Microplate Reader, and the inhibition rate was calculated with the absorbance at 650nm as a reference.

(5) Data processing

The inhibition rate of the drug on the growth of tumor cells was calculated according to the following formula: the tumor cell growth inhibition rate is [ (Ac-As)/(Ac-Ab) ]. times.100%

As OA of sample (cell + CCK-8+ test Compound)

Ac negative control OA (cell + CCK-8+ DMSO)

Ab: OA of Positive control (Medium + CCK-8+ DMSO)

(6) Results of the experiment

The test tests the cytotoxic effect of the candidate compounds on SK-MEL28 human skin malignant melanoma cell line. The final concentration of the compound was from 10. mu.M to 0. mu.M, and three-fold gradient dilution was performed for 10 concentration points.

IC₅₀Values are detailed in table 2:

TABLE 2

Compound numbering	Median inhibitory concentration (uM)	Compound numbering	Median inhibitory concentration (uM)
				Control Vemurafenib	0.1826	11	0.5112
1	>10	12	1.181
				2	>10	13	1.373
3	>10	14	0.2096
				4	>10	15	Not testing
5	8.042	16	Not testing
				6	0.7802	17	0.1376
7	5.572	18	0.1245
				8	>10	19	0.4506

9	>10	20	0.6345
				10	5.572	/	/

As can be seen from the above table: the diaryl-substituted pyrazolocyclic derivatives provided by the invention have obvious inhibition effect on the human skin malignant melanoma SK-MEL28 cell strain with high mutation expression of B-RAF V600E, and show better development and application prospects.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Claims

1. A compound of formula I, a pharmaceutically acceptable salt thereof:

I

in the formula:

n is 1 or 2;

x is N;

y is NR₂R₃；

R₁Selected from the group consisting of: substituted or unsubstituted C₁-C₈Alkyl, substituted or unsubstituted C₆-C₁₀Aryl, wherein said substitution is with a substituent selected from the group consisting of: c₁-C₃Alkyl, halogen;

R₂selected from the group consisting of: -L₁NHC(O)OR_1a、-L₁NHC(O)NHR_1aSubstituted or unsubstituted C₃-C₆Cycloalkyl, substituted or unsubstitutedC of (A)₁-C₃An alkyl group;

wherein, the "substituted" means having a substituent selected from the group consisting of: cyano or C₁-C₆An alkyl group;

L₁is optionally substituted by 1-3 independent C₁-C₄Alkyl substituted C₁-C₄An alkylene group;

R_1ais H or C₁-C₄An alkyl group;

R₃is H or methyl;

R₄is H, halogen, or C₁An alkyl group;

R₅is H or halogen;

R₆is H, halogen, or C₁An alkyl group;

R₇is halogen, H or C₁An alkyl group.

2. A compound of formula I according to claim 1, wherein R is₂Selected from the group consisting of: -L₁NHC(O)OR_1aSubstituted or unsubstituted C₁-C₃An alkyl group.

3. A compound of formula I according to claim 1, wherein R is₂is-L₁NHC(O)OR_1a(ii) a Wherein L is₁Is optionally substituted by 1-3 independent C₁-C₄Alkyl substituted C₁-C₄An alkylene group; r_1aIs C₁-C₄An alkyl group.

4. A compound of formula I according to claim 1, wherein R is₂Is substituted or unsubstituted C₁-C₃An alkyl group; wherein, the "substituted" means having a substituent selected from the group consisting of: a cyano group.

5. A compound of formula I according to claim 1, wherein R is₃Is H.

6. A compound of formula I according to claim 1,

R₂is substituted or unsubstituted C₁-C₃An alkyl group; wherein, the "substituted" means having a substituent selected from the group consisting of: a cyano group; or R₂is-L₁NHC(O)OR_1a(ii) a Wherein L is₁Is optionally substituted by 1-3C₁-C₄Alkyl substituted C₁-C₄An alkylene group; r_1aIs C₁-C₄An alkyl group;

and R is₁Is substituted or unsubstituted C₆-C₁₀Aryl, wherein said substitution is with a substituent selected from the group consisting of: c₁-C₃Alkyl, halogen.

7. A compound of formula I according to claim 1, wherein R is₁Is substituted or unsubstituted C₆-C₁₀Aryl, wherein said substitution is with a substituent selected from the group consisting of: a halogen.

8. The compound of formula I according to claim 1, selected from the group consisting of:

9. The compound of formula I according to claim 1, selected from the group consisting of:

n- (3- {3- [2- (2-cyanoethylamino) -pyrimidin-4-yl ] -5, 6-dihydro-4H-pyrrolo [1,2-b ] pyrazol-2-yl } -2, 6-difluorophenyl) -2, 6-difluorobenzenesulfonamide.

10. A process for the preparation of a compound of formula I according to claim 1, characterized in that it comprises the steps of:

；

V I

wherein R is₁、R₄、R₅、R₆、R₇X, Y and n are as defined in claim 1.

11. A pharmaceutical composition comprising a therapeutically effective amount of a compound of formula I as claimed in claim 1, or a pharmaceutically acceptable salt thereof.

12. The pharmaceutical composition of claim 11, wherein the pharmaceutical composition is for use in the preparation of a medicament for treating a disease associated with mutation of B-RAF V600E or for use in the preparation of a medicament for preventing a disease associated with mutation of B-RAF 600E.

13. The pharmaceutical composition of claim 11, wherein:

the pharmaceutically acceptable salt is a salt of a compound of formula I selected from the group consisting of: inorganic acid salts, organic acid salts, or combinations thereof.

14. The pharmaceutical composition of claim 13, wherein: the organic acid salt is selected from the following group: alkyl sulfonates, aryl sulfonates, or combinations thereof.

15. The pharmaceutical composition of claim 13, wherein: the salt is selected from the group consisting of: hydrochloride, hydrobromide, nitrate, sulfate, phosphate, formate, acetate, propionate, benzoate, maleate, fumarate, succinate, tartrate, citrate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, or a combination thereof.

16. A B-RAF kinase inhibitor comprising an inhibitory effective amount of a compound of formula I as defined in claim 1, or a pharmaceutically acceptable salt thereof.

17. The inhibitor of claim 16, wherein:

18. The pharmaceutical composition of claim 17, wherein: the organic acid salt is selected from the following group: alkyl sulfonates, aryl sulfonates, or combinations thereof.

19. The inhibitor of claim 17, wherein:

the salt is selected from the group consisting of: hydrochloride, hydrobromide, nitrate, sulfate, phosphate, formate, acetate, propionate, benzoate, maleate, fumarate, succinate, tartrate, citrate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, or a combination thereof.

20. Use of a compound of formula I according to claim 1 for:

(a) preparing a B-RAF inhibitor;

(b) for non-therapeutically inhibiting the activity of SK-MEL28 cells in vitro;

(c) for non-therapeutically inhibiting tumor cell growth in vitro;

21. An in vitro non-therapeutic method of inhibiting B-RAF kinase, comprising the steps of:

22. Use of a compound of formula I according to claim 1, or a pharmaceutically acceptable salt thereof, for the preparation of a medicament for the treatment of a disease associated with mutation of BRAF V600E.