CN114437113A - Thiazolopyridine ring-linked triazole compound and preparation method and application thereof - Google Patents

Abstract

The invention discloses a thiazolopyridine ring-linked triazole compound, and a preparation method and application thereof. The halogenated substituent introduced on the end benzene ring can expand the structural diversity of the Bcr-Abl kinase inhibitor, and meanwhile, an activity test shows that the thiazolopyridine ring has an important effect on the kinase inhibition activity of the compound, can improve the affinity between a receptor and the compound, and can be used as a pharmacodynamic fragment of the Bcr-Abl tyrosine kinase inhibitor.

Description

Thiazolopyridine ring-linked triazole compound and preparation method and application thereof

Technical Field

The invention belongs to the technical field of inhibitor preparation, and relates to a thiazolopyridine cyclotriazole compound, and a preparation method and application thereof.

Background

Chronic Myeloid Leukemia (CML), a malignant clonal proliferative disease that occurs in myeloid hematopoietic stem cells, accounts for up to 15% -20% of adult leukemia patients and is characterized by the fact that CmL patients detect the Ph chromosome. Ph chromosome is a breakpoint cluster-Elbeson (Bcr-Abl) fusion gene formed by the mutual translocation of the normal human chromosome 22 and the normal human chromosome 9, and the fusion gene codes a Bcr-Abl fusion protein generating continuous activation of tyrosine kinase activity. Small molecule tyrosine kinase inhibitors aiming at Bcr-Abl as targets are marketed in the market at present, but the small molecule tyrosine kinase inhibitors have the problems of drug resistance, other clinical adverse reactions and the like. Accordingly, research and development of novel Bcr-Abl tyrosine kinase inhibitors have become one of hot spots in the pharmaceutical field.

Disclosure of Invention

The invention aims to provide a thiazolopyridine bitriazole compound as well as a preparation method and application thereof.

In order to achieve the purpose, the invention adopts the following technical scheme:

a thiazolopyridine bitriazole compound has the following structural formula:

wherein R is₁、R₂And R' are specifically as follows:

a preparation method of the thiazolopyridine bitriazole compound comprises the following steps:

1) condensing the L-hydroxyproline protected by the tert-butyloxycarbonyl group and an aniline compound containing a substituent group to generate an aminated Boc-L-hydroxyproline compound;

2) the aminated Boc-L-hydroxy proline compound and methylsulfonyl chloride are subjected to substitution reaction to generate an aminated Boc-L-methylsulfonyl proline compound;

3) under the protection of nitrogen, carrying out substitution reaction on an aminated Boc-L-methylsulfonyl proline compound and sodium azide to generate an aminated Boc-L-azidoprolinic acid compound;

4) condensing 5-ethynylthiazolo [5,4-b ] pyridine-2-amine and aminated Boc-L-azidoproline compounds under the action of sodium ascorbate and copper sulfate pentahydrate to obtain Boc-protected thiazolopyridine-triazole compounds;

5) reacting a plurality of acyl-containing compounds with a thiazolopyridine triazole compound protected by Boc, and removing the Boc protecting group by trifluoroacetic acid to obtain the indazole cyclic triazole compound.

In a further improvement of the invention, in step 1), the tert-butoxycarbonyl protected L-hydroxyproline is prepared by the following process: dissolving L-hydroxyproline in tetrahydrofuran and sodium hydroxide solution at 0 ℃, adding di-tert-butyl dicarbonate in an ice water bath, and then reacting for 6 hours at 25-30 ℃ to prepare the L-hydroxyproline protected by tert-butoxycarbonyl.

In a further development of the invention, in step 4) the 5-ethynylthiazolo [5,4-b ] pyridin-2-amine is prepared by:

a) under the protection of nitrogen, 6-bromopyridine-3-amine reacts with potassium thiocyanate to prepare 5-bromothiazolo [5,4-b ] pyridine-2-amine;

b) under the protection of nitrogen, 5-bromothiazolo [5,4-b ] pyridin-2-amine reacts with trimethylsilylacetylene to prepare 5- ((trimethylsilyl) ethynyl) thiazolo [5,4-b ] pyridin-2-amine;

c) 5- ((trimethylsilyl) ethynyl) thiazolo [5,4-b ] pyridin-2-amine was trimethylsilyl-removed with sodium hydroxide in methanol to give 5-ethynylthiazolo [5,4-b ] pyridin-2-amine.

The further improvement of the invention is that the specific process of the step a) is as follows: dissolving KSCN in acetic acid in nitrogen, cooling to 0 ℃, adding 6-bromopyridine-3-amine, cooling to-5 ℃, dropwise adding acetic acid containing bromine, heating to 25-30 ℃ after dropwise adding, and stirring to obtain 5-bromothiazolo [5,4-b ] pyridine-2-amine;

the specific process of the step b) is as follows: adding 5-bromothiazolo [5,4-b ] pyridine-2-amine, copper iodide and tetrakis (triphenylphosphine) palladium into a reaction vessel, sealing the vessel by using a rubber plug, vacuumizing and backfilling with nitrogen, adding triethylamine, then adding trimethylsilyl acetylene, and heating and refluxing for reaction to obtain 5- ((trimethylsilyl) ethynyl) thiazolo [5,4-b ] pyridine-2-amine;

the concrete process of the step c) is as follows: dissolving 5- ((trimethylsilyl) ethynyl) thiazolo [5,4-b ] pyridine-2-amine in dichloromethane, adding a methanol solution of sodium hydroxide under stirring, and reacting at 25-30 ℃ to obtain 5-ethynylthiazolo [5,4-b ] pyridine-2-amine.

The further improvement of the invention is that the specific process of the step 1) is as follows: dissolving Boc-L-hydroxyproline in dichloromethane, adding triethylamine, cooling to 0 ℃, dropwise adding a dichloromethane solution containing ethyl chloroformate, and reacting for 30 min-1 h to generate an active intermediate; dropwise adding the aniline compound solution containing the substituent group into the active intermediate at 0 ℃, and stirring at 25-30 ℃ to obtain an aminated Boc-L-hydroxy proline compound;

the specific process of the step 2) is as follows: dissolving the aminated Boc-L-hydroxyproline compound in dichloromethane, cooling to 0 ℃, adding triethylamine, stirring uniformly, dropwise adding methylsulfonyl chloride, and reacting at 25-30 ℃ to obtain the aminated Boc-L-methylsulfonyl prolinic acid compound.

The invention has the further improvement that the aniline compound containing the substituent group is 4-chloro-3-trifluoromethyl aniline, 2, 4-dichloroaniline, 4-bromoaniline or 3, 4-difluoroaniline;

the specific process of the step 3) is as follows: dissolving an aminated Boc-L-methylsulfonyl proline compound in DMF, adding sodium azide, and reacting at 65-70 ℃ under the protection of nitrogen to obtain an aminated Boc-L-azidoproline compound;

the specific process of the step 4) is as follows: dissolving 5-ethynylthiazolo [5,4-b ] pyridine-2-amine and aminated Boc-L-azido proline compound in a mixed solvent of ethanol and water, then adding sodium ascorbate and copper sulfate pentahydrate, and stirring at 30 ℃ to obtain the thiazolopyridinitride compound protected by the Boc protecting group.

The further improvement of the invention is that the specific process of the step 5) is as follows: and dissolving the indazole-triazole compound protected by the Boc protecting group in anhydrous dichloromethane, adding triethylamine, stirring at 0 ℃ for 30min, dropwise adding a dichloromethane solution containing a chloride compound, and reacting at 25-30 ℃ to obtain the indazole-triazole compound with the Boc protecting group removed.

The application of the thiazolopyridine ring triazole compound in preparing a Bcr-Abl kinase inhibitor.

Further, the Bcr-Abl kinase is a wild-Abl kinase or a T315I mutant Abl kinase.

The application of the thiazolopyridine ring-linked triazole compound in preparing an antitumor drug.

Furthermore, the anti-tumor drug is a drug for resisting leukemia cells.

Compared with the prior art, the invention has the following beneficial effects:

the invention utilizes the reaction of the ring-pulling head, the click chemistry, the acylation, the condensation and the like to synthesize the target compound and constructs a compound library, the compound is a Bcr-Abl small molecule tyrosine kinase inhibitor with a novel molecular structure, and the structure of the target compound is represented by means of MS, NMR and the like. Based on the previous Bcr-Abl tyrosine kinase inhibitor, the interaction analysis of Bcr-Abl protein and ligand and other researches, a fragment-based drug design strategy is adopted, thiazolopyridine triazole is taken as a hinge region binding fragment, L-proline is taken as a flexible Linker, so that a small molecule compound library with kinase inhibition activity is constructed, and the tyrosine kinase inhibitor with Bcr-Abl kinase inhibition activity is found through ADP-Glo kinase activity screening. Kinase screening test shows that the compounds have certain inhibitory activity on both Abl kinase and T315I mutant Abl kinaseWherein R is₁Is 2, 4-dichloro, R₂The cyclopropyl formyl group is the most active for Abl kinase. Cell proliferation tests show that most compounds have certain inhibitory activity on K562 cells. Wherein also when R₁Is 2, 4-dichloro, R₂In the case of cyclopropylformyl, the antiproliferative activity is the best. The analysis of the structure-activity relationship finds that: the derivative introduced with the L-proline has good spatial matching with the ATP site of the Abl kinase, and the action mode is consistent with that of a reference small molecule imatinib. The thiazolopyridine bitriazole is important for improving the affinity of the compound to kinase, and meanwhile, the amide side chain is introduced to the thiazolopyridine ring, so that the affinity of small molecules and receptors can be further improved, and the thiazolopyridine bitriazole can be used as a novel drug effect fragment for tyrosine kinase inhibition with Bcr-Abl as a target.

Drawings

FIG. 1 is a synthetic route diagram of the present invention.

Detailed Description

The present invention will be described in detail with reference to the accompanying drawings.

The structural formula of the thiazolopyridine bitriazole compound is as follows:

wherein R is₁、R₂R' is specifically as follows:

referring to fig. 1, the preparation method of the thiazolopyridine bitriazole compound as described above includes the following steps:

1) under the protection of nitrogen, 6-bromopyridine-3-amine reacts with potassium thiocyanate to prepare 5-bromothiazolo [5,4-b ] pyridine-2-amine;

2) under the protection of nitrogen, 5-bromothiazolo [5,4-b ] pyridin-2-amine reacts with trimethylsilylacetylene to prepare 5- ((trimethylsilyl) ethynyl) thiazolo [5,4-b ] pyridin-2-amine;

3) desilylation of compound 5- ((trimethylsilyl) ethynyl) thiazolo [5,4-b ] pyridin-2-amine with sodium hydroxide in methanol afforded 5-ethynylthiazolo [5,4-b ] pyridin-2-amine;

4) carrying out substitution reaction on L-hydroxyproline and di-tert-butyl dicarbonate in an ice-water bath to prepare L-hydroxyproline (Boc-L-hydroxyproline) protected by tert-butoxycarbonyl;

5) condensing Boc-L-hydroxyproline and aniline compounds containing substituent groups to generate aminated Boc-L-hydroxyproline compounds; wherein the aniline compound containing the substituent group is 4-chloro-3-trifluoromethylaniline, 2, 4-dichloroaniline, 4-bromoaniline or 3, 4-difluoroaniline.

6) The aminated Boc-L-hydroxy proline compound and methylsulfonyl chloride are subjected to substitution reaction to generate an aminated Boc-L-methylsulfonyl proline compound;

7) under the protection of nitrogen, the ammoniated Boc-L-methylsulfonyl proline compound and sodium azide are subjected to substitution reaction to generate the ammoniated Boc-L-azido proline compound.

8) Under the action of sodium ascorbate and copper sulfate pentahydrate, 5-ethynylthiazolo [5,4-b ] pyridine-2-amine and aminated Boc-L-azidoproline compounds are condensed to obtain the Boc-protected thiazolopyridine-bis-triazole compounds.

9) Reacting a plurality of acyl-containing compounds with a thiazolopyridine triazole compound protected by Boc, and removing the Boc protecting group by trifluoroacetic acid to obtain the indazole cyclic triazole compound.

The specific process of the step 1) is as follows: KSCN was dissolved in acetic acid under nitrogen, cooled to 0 ℃ and 6-bromopyridin-3-amine (10g, 57.8mmol) was added. The mixture was further cooled to-5 ℃ and bromine-containing acetic acid was added dropwise while maintaining the bath temperature below 10 ℃. The reaction mixture was warmed to room temperature and stirred for 12 h. After work-up, 5-bromothiazolo [5,4-b ] pyridin-2-amine was obtained as a yellow solid.

The specific process of the step 2) is as follows: 5-bromothiazolo [5,4-b ] pyridin-2-amine, copper iodide, and tetrakis (triphenylphosphine) palladium were added to a reaction vessel. The vessel was then sealed with a rubber stopper, evacuated and backfilled with nitrogen. Triethylamine is used as a base and a solvent, and the injection is carried out by using an injector. And (3) after 5min at room temperature, adding trimethylsilyl acetylene, heating and refluxing for reaction for 12h, and recording the reaction completion by thin layer chromatography. And purifying by post-treatment to obtain 5- ((trimethylsilyl) ethynyl) thiazolo [5,4-b ] pyridine-2-amine.

The specific process of the step 3) is as follows: the compound 5- ((trimethylsilyl) ethynyl) thiazolo [5,4-b ] pyridin-2-amine was dissolved in dichloromethane and a solution of sodium hydroxide in methanol was added slowly while stirring. The mixture was then reacted at room temperature for 2h, and then the organic solvent was removed. And purifying by post-treatment to obtain 5-ethynylthiazolo [5,4-b ] pyridine-2-amine.

The specific process of the step 4) is as follows: l-hydroxyproline was dissolved in tetrahydrofuran and sodium hydroxide solution at 0 ℃. Di-tert-butyl dicarbonate is added to the ice-water bath. The mixture was allowed to react at room temperature for 6h until complete reaction was detected by thin layer chromatography. The reaction solution was concentrated in vacuo, with continued ice-water bath. And carrying out substitution reaction on the L-hydroxyproline and di-tert-butyl dicarbonate in an ice-water bath of the concentrated product to prepare the tert-butoxycarbonyl protected L-hydroxyproline, and continuously using the tert-butoxycarbonyl protected L-hydroxyproline without purification.

The specific process of the step 5) is as follows: Boc-L-hydroxyproline was dissolved in dichloromethane and triethylamine was added. The solution was cooled to 0 ℃ and a solution of ethyl chloroformate in dichloromethane was added dropwise. After reacting for 30 min-1 h, generating an active intermediate. Then, the substituted aniline compound-containing solution was added dropwise to the above solution at 0 ℃ and stirred at room temperature for 12 hours. And carrying out post-treatment to obtain an aminated Boc-L-hydroxy proline compound.

The specific process of the step 6) is as follows: the aminated Boc-L-hydroxyproline compound was dissolved in dichloromethane and cooled to 0 ℃. Triethylamine was added and stirred for 15 min. Methanesulfonyl chloride was added dropwise and reacted at room temperature for 12 hours. The reaction product is post-treated to obtain the aminated Boc-L-methylsulfonyl proline compound.

The specific process of the step 7) is as follows: the aminated Boc-L-methylsulfonylproline compound was dissolved in DMF and sodium azide was added. The mixture reacts for 16 hours under the protection of nitrogen at 65-70 ℃. The reaction solution was then cooled to room temperature, and a solid precipitated in ice water. Then the Boc-L-azidoproline compound is obtained after treatment and purification.

The specific process of the step 8) is as follows: dissolving a compound 5-ethynylthiazolo [5,4-b ] pyridin-2-amine and a compound aminated Boc-L-azidoproline compound in a mixed solvent of ethanol and water in equal proportion. Sodium ascorbate and copper sulfate pentahydrate were then added. And stirring the suspension for 24h at 30 ℃, concentrating the reaction solution in vacuum, and carrying out aftertreatment purification to obtain the thiazolopyridine bitriazole compound protected by the Boc protecting group.

The specific process of the step 9) is as follows: and dissolving the indazole-bis-triazole compound protected by the Boc protecting group in anhydrous dichloromethane, and adding triethylamine. The solution was stirred at 0 ℃ for 30min, and a dichloromethane solution containing an acyl chloride compound was added dropwise. The solution reacts for 2h at room temperature, dichloromethane is removed in vacuum, and the product is obtained after chromatographic purification by ethyl acetate. Then dissolved in 30mL of anhydrous dichloromethane. Trifluoroacetic acid was added dropwise at 0 ℃. The solution is stirred for 2h at room temperature, and the indazole cyclic triazole compound with the Boc protecting group removed is obtained after purification. Wherein the acyl chloride compound is acetyl chloride or cyclopropane formyl chloride.

The thiazolopyridine bitriazole compound is applied to preparation of medicines for inhibiting activity of Abl kinase and T315I mutant Abl kinase.

The application of the thiazolopyridine bitriazole compound in preparation of antitumor drugs.

The antitumor drug is an anti-leukemia drug.

The following description will be made by way of specific examples.

The room temperature in the invention is 25-30 ℃, and the overnight time is 12 h.

Example 1

Thiazolopyridine ring-linked triazole compound R₁Is 4-Cl-3-CF₃，R₂When the H is shown in the specification and R' is Boc or H, the preparation method comprises the following steps:

1) 5-bromothiazolo [5,4-b ]]Synthesis of pyridin-2-amine: KSCN (28g, 288mmol) was charged to a 500mL three-necked flask under nitrogen, followed by 75mL of acetic acid. Cooled to 0 ℃ and 6-bromopyridin-3-amine (10g, 57.8mmol) was added slowly. The mixture was further cooled to-5 ℃ and 4mL of acetic acid containing bromine was added dropwise while maintaining the bath temperature below 10 ℃. Next, the reaction mixture was warmed to room temperature (25 ℃ C.) and stirred overnight (12 h). The precipitate was filtered, the filtrate was cooled to 0 ℃ and 100mL of water was added. Stirring for 5min, collecting precipitate, and washing with water. The solid was dried under vacuum overnight (12h) to give 6.8g of a yellow solid in 51% yield. Mp 218-220 ℃; EI-MS (M/z)231.85[ M + H]⁺,229.95[M-H]^-。

2)5- ((trimethylsilyl) ethynyl) thiazolo [5,4-b]Synthesis of pyridin-2-amine: reacting 5-bromothiazolo [5,4-b ]]Pyridin-2-amine (3.42g,16.12mmol), CuI (10%, 0.31g,1.61mmol), and Pd (PPh)₃)₄(10%, 1.86g,1.61mmol) was charged to a 100mL two-necked round bottom flask with a condenser and magnetic stirring. The vessel was then sealed with a rubber stopper, evacuated and backfilled with nitrogen (3 times). Triethylamine (30mL) was used as the base and solvent and injected with a syringe. After 5min at room temperature, trimethylsilylacetylene (4.74mL,48.36mmol) was added and the reaction heated to reflux overnight and was recorded to completion by Thin Layer Chromatography (TLC). The reaction was cooled to room temperature and quenched with water (50 mL). The solution was then diluted with ethyl acetate (50mL) and filtered. The filtrate was washed with water until no blue color of the copper complex was visible in the aqueous phase. The combined aqueous layers were extracted with ethyl acetate (30 mL. times.3). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and the solvent was evaporated. The crude product was purified by flash column chromatography (volume ratio petroleum ether: ethyl acetate: 5: 1) to give the product in 72% yield. EI-MS (M/z)248.05[ M + H]⁺,246.30[M-H]^-。

3) 5-ethynylthiazolo [5,4-b ]]Synthesis of pyridin-2-amine: compound 5- ((trimethylsilyl) ethynyl) thiazolo [5,4-b]Pyridin-2-amine (2.77g, 13)16mmol) was dissolved in 30mL of dichloromethane and 30mL of NaOH in methanol (3% strength by mass) were slowly added while stirring. The mixture was then reacted at room temperature for 2h, and then the organic solvent was removed. The residue was further purified by chromatography (volume ratio PE: EtOAc ═ 1: 1) to give compound 1.98g as a yellow solid in 86% yield. Mp 225-227 ℃; EI-MS (M/z)176.15[ M + H]⁺,174.20[M-H]^-。

4) Preparation of Boc-L-hydroxyproline: l-hydroxyproline (9.0g, 68.7mmol) was dissolved in tetrahydrofuran (82.5mL) and sodium hydroxide solution (1mol/L, 82.5mL) at 0 ℃. Di-tert-butyl dicarbonate (15.75mL, 68.7mmol) was added to the ice-water bath. The mixture was allowed to react at room temperature for 6h until complete reaction was detected by thin layer chromatography. The reaction solution is concentrated in vacuum to about 80mL, cooled in an ice water bath, acidified to pH 2-3 with 1mol/L hydrochloric acid, and diluted with 100mL ethyl acetate. Extract 3 times with 50mL of ethyl acetate. The combined organic layers were washed 2 times with 50mL of brine and then dried over anhydrous sodium sulfate to give a colorless oil that was used without further purification.

5) Preparation of tert-butyl (2S,4R) -2- ((4-chloro-3- (trifluoromethyl) phenyl) carbamoyl) -4-hydroxypyrrole-1-carboxylate: Boc-L-hydroxyproline (3.48g, 15.31mmol) was dissolved in 50mL of dichloromethane and triethylamine (1.5mL, 15.31mmol) was added. The solution was cooled to 0 deg.C and a solution of ethyl chloroformate (2mL, 15.31mmol) in 2mL of methylene chloride was added dropwise. After reacting for 30 min-1 h, generating an active intermediate. Then, 5-amino-2-chlorotrifluoromethylbenzene (2.7g, 13.92mmol) dissolved in 10mL of dichloromethane was added dropwise to the above solution at 0 ℃ and stirring was continued at room temperature overnight. The reaction solution was diluted with 50mL of dichloromethane, washed twice with saturated sodium bicarbonate solution (30 mL. times.2), twice with water (30mL) and 1 time with brine (30 mL). The organic phase was dried over anhydrous sodium sulfate, filtered and chromatographed (volume ratio petroleum ether: ethyl acetate: 3: 1) to give 4.26g of product in 75% yield.

6) Preparation of tert-butyl (2S,4R) -2- ((4-chloro-3- (trifluoromethyl) phenyl) carbamoyl) -4-methanesulfonyloxypyrrole-1-carboxylate: the compound (2S,4R) -2- ((4-chloro-3- (trifluoromethyl) phenyl) aminoFormyl) -4-hydroxypyrrole-1-carboxylic acid tert-butyl ester (3.79g, 9.27mmol) was dissolved in 80mL of dichloromethane and cooled to 0 ℃. Triethylamine (1.55mL, 11.12mmol) was added and stirred for 15 min. Methanesulfonyl chloride (0.86mL, 11.12mmol) was added dropwise and reacted at room temperature overnight. The reaction was quenched with 20mL of water and washed once with 30mL of brine. The organic phase was dried over anhydrous sodium sulfate, filtered and evaporated to give the product 3.87g, 86% yield. EI-MS (M/z)487.05.10[ M + H]⁺,485.00[M-H]^-。

7) Preparation of tert-butyl (2S,4S) -4-azido-2- ((4-chloro-3- (trifluoromethyl) phenyl) carbamoyl) pyrrolidine-1-carboxylate: the compound (2S,4R) -2- ((4-chloro-3- (trifluoromethyl) phenyl) carbamoyl) -4-methanesulfonyloxypyrrole-1-carboxylic acid tert-butyl ester (3.78g, 7.76mmol) was dissolved in 10mL of DMF and sodium azide (0.99g, 15.52mmol) was added. The mixture is reacted for 16 hours under the protection of nitrogen at 65-70 ℃. The reaction solution was then cooled to room temperature and poured into 100mL of ice water to precipitate a white solid. The reaction mixture was extracted 3 times with 80mL of ethyl acetate. The organic phases were combined, washed 1 time with 50mL of brine, dried over anhydrous sodium sulfate and purified by chromatography (vol.% petroleum ether: ethyl acetate: 5: 1) to give 2.69g of product in 80% yield. Melting point 57.6-58.2 deg.C, EI-MS (M/z)434[ M + H]⁺，432[M-H]^-。

8) (2S,4S) -4- (4- (2-aminothiazolo [5, 4-b)]Preparation of pyridin-5-yl) -1H-1,2, 3-triazol-1-yl) -2- ((4-chloro-3- (trifluoromethyl) phenyl) carbamoyl) pyrrolidine-1-carboxylic acid tert-butyl ester (SPZ 1): a100 mL round bottom flask was charged with a mixture of 40mL ethanol and 40mL water. Then adding the compound 5-ethynylthiazolo [5,4-b ]]Pyridin-2-amine (0.4g, 2.28mmol) and the compound (2S,4S) -4-azido-2- ((4-chloro-3- (trifluoromethyl) phenyl) carbamoyl) pyrrolidine-1-carboxylic acid tert-butyl ester (0.99g, 2.28 mmol). Sodium ascorbate (0.1g, 0.5mmol) and copper sulfate pentahydrate (0.055g, 0.25mmol) were then added. The suspension is stirred at 30 ℃ for 24h, the reaction is concentrated in vacuo to 40mL and extracted 3 times with 30mL portions of ethyl acetate. Drying over anhydrous sodium sulfate, filtration, chromatography (ethyl acetate) gave the product, SPZ10.65g, in 47% yield. Mp 145-147 ℃; EI-MS (M/z)609.25[ M + H]⁺,607.30[M-H]^-。HRMSm/z to C₂₅H₂₅ClF₃N₈O₃S([M+H]⁺) The calculated value was 609.14109, found 609.14184.¹H NMR(400MHz,DMSO-d₆)δ10.46(s,1H),8.69(d,J＝7.6Hz,1H),8.12(s,1H),7.85(d,J＝8.2Hz,4H),7.70–7.62(m,2H),5.38–5.28(m,1H),4.42–4.38(m,1H),4.18–4.09(m,1H),3.87–3.79(m,1H),3.02–2.91(m,1H),2.66–2.61(m,1H),1.34(s,3H),1.28(s,6H)。

9) (2S,4S) -4- (4- (2-aminothiazolo [5, 4-b)]Preparation of pyridin-5-yl) -1H-1,2, 3-triazol-1-yl) -N- (4-chloro-3- (trifluoromethyl) phenyl) pyrrolidine-2-carboxamide (SPZ 2): the compound (2S,4S) -4- (4- (2-aminothiazolo [5, 4-b)]Pyridin-5-yl) -1H-1,2, 3-triazol-1-yl) -2- ((4-chloro-3- (trifluoromethyl) phenyl) carbamoyl) pyrrolidine-1-carboxylic acid tert-butyl ester was dissolved in 30mL of anhydrous dichloromethane. At 0 deg.C, 2mL of trifluoroacetic acid was added dropwise. The solution was stirred at room temperature for 2h, diluted with 50mL of dichloromethane, adjusted to pH 8 with sodium bicarbonate solution and washed 3 times with 30mL of water. The organic layer was dried over anhydrous sodium sulfate, filtered, and purified by chromatography (vol. petrol ether: ethyl acetate 1; 3) to give the product (SPZ2) in 36% yield. Mp 123-125 ℃; EI-MS (M/z)509.15[ M + H]⁺,507.15[M-H]^-. HRMS m/z to C₂₀H₁₇ClF₃N₈OS([M+H]⁺) The calculated value was 509.08867, found 509.09129.¹H NMR(400MHz,DMSO-d₆)δ10.40(s,1H),8.65(s,1H),8.23(s,1H),7.89–7.80(m,3H),7.74–7.71(m,1H),7.68–7.66(m,1H),7.65–7.59(m,1H),5.23–5.14(m,1H),4.03–3.93(m,1H),3.53–3.45(m,2H),2.91–2.73(m,1H),2.48–2.43(m,1H),1.91(s,1H)。

Example 2

Thiazolopyridine ring-linked triazole compound R₁Is 4-Cl-3-CF₃，R₂When the compound is acetyl and R' is H, the preparation method comprises the following steps:

step 1) to step 8) in the same manner as in example 1, tert-butyl (2S,4S) -4- (4- (2-aminothiazolo [5,4-b ] pyridin-5-yl) -1H-1,2, 3-triazol-1-yl) -2- ((4-chloro-3- (trifluoromethyl) phenyl) carbamoyl) pyrrolidine-1-carboxylate was obtained.

9) (2S,4S) -4- (4- (2-acetamido-thiazolo [5, 4-b)]Preparation of pyridin-6-yl) -1H-1,2, 3-triazol-1-yl) -N- (4-chloro-3- (trifluoromethyl) phenyl) pyrrolidine-2-carboxamide (SPZ 3): compound (2S,4S) -4- (4- (2-aminothiazolo [5, 4-b)]Pyridin-5-yl) -1H-1,2, 3-triazol-1-yl) -2- ((4-chloro-3- (trifluoromethyl) phenyl) carbamoyl) pyrrolidine-1-carboxylic acid tert-butyl ester was dissolved in 30mL of anhydrous dichloromethane and triethylamine (0.37mL, 2.7mmol) was added. The solution was stirred at 0 ℃ for 30min and dichloromethane (2mL) containing acetyl chloride was added dropwise. The solution reacts for 2h at room temperature, dichloromethane is removed in vacuum, and the product (2S,4S) -4- (4- (2-acetamido thiazolo [5, 4-b) is obtained after purification by chromatography]Pyridin-6-yl) -1H-1,2, 3-triazol-1-yl) -2- ((4-chloro-3- (trifluoromethyl) phenyl) carbamoyl) pyrrolidine-1-carboxylic acid tert-butyl ester. The product was then dissolved in 30mL of anhydrous dichloromethane. At 0 deg.C, 2mL of trifluoroacetic acid was added dropwise. The solution was stirred at room temperature for 2h, diluted with 50mL of dichloromethane, adjusted to pH 8 with sodium bicarbonate solution and washed 3 times with 30mL of water. The organic layer was dried over anhydrous sodium sulfate, filtered, and purified by chromatography (volume ratio petroleum ether: ethyl acetate: 1:3) to give compound (SPZ3) in 30% yield. Mp 137-139 ℃; EI-MS (M/z)551.15[ M + H]⁺. HRMS m/z to C₂₂H₁₉ClF3N₈O₂S([M+H]⁺) The calculated value was 551.09923, found 551.09943.¹H NMR(400MHz,DMSO-d₆)δ12.52(s,1H),10.39(s,1H),8.81(s,1H),8.20(d,J＝2.4Hz,1H),8.14(d,J＝8.5Hz,1H),8.05(d,J＝8.5Hz,1H),7.93(dd,J＝8.8,2.2Hz,1H),7.59(d,J＝8.8Hz,1H),5.24–5.17(m,1H),4.01–3.97(m,1H),3.53–3.48(m,1H),3.43-3.41(m,1H),2.87–2.77(m,1H),2.55-2.53(m,1H),2.23(s,4H)。

Example 3

Thiazolopyridine ring-linked triazole compound R₁Is 4-Cl-3-CF₃，R₂When the compound is cyclopropylformyl and R' is H, the preparation process is the same as in example 2 except that acetyl chloride in example 2 is replaced by cyclopropylformyl chloride, thereby obtaining (2S,4S) -N- (4-chloro-3- (trifluoromethyl) phenyl) -4- (4- (2- (cyclopropanecarboxamido) thiazolo [5, 4-b)]Pyridin-6-yl) -1H-1,2, 3-triazol-1-yl) pyrrolidine-2-carboxamide (SPZ4) in 41% yield after chromatographic purification (1: 2 by volume with petroleum ether: ethyl acetate). Mp 159-161 ℃; EI-MS (M/z)577.15[ M + H]⁺,575.20[M-H]^-. HRMS m/z to C₂₄H₂₁ClF₃N₈O₂S([M+H]⁺) The calculated value was 577.11488, found 577.11560.¹H NMR(400MHz,DMSO-d₆)δ12.84(s,1H),10.37(s,1H),8.82(s,1H),8.21(s,1H),8.14(d,J＝8.4Hz,1H),8.05(d,J＝8.5Hz,1H),7.93(d,J＝9.0Hz,1H),7.59(d,J＝8.7Hz,1H),5.23–5.14(m,1H),3.99–3.95(m,1H),3.51–3.47(m,2H),2.85–2.78(m,1H),2.47–2.45(m,1H),2.05–2.01(m,1H),1.91(s,1H),1.00(d,J＝7.9Hz,4H)。

Example 4

Thiazolopyridine ring-linked triazole compound R₁Is 2,4-di-Cl, R₂When H and R' are Boc or H, the procedure is as in example 1 except that 5-amino-2-chlorotrifluoromethylbenzene in example 1 is replaced with 2, 4-dichloroaniline to prepare (2S,4S) -4- (4- (2-aminothiazolo [5,4-b ]]Pyridin-5-yl) -1H-1,2, 3-triazol-1-yl) -2- ((2, 4-dichlorophenyl) carbamoyl) pyrrolidine-1-carboxylic acid tert-butyl ester (SPZ5) and (2S,4S) -4- (4- (2-aminothiazolo [5, 4-b)]Pyridin-5-yl) -1H-1,2, 3-triazol-1-yl) -N- (2, 4-dichlorophenyl) pyrrolidine-2-carboxamide (SPZ 6).

The compound SPZ5 was purified by ethyl acetate chromatography to give 0.80g of a solid compound in 66% yield. Mp 134-136 ℃; EI-MS (M/z)573.25[ M-H]^-HRMS m/z vs C₂₄H₂₅Cl₂N₈O₃S([M+H]⁺) The calculated value was 575.11474, found 575.11529.¹H NMR(400MHz,DMSO-d₆)δ9.72(s,1H),8.70(s,1H),8.18–8.09(m,1H),7.88(s,2H),7.71–7.66(m,3H),7.37(s,1H),5.37–5.31(m,1H),4.59–4.56(m,1H),4.15–4.12(m,1H),3.87–3.84(m,1H),3.06–2.93(m,1H),2.76–2.64(m,1H),1.46(s,3H),1.36(s,6H)。

Compound SPZ6 was purified by chromatography (vol.% petroleum ether: ethyl acetate: 1: 5) to give the compound as a solid in 51% yield. Mp is 200-202 ℃; EI-MS (M/z)475.15[ M + H]⁺,473.05[M-H]^-. HRMS m/z to C₁₉H₁₇Cl₂N₈OS([M+H]⁺) The calculated value was 475.06231, found 475.06292.¹H NMR(400MHz,DMSO-d₆)δ10.33(s,1H),8.63(s,1H),8.26(d,J＝8.9Hz,1H),7.85(s,2H),7.79(d,J＝8.3Hz,1H),7.65(d,J＝8.3Hz,1H),7.59(d,J＝2.4Hz,1H),7.37(dd,J＝8.9,2.4Hz,1H),5.18–5.10(m,1H),4.06–3.99(m,1H),3.57–3.52(m,1H),3.20–3.12(m,1H),2.87–2.81(m,1H),2.55–2.52(m,1H),1.88(s,1H)。

Example 5

Thiazolopyridine ring-linked triazole compound R₁Is 2,4-di-Cl, R₂For acetyl, R' is H, the procedure is as in example 2. Preparation of (2S,4S) -4- (4- (2-acetylaminothiazolo [5, 4-b) by substituting 5-amino-2-chlorotrifluoromethylbenzene in example 2 with 2, 4-dichloroaniline]Pyridin-5-yl) -1H-1,2, 3-triazol-1-yl) -N- (2, 4-dichlorophenyl) pyrrolidine-2-carboxamide (SPZ 7). Purification by chromatography (vol/vol ethyl acetate: methanol-1: 1) gave the product in 84% yield. Mp 198-200 ℃; EI-MS (M/z)517.20[ M + H]⁺,515.25[M-H]^-. HRMS m/z to C₂₁H₁₉Cl₂N₈O₂S([M+H]⁺) The calculated value was 517.07287, found 517.07356.¹H NMR(400MHz,DMSO-d₆)δ12.53(s,1H),10.34(s,1H),8.79(s,1H),8.22(d,J＝8.6Hz,1H),8.13(d,J＝8.5Hz,1H),8.02(d,J＝8.5Hz,1H),7.56(d,J＝2.3Hz,1H),7.35(dd,J＝8.9,2.3Hz,1H),5.22–5.16(m,1H),4.14–4.02(m,1H),3.60–3.56(m,1H),3.48–3.46(m,1H),2.93–2.82(m,1H),2.63–2.55(m,1H),2.23(s,3H),1.91(s,1H)。

Example 6

Thiazolopyridine cyclotriazole compound R₁Is 2,4-di-Cl, R₂For cyclopropanecarbonyl, R' is H, the procedure is as in example 3. Preparation of (2S,4S) -4- (4- (2- (cyclopropanecarboxamido) thiazolo [5, 4-b) by substituting 5-amino-2-chlorotrifluoromethylbenzene in example 3 with 2, 4-dichloroaniline]Pyridin-6-yl) -1H-1,2, 3-triazol-1-yl) -N- (2, 4-dichlorophenyl) pyrrolidine-2-carboxamide (SPZ 8). Purifying by chromatography (volumetric to acetic acid)Ethyl ester: methanol 1: 1) the product was obtained in 55% yield. Mp 202-204 ℃; EI-MS (M/z)543.20[ M + H]⁺,541.25[M-H]^-. HRMS m/z to C₂₃H₂₁Cl₂N₈O₂S([M+H]⁺) The calculated value was 543.08852, found 543.08965.¹H NMR(400MHz,DMSO-d₆)δ12.84(s,1H),10.33(s,1H),8.79(s,1H),8.26(d,J＝8.9Hz,1H),8.13(d,J＝8.5Hz,1H),8.01(d,J＝8.5Hz,1H),7.54(d,J＝2.4Hz,1H),7.34(dd,J＝8.9,2.4Hz,1H),5.19–5.13(m,1H),4.08–3.99(m,1H),3.62–3.51(m,2H),2.91–2.80(m,1H),2.60–2.55(m,1H),2.07–2.00(m,1H),1.91(s,1H),1.01–0.98(m,4H)。

Example 7

Thiazolopyridine ring-linked triazole compound R₁Is 4-Br, R₂For H, R' Boc or H, preparation was as in example 1. Preparation of (2S,4S) -4- (4- (2-aminothiazolo [5, 4-b) by substituting 5-amino-2-chlorotrifluoromethylbenzene in example 1 with 4-bromoaniline]Pyridin-5-yl) -1H-1,2, 3-triazol-1-yl) -2- ((4-bromophenyl) carbamoyl) pyrrolidine-1-carboxylic acid tert-butyl ester (SPZ9) and (2S,4S) -4- (4- (2-aminothiazolo [5, 4-b)]Pyridin-5-yl) -1H-1,2, 3-triazol-1-yl) -N- (4-bromophenyl) pyrrolidine-2-carboxamide (SPZ 10).

The compound SPZ9 was purified by ethyl acetate chromatography to give the product 0.60g in 67% yield. MP 176-178 ℃; EI-MS (M/z)583.25[ M-H]^-HRMS m/z vs C₂₄H₂₆BrN₈O₃S([M+H]⁺) The calculated value was 585.10320, found 585.10303.¹H NMR(400MHz,DMSO-d₆)δ10.24(s,1H),8.71(s,1H),7.88(s,3H),7.69(d,J＝8.4Hz,1H),7.59-7.55(m,2H),7.49(d,J＝8.5Hz,2H),5.37-5.30(m,1H),4.44–4.38(m,1H),4.19–4.10(m,1H),3.83–3.72(m,1H),3.04–2.92(m,1H),2.62–2.55(m,1H),1.43(s,3H),1.29(s,6H)。

The compound SPZ10 was purified by chromatography (vol.% petroleum ether: ethyl acetate: 1:3) to give the compound as a solid in 60% yield. Mp 152-154 ℃; EI-MS (M/z)485.10[ M + H]⁺,483.10[M-H]^-. HRMS m/z to C₁₉H₁₈BrN₈OS([M+H]⁺) The calculated value was 485.05077, found 485.05200.¹H NMR(400MHz,DMSO-d₆)δ10.11(s,1H),8.66(s,1H),7.91–7.83(m,3H),7.68(d,J＝8.3Hz,1H),7.62(d,J＝8.8Hz,2H),7.45(d,J＝8.8Hz,2H),5.21–5.15(m,1H),3.95–3.91(m,1H),3.48–3.43(m,1H),3.35–3.29(m,1H),2.83–2.77(m,1H),2.42–2.33(m,1H),1.90(s,1H)。

Example 8

Thiazolopyridine ring-linked triazole compound R₁Is 4-Br, R₂For acetyl, R' is H, the procedure is as in example 2. Preparation of (2S,4S) -4- (4- (2-acetylaminothiazolo [5, 4-b) by substituting 5-amino-2-chlorotrifluoromethylbenzene in example 2 with 4-bromoaniline]Pyridin-6-yl) -1H-1,2, 3-triazol-1-yl) -N- (4-bromophenyl) pyrrolidine-2-carboxamide (SPZ 11). The product was obtained in 39% yield after purification by chromatography (vol.% petroleum ether: ethyl acetate: 1: 2). Mp 173-175 ℃; EI-MS (M/z)526.95[ M + H]⁺,525.10[M-H]^-HRMS m/z vs C₂₁H₂₀BrN₈O₂S([M+H]⁺) The calculated value was 527.06133, found 527.06202.¹H NMR(400MHz,DMSO-d₆)δ12.54(s,1H),10.12(s,1H),8.82(s,1H),8.16(d,J＝8.4Hz,1H),8.08(d,J＝8.4Hz,1H),7.62(d,J＝8.8Hz,2H),7.44(d,J＝8.8Hz,2H),5.26–5.18(m,1H),3.97–3.93(m,1H),3.49–3.46(m,2H),2.87–2.79(m,1H),2.47–2.39(m,1H),2.23(s,3H),1.91(s,1H)。

Example 9

Thiazolopyridine cyclotriazole compound R₁Is 4-Br, R₂For cyclopropanecarbonyl, the procedure is as in example 3. Preparation of (2S,4S) -N- (4-bromophenyl) -4- (4- (2- (cyclopropanecarboxamido) thiazolo [5, 4-b) by substituting 5-amino-2-chlorotrifluoromethylbenzene in example 3 with 4-bromoaniline]Pyridin-6-yl) -1H-1,2, 3-triazol-1-yl) pyrrolidine-2-carboxamide (SPZ 12). Purification by chromatography (volume ratio petroleum ether: ethyl acetate 1:3) gave the product as a solid in 43% yield. Mp 156-158 ℃; EI-MS (M/z)553.10[ M + H]⁺,551.15[M-H]^-. HRMS m/z to C₂₃H₂₂BrN₈O₂S([M+H]⁺) The calculated value was 553.07698, found 553.07346.¹H NMR(400MHz,DMSO-d₆)δ12.86(s,1H),10.11(s,1H),8.83(s,1H),8.16(d,J＝8.4Hz,1H),8.08(d,J＝8.4Hz,1H),7.61(d,J＝8.8Hz,2H),7.44(d,J＝8.8Hz,2H),5.23–5.17(m,1H),3.96–3.92(m,1H),3.48–3.44(m,2H),2.86–2.79(m,1H),2.46–2.38(m,1H),2.07–2.01(m,1H),1.91(s,1H),1.01–0.98(m,4H)。

Example 10

Thiazolopyridine ring-linked triazole compound R₁Is 3,4-di-F, R₂For H, R' is Boc or H, the procedure is as in example 1. (2S,4S) -4- (4- (2-Aminothiazolo [5, 4-b) was prepared by substituting 5-amino-2-chlorotrifluoromethylbenzene in example 1 with 3, 4-difluoroaniline]Pyridin-5-yl) -1H-1,2, 3-triazol-1-yl) -2- ((3, 4-difluorophenyl) carbamoyl) pyrrolidine-1-carboxylic acid tert-butyl ester (SPZ13) and (2S,4S) -4- (4- (2-aminothiazolo [5, 4-b)]Pyridin-5-yl) -1H-1,2, 3-triazol-1-yl) -N- (3, 4-difluorophenyl) pyrrolidine-2-carboxamide (SPZ 14).

Purification of compound SPZ13 with ethyl acetate provided the product in 0.70g, 84% yield. Mp 137-139 ℃; EI-MS (M/z)541.30[ M-H]^-HRMS m/z vs C₂₄H₂₅F₂N₈O₃S([M+H]⁺) The calculated value was 543.17384, found 543.17396.¹H NMR(400MHz,DMSO-d₆)δ10.13(s,1H),8.69(s,1H),7.92(d,J＝5.0Hz,1H),7.71(d,J＝5.8Hz,2H),7.59–7.54(m,1H),7.51(d,J＝8.3Hz,1H),7.20–7.13(m,1H),7.12–7.08(m,1H),5.24–5.11(m,1H),4.30–4.17(m,1H),4.01–3.94(m,1H),3.67–3.59(m,1H),2.86–2.75(m,1H),2.47–2.33(m,1H),1.25(s,3H),1.12(s,6H)。

Compound SPZ14 was purified by chromatography (vol.% petroleum ether: ethyl acetate: 1) to give the compound as a solid in 74% yield. MP 155-157 ℃; EI-MS (M/z)443.05[ M + H]⁺,441.20[M-H]^-HRMS m/z vs C₁₉H₁₇F₂N₈OS([M+H]⁺) The calculated value was 443.12141, found 443.12341.¹H NMR(400MHz,DMSO-d₆)δ10.19(s,1H),8.64(s,1H),7.88–7.83(m,3H),7.83–7.77(m,1H),7.67(d,J＝8.3Hz,1H),7.42(d,J＝9.0Hz,1H),7.37–7.31(m,1H),5.24–5.13(m,1H),3.95–3.92(m,1H),3.52–3.44(m,1H),3.33–3.29(m,1H),2.85–2.77(m,1H),2.44–2.37(m,1H),1.90(s,1H)。

Example 11

Thiazolopyridine ring-linked triazole compound R₁Is 3,4-di-F, R₂For acetyl, R' is H, the procedure is as in example 2. (2S,4S) -4- (4- (2-acetamido-thiazolo [5, 4-b) was prepared by substituting 5-amino-2-chlorotrifluoromethylbenzene in example 2 with 3, 4-difluoroaniline]Pyridin-5-yl) -1H-1,2, 3-triazol-1-yl) -N- (3, 4-difluorophenyl) pyrrolidine-2-carboxamide (SPZ 15). The product was obtained in 50% yield after purification by chromatography (vol.% petroleum ether: ethyl acetate: 1: 2). Mp 129-131 ℃; EI-MS (M/z)485.25[ M + H]⁺,483.30[M-H]^-. HRMS m/z to C₂₁H₁₉F₂N₈O₂S([M+H]⁺) The calculated value was 485.13197, found 485.12879.¹H NMR(400MHz,DMSO-d₆)δ12.54(s,1H),10.20(s,1H),8.81(s,1H),8.16(d,J＝8.5Hz,1H),8.08(d,J＝8.7Hz,1H),7.82–7.77(m,1H),7.43–7.38(m,1H),7.37-7.35(m,1H),5.25–5.16(m,1H),3.97-3.93(m,1H),3.51–3.47(m,2H),2.86–2.79(m,1H),2.51–2.42(m,1H),2.23(s,3H),1.91(s,1H)。

Example 12

Thiazolopyridine ring-linked triazole compound R₁Is 3,4-di-F, R₂For cyclopropanecarbonyl, R' is H, the procedure is as in example 3. Preparation of (2S,4S) -4- (4- (2- (cyclopropanecarboxamido) thiazolo [5, 4-b) by substituting 5-amino-2-chlorotrifluoromethylbenzene in example 3 with 3, 4-difluoroaniline]Pyridin-5-yl) -1H-1,2, 3-triazol-1-yl) -N- (3, 4-difluorophenyl) pyrrolidine-2-carboxamide (SPZ 16). Purification by chromatography (vol.% petroleum ether: ethyl acetate 1: 2) gave the product as a solid in 69% yield. Mp 133-135 ℃; EI-MS (M/z)511.20[ M + H]⁺,509.30[M-H]^-. HRMS m/z to C₂₃H₂₁F₂N₈O₂S([M+H]⁺) Calculated value of 511.14762, found value of 511.14361.¹H NMR(400MHz,DMSO-d₆)δ12.87(s,1H),10.46(s,1H),8.88(s,1H),8.18(d,J＝8.5Hz,1H),8.10(d,J＝8.5Hz,1H),7.77–7.72(m,1H),7.40–7.38(m,1H),7.37-7.35(m,1H),5.39–5.33(m,1H),4.28–4.20(m,1H),3.71–3.58(m,2H),3.01–2.93(m,1H),2.65–2.59(m,1H),2.06–2.00(m,1H),1.91(s,1H),1.03–0.95(m,4H)。

The screening of Bcr-Abl kinase inhibitory activity is performed on the thiazolopyridine bitriazole compound with anti-tumor activity prepared by the invention.

The determination method specifically comprises the following steps:

the kinases Bcr-Abl, Bcr-Abl (T315I) and substrate Abltide were purchased from Signal-Chem, and selected from ADP-Glo of Promega^TMThe enzyme inhibitory activity of the target compound is detected by the Kinase Assays detection kit, and the operation method is carried out according to the kit instructions.

In the Abl experiment, buffer (2X) (Tris 80mM, MgCl) was used₂20mM, BSA 0.2mg/mL, DTT 2mM) ATP (1mM) was diluted 80-fold and prepared as a buffer (2X) solution of ATP (125. mu.M); then preparing a mixed solution of ATP (62.5 mu M) -Abltide (0.5 mu g/mu L) according to the volume of 1:1 for later use by using the ATP (125 mu M) solution and the Abltide solution; with buffer (1X) (Tris 40mM, MgCl)₂10mM, BSA 0.1mg/mL, DTT 1mM) the Abl kinase solution was diluted 100-fold and prepared as a buffer (1X) solution of Abl (1 ng/. mu.L) for use.

In the Abl (T315I) experiment, the procedures for ATP-Abltide and Abl (T315I) were as above, except that the concentrations of ATP and Abl (T315I) were 12.5. mu.M and 2 ng/. mu.L, respectively.

Four target compounds were each formulated with buffer (1X) to 1.5X 10^-5，1.5×10^-6，1.5×10^-7，1.5×10^-8，1.5×10^-9，1.5×10^-10Adding 2 mu L of ATP-Abltide mixed solution, 1 mu L of sample solution and 2 mu L of enzyme solution into each well of a 384-well plate in turn according to the mol/L concentration gradient sample solution; adding 3 mu L of buffer solution and 2 mu L of ATP-Abltide mixed solution into a blank hole; adding 2. mu.L of ATP-Abltide mixed solution, 1. mu.L of buffer solution and 2. mu.L of enzyme solution into each control well, and incubating at 30 ℃ for 60min after the addition is finished; adding 5 μ L of ADP-Glo reagent, and incubating at 25 deg.C for 40 min; most preferablyThen Kinase detection reagent was added and incubated at 25 ℃ for 30 min. Measuring the luminescence value of each hole by using a chemiluminescence module of a PerkinElmer multifunctional microplate reader, and calculating the inhibition rate and IC (integrated Circuit) of the compound on Abl₅₀。

The structural formula of the thiazolopyridine bitriazole compound is as follows:

the kinase inhibitory activity of the thiazolopyridine bitriazole compounds of the above structural formula is shown in Table 1

TABLE 1 Thiazolopyridine bitriazole compounds for Bcr-Abl^WTAnd Bcr-Abl^T315IInhibitory Activity of IC₅₀(nM)

The compound SPZ1-SPZ16 against Bcr-Abl^WTAnd Bcr-Abl^T315IThe results of the kinase inhibitory activity of (2) are summarized in Table 1. It can be seen that when thiazolo [5,4-b ]]When pyridine is used as a core structure and interacts with different binding groups, most compounds have better inhibitory activity on Bcr-Abl kinase, wherein the best activity is compounds SPZ2, SPZ8 and SPZ12 and IC thereof₅₀Values were 1.60nM, 0.60nM and 3.58nM, respectively. And in Bcr-Abl^T315IThe kinases, compounds SPZ2, SPZ3, SPZ9 and SPZ12, showed better activity, their IC₅₀Values were 9.69nM, 59.62nM, 25.21nM and 49.95 nM. From the data in the above table, it can be seen that amino acetylation favors thiazolo [5,4-b ]]Activity of pyridine derivatives on Bcr-Abl kinase (SPZ3, SPZ4, SPZ7, SPZ8, SPZ11, SPZ12, SPZ15, SPZ16>SPZ2, SPZ6, SPZ10, SPZ 14). In general, better cyclopropylformylation than acetylated amino groups can increase thisThe activity of the series. At the same time, halogenated substituents are also introduced on the terminal benzene ring, these substituents also influence the activity on kinases, and the relationship between the various substituents and inhibitory activity can be summarized roughly as: 2, 4-dichloro substituents>4-bromo-substituted radical>4-chloro-3-trifluoromethyl substituent>3, 4-difluoro substituents. The activity results show that the difference of the substituent can directly influence the inhibition activity of the compound on the kinase.

Next, the proliferation inhibitory activity of the thiazolopyridine bitriazole compounds on tumor cells was measured.

The MTT method is adopted to determine the proliferation inhibition activity of the thiazolopyridine bitriazole compounds on tumor cells.

The thiazolopyridine bitriazole compound provided by the invention has an anti-tumor effect. Has effect in inhibiting proliferation activity of tumor cells in vitro and in human leukemia cells (K562 cells), and can be used for treating leukemia.

Diluting human leukemia cells (K562 cells) in growth exponential phase to 10 with RPMI1640 medium⁴Cell solutions of the order of one/mL were plated in parallel in 96-well plates (2000-4000/well) at a volume of 180. mu.L/well and 5% CO at 37 ℃ in a culture medium₂Culturing for 12 h;

20 μ L of test compound was added to each well at different concentrations to give final concentrations of compound in the wells: 1.5X 10^{- 7}mol/L，1.5×10^-6mol/L，1.5×10^-5mol/L，1.5×10^-4mol/L, setting 3 multiple wells for each concentration, setting 6 multiple wells for negative control, adding cells without compound in each well, taking nilotinib or imatinib as positive control, continuing to perform reaction at 37 deg.C and 5% CO₂Culturing for 48 h;

mu.L of MTT (5mg/mL) was added to each well to give a final concentration of 0.5mg/mL MTT in each well at 37 ℃ with 5% CO₂Culturing for 4 hr, carefully removing supernatant, adding DMSO 150 μ L into each well, shaking for 15min, measuring ultraviolet absorption (OD) at 490nm of each well with ELISA detector, calculating cell inhibition rate, and calculating by linear regression method according to the inhibition rateIC of compound₅₀A value;

the formula for calculating the cell inhibition rate is as follows:

percent inhibition is (average OD value in control well-average OD value in drug group)/average OD value in control well × 100%;

and (3) displaying a detection result: the thiazolopyridine bitriazole compounds have different degrees of in vitro inhibition effects on the above-mentioned tumor cells, as compared with the negative control group, as shown in table 2.

K562 cell proliferation activity:

TABLE 2 contains the inhibitory activity IC of thiazolopyridine-bis-triazole compounds on K562 cells₅₀(μM)

The cell activity screening test shows that the compound SPZ1-SPZ16 has certain cell proliferation inhibiting activity on K562 cells, and the IC of the compound₅₀Values were at the micromolar level, ranging from 0.74. mu.M to 62.96. mu.M. Among the most active compounds is SPZ8, IC₅₀The value was 0.74. mu.M. P-thiazolo [5,4-b ]]Pyridine derivatives, when halogenated substituents are introduced on the terminal benzene ring, have a significant effect on the antiproliferative activity of tumor cells, with compounds in which the substituent is 2, 4-dichloro being the most active. In addition, the compounds SPZ2, SPZ12, and SPZ13 also have a good cytostatic activity against K562 cells, and further intensive studies can be conducted.

The invention adopts a fragment-based drug design strategy, introduces halogenated substituent groups on a benzene ring at the end part while carrying out amino acylation on a thiazolopyridine ring to construct a thiazolopyridine bitriazole compound library with kinase inhibitory activity, and discovers tyrosine kinase inhibitors SPZ2, SPZ8, SPZ12 and SPZ13 with Bcr-Abl kinase inhibitory activity through kinase activity screening. The four compounds can be used for preparing anti-tumor (chronic granulocytic leukemia) medicaments, and have certain inhibition on Bcr-Abl and Bcr-Abl^T315IKinase activity and certain cell proliferation inhibiting activity on K562 cells. Terminal endThe halogenated substituent introduced on the benzene ring can expand the structural diversity of the Bcr-Abl kinase inhibitor, and meanwhile, an activity test shows that the thiazolopyridine ring has an important effect on the kinase inhibition activity of the compound, can improve the affinity between a receptor and the compound, and can be used as a pharmacodynamic fragment of the Bcr-Abl tyrosine kinase inhibitor.

Claims (10)

1. A thiazolopyridine bitriazole compound is characterized in that the structural formula of the compound is as follows:

wherein R is₁、R₂And R' are specifically as follows:

2. a process for the preparation of a thiazolopyridine bitriazole compound according to claim 1, which comprises the steps of:

1) condensing the L-hydroxyproline protected by the tert-butyloxycarbonyl group and an aniline compound containing a substituent group to generate an aminated Boc-L-hydroxyproline compound;

2) the aminated Boc-L-hydroxy proline compound and methylsulfonyl chloride are subjected to substitution reaction to generate an aminated Boc-L-methylsulfonyl proline compound;

3) under the protection of nitrogen, carrying out substitution reaction on an aminated Boc-L-methylsulfonyl proline compound and sodium azide to generate an aminated Boc-L-azidoprolinic acid compound;

4) condensing 5-ethynylthiazolo [5,4-b ] pyridine-2-amine and aminated Boc-L-azidoproline compounds under the action of sodium ascorbate and copper sulfate pentahydrate to obtain Boc-protected thiazolopyridine-triazole compounds;

5) reacting a plurality of acyl-containing compounds with a thiazolopyridine triazole compound protected by Boc, and removing the Boc protecting group by trifluoroacetic acid to obtain the indazole cyclic triazole compound.

3. The method for producing a thiazolopyridine bitriazole compound according to claim 2, wherein in step 1), the t-butoxycarbonyl-protected L-hydroxyproline is produced by: dissolving L-hydroxyproline in tetrahydrofuran and sodium hydroxide solution at 0 ℃, adding di-tert-butyl dicarbonate in an ice water bath, and then reacting for 6 hours at 25-30 ℃ to prepare the L-hydroxyproline protected by tert-butoxycarbonyl.

4. The process for producing a thiazolopyridinetriazole compound according to claim 2, characterized in that 5-ethynylthiazolo [5,4-b ] pyridin-2-amine in step 4) is produced by:

a) under the protection of nitrogen, 6-bromopyridine-3-amine reacts with potassium thiocyanate to prepare 5-bromothiazolo [5,4-b ] pyridine-2-amine;

b) under the protection of nitrogen, 5-bromothiazolo [5,4-b ] pyridin-2-amine reacts with trimethylsilylacetylene to prepare 5- ((trimethylsilyl) ethynyl) thiazolo [5,4-b ] pyridin-2-amine;

c) 5- ((trimethylsilyl) ethynyl) thiazolo [5,4-b ] pyridin-2-amine was trimethylsilyl-removed with sodium hydroxide in methanol to give 5-ethynylthiazolo [5,4-b ] pyridin-2-amine.

5. The preparation method of a thiazolopyridine bitriazole compound according to claim 4, wherein the specific process in step a) is as follows: dissolving KSCN in acetic acid in nitrogen, cooling to 0 ℃, adding 6-bromopyridine-3-amine, cooling to-5 ℃, dropwise adding acetic acid containing bromine, heating to 25-30 ℃ after dropwise adding, and stirring to obtain 5-bromothiazolo [5,4-b ] pyridine-2-amine;

the specific process of the step b) is as follows: adding 5-bromothiazolo [5,4-b ] pyridine-2-amine, copper iodide and tetrakis (triphenylphosphine) palladium into a reaction vessel, sealing the vessel by using a rubber plug, vacuumizing and backfilling with nitrogen, adding triethylamine, then adding trimethylsilyl acetylene, and heating and refluxing for reaction to obtain 5- ((trimethylsilyl) ethynyl) thiazolo [5,4-b ] pyridine-2-amine;

the concrete process of the step c) is as follows: dissolving 5- ((trimethylsilyl) ethynyl) thiazolo [5,4-b ] pyridine-2-amine in dichloromethane, adding a methanol solution of sodium hydroxide under stirring, and reacting at 25-30 ℃ to obtain 5-ethynylthiazolo [5,4-b ] pyridine-2-amine.

6. The method for preparing a thiazolopyridine bitriazole compound according to claim 2, wherein the specific process in step 1) is as follows: dissolving Boc-L-hydroxyproline in dichloromethane, adding triethylamine, cooling to 0 ℃, dropwise adding a dichloromethane solution containing ethyl chloroformate, and reacting for 30 min-1 h to generate an active intermediate; dropwise adding the aniline compound solution containing the substituent group into the active intermediate at 0 ℃, and stirring at 25-30 ℃ to obtain an aminated Boc-L-hydroxy proline compound;

the specific process of the step 2) is as follows: dissolving the aminated Boc-L-hydroxyproline compound in dichloromethane, cooling to 0 ℃, adding triethylamine, stirring uniformly, dropwise adding methylsulfonyl chloride, and reacting at 25-30 ℃ to obtain the aminated Boc-L-methylsulfonyl prolinic acid compound.

7. The method for producing a thiazolopyridine bitriazole compound according to claim 2, characterized in that the substituent-containing aniline compound is 4-chloro-3-trifluoromethylaniline, 2, 4-dichloroaniline, 4-bromoaniline, or 3, 4-difluoroaniline;

the specific process of the step 3) is as follows: dissolving an aminated Boc-L-methylsulfonyl proline compound in DMF, adding sodium azide, and reacting at 65-70 ℃ under the protection of nitrogen to obtain an aminated Boc-L-azidoproline compound;

the specific process of the step 4) is as follows: dissolving 5-ethynylthiazolo [5,4-b ] pyridine-2-amine and aminated Boc-L-azido proline compound in a mixed solvent of ethanol and water, then adding sodium ascorbate and copper sulfate pentahydrate, and stirring at 30 ℃ to obtain the thiazolopyridinitride compound protected by the Boc protecting group.

8. The method for preparing a thiazolopyridine bitriazole compound according to claim 2, wherein the specific process of the step 5) is as follows: and dissolving the indazole-triazole compound protected by the Boc protecting group in anhydrous dichloromethane, adding triethylamine, stirring at 0 ℃ for 30min, dropwise adding a dichloromethane solution containing a chloride compound, and reacting at 25-30 ℃ to obtain the indazole-triazole compound with the Boc protecting group removed.

9. Use of a thiazolopyridine ring triazole compound of claim 1 in the preparation of a Bcr-Abl kinase inhibitor.

10. Use of the thiazolopyridine ring-linked triazole compound according to claim 1 in preparation of an antitumor drug.

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