A preparation method of trilaciclib and its intermediates

Abstract

A preparation method of trilaciclib and its intermediates belongs to the technical field of pharmaceutical chemistry. The purpose of the present invention is to provide a preparation method of trilaciclib and its intermediates that is environmentally friendly, low-cost, easy to operate, and suitable for industrial production. The main raw materials involved are 5-bromo-2,4-dichloropyrimidine, pyruvic acid, bromoacetonitrile, 1,5-dibromopentane, and 5-bromo-2-nitropyridine. Compared with the synthetic method reported in the literature that first introduces a spiro ring and then generates a pyrrolopyrimidine ring, the route of the present invention selects commercially available 5-bromo-2,4-dichloropyrimidine as the starting material, which is inexpensive and easily available, reducing the synthesis cost. Moreover, the reaction conditions of each step are mild, and the intermediates and end products are easy to purify and preserve. And this method uses new intermediates for synthetic research and has the potential for industrial production.

Description

Technical Field

[0001] The present invention belongs to the technical field of medicinal chemistry, and particularly relates to a preparation method of trilaciclib and its intermediates. Background Art

[0002] Trilaciclib is a cyclin-dependent kinase (CDK4 / 6) inhibitor developed by G1 Therapeutics, and is used to reduce myelosuppression in patients with advanced extensive-stage small cell lung cancer undergoing chemotherapy. The drug was approved by the US FDA for marketing on February 12, 2021, and it is the only myeloprotective agent approved by the FDA, with the trade name Cosela. As the first short-acting small molecule CDK4 / 6 inhibitor, after intravenous injection, trilaciclib binds to and inhibits the activity of CDK4 / 6, thereby blocking the phosphorylation of retinoblastoma protein (Rb) in the early G1 phase. This prevents the G1 / S phase transition, causes cell cycle arrest in the G1 phase, induces apoptosis, and inhibits the proliferation of tumor cells with overexpression of CDK4 / 6.

[0003] The chemical name of trilaciclib is 2’-{[5-(4-methylpiperazin-1-yl)pyridin-2-yl]amino}-7',8'-dihydro-6'H-spiro[cyclohexane-1,9'-pyrazino[1',2':1,5]pyrrolo[2,3-d]pyrimidin]-6'-one, and its structural formula is:

[0004]

[0005] Currently, the preparation methods of trilaciclib include the trilaciclib synthesis method reported in its original research patent WO2012061156A1: using benzyl N-[1-(aminomethyl)cyclohexyl]carbamate as the raw material, through Boc protection and debenzylation to obtain the intermediate N-[(1-aminocyclohexyl)methyl]carbamic acid tert-butyl ester, and then reacting with 5-bromo-2',4-dichloropyrimidine through nucleophilic substitution, coupling, cyclization, hydrolysis, oxidation and other reactions to obtain the intermediate 2’-chloro-7',8'-dihydro-6'H-spiro[cyclohexane-1,9'-pyrazino[1',2':1,5]pyrrolo[2,3-d]pyrimidin]-6'-one, and then reacting with 5-(4-methylpiperazin-1-yl)pyridin-2-amine through Buchwald-Hartwig coupling to generate trilaciclib. The yields of each step of this route are not given, the raw materials are not commercially available, and there are few reports on the synthesis method. In addition, column chromatography purification is required for multiple steps of this route, increasing the production difficulty, and further process optimization is needed. The method of coupling reaction to form the pyrrolopyrimidine ring is worth referring to.

[0006] The process route of the original research patent WO2012061156A1 is as follows:

[0007]

[0008] A preparation method of trilaciclib disclosed in Patent WO2018005865A1 uses 4-chloro-2-(methylthio)pyrimidine-5-carbaldehyde as a raw material to undergo a substitution reaction with 1,4-diazaspiro[5.5]undecan-3-one to obtain the intermediate 2-(methylthio)-4-(3-oxo-1,4-diazaspiro[5.5]undec-1-yl)pyrimidine-5-carbaldehyde, which is then subjected to amino protection, cyclization, de-Boc, and oxidation to obtain the intermediate 2'-(methylsulfonyl)-7',8'-dihydro-6'H-spiro[cyclohexane-1,9'-pyrazino[1',2':1,5]pyrrolo[2,3-d]pyrimidin]-6'-one, and then trilaciclib is obtained through Buchwald-Hartwig coupling reaction. The total yield of the route is 12.2%. This synthetic route is relatively short and the reaction conditions are mild. However, the starting material is expensive, and there are many by-products in the first step reaction, making purification difficult and the yield only 30.2%. The product of the second step reaction must be purified by column chromatography, increasing the production difficulty and cost.

[0009]

[0010] The author improved this route. Using ethyl 4-chloro-2-(methylthio)pyrimidine-5-carboxylate as a raw material, it undergoes a substitution reaction with 1,4-diazaspiro[5.5]undecan-3-one to obtain the intermediate ethyl 2-(methylthio)-4-(3-oxo-1,4-diazaspiro[5.5]undec-1-yl)pyrimidine-5-carboxylate, and then trilaciclib is obtained through a total of eight steps of reaction including amino protection, cyclization, sulfonation, reduction, de-Boc, oxidation, and coupling. The total yield of the route is 16.8%. Using ethyl 4-chloro-2-(methylthio)pyrimidine-5-carboxylate instead of 4-chloro-2-(methylthio)pyrimidine-5-carbaldehyde as the starting material reduces the cost. Although the number of reaction steps increases, the selectivity is improved, reducing the generation of by-products, and column chromatography purification is not required for each step of the reaction.

[0011] A preparation method of trilaciclib disclosed in Patent CN114014863 uses methyl 2-(methylthio)-7H-pyrrolo[2,3-d]pyrimidine-6-carboxylate as a raw material to obtain trilaciclib through substitution, hydrolysis, condensation, and coupling reactions. The total yield of the route is 61.4%. This synthetic route is relatively short, the reaction conditions for each step are mild, column chromatography purification is not required, and it has good yield and purity at the same time. However, the synthesis method of the key intermediate used is not given, and the raw material is not commercially available.

[0012]

[0013] A preparation method of trilaciclib disclosed in Patent CN114014864 uses 1-(aminocyclohexyl)methanamine as a raw material. Through cyclization and amino protection, tert-butyl 3-oxo-1,4-diazaspiro[5.5]undecane-4-carboxylate is obtained; using uracil as a raw material, through chlorination and formylation reactions, 2,4-dichloropyrimidine-5-carbaldehyde is obtained; these two intermediates then undergo substitution, cyclization, de-Boc, and coupling reactions to obtain trilaciclib, with an overall yield of the route of 42.9%. This synthetic route is shorter and has a higher yield, but the raw materials are expensive, and phosphorus oxychloride is used as a halogenating reagent, which causes greater environmental pollution and requires further process optimization.

[0014]

[0015] A preparation method of trilaciclib disclosed in Patent CN115477653 uses 2-amino-4-chloropyrimidine-5-carbaldehyde as a raw material. Through amino protection, substitution, cyclization, de-Boc, and coupling, trilaciclib is obtained, with an overall yield of the route of 74.4%. This synthetic route has fewer steps, and the reaction conditions for each step are mild. There is no need to use expensive palladium catalysts and hazardous reagents, but the synthesis method of the key intermediate used is not given, and the raw materials are expensive, making the cost of the whole route high.

[0016] Summary of the Invention

[0017] The object of the present invention is to provide a preparation method of trilaciclib and its intermediates that is environmentally friendly, low-cost, easy to operate, and suitable for industrial production. The main raw materials involved are 5-bromo-2,4-dichloropyrimidine, pyruvic acid, bromoacetonitrile, 1,5-dibromopentane, and 5-bromo-2-nitropyridine. Compared with the synthetic method reported in the literature that first introduces a spiro ring and then forms a pyrrolopyrimidine ring, the route of the present invention selects commercially available 5-bromo-2,4-dichloropyrimidine as the starting material, which is inexpensive and easily available, reducing the synthesis cost. Moreover, the reaction conditions for each step are mild, and the intermediates and end products are easy to purify and store. And this method uses a new intermediate for synthetic research and has the potential for industrial production.

[0018] The technical solution of the present invention is achieved through the following technical solutions:

[0019] An intermediate of trilaciclib of the present invention is as shown in the following formula, which is ethyl 2-chloro-7-cyanomethyl-7H-pyrrolo[2,3-d]pyrimidine-6-carboxylate, and its structural formula is:

[0020]

[0021] The preparation method of the trilaciclib intermediate includes using 5-bromo-2,4-dichloropyrimidine as a raw material, reacting with ammonia water, followed by coupling, esterification, and N-alkylation to obtain the trilaciclib intermediate ethyl 2-chloro-7-cyanomethyl-7H-pyrrolo[2,3-d]pyrimidine-6-carboxylate.

[0022] The preparation method of the ethyl 2-chloro-7-cyanomethyl-7H-pyrrolo[2,3-d]pyrimidine-6-carboxylate specifically includes the following steps:

[0023] Step (1): Mix and react 5-bromo-2,4-dichloropyrimidine ② with ammonia water to prepare 4-amino-5-bromo-2-chloropyrimidine ③;

[0024] Step (2): Carry out a coupling reaction between 4-amino-5-bromo-2-chloropyrimidine ③ and pyruvic acid to obtain 2-chloro-7H-pyrrolo[2,3-d]pyrimidine-6-carboxylic acid ④;

[0025] Step (3): Carry out an esterification reaction between 2-chloro-7H-pyrrolo[2,3-d]pyrimidine-6-carboxylic acid ④ and ethanol to obtain ethyl 2-chloro-7H-pyrrolo[2,3-d]pyrimidine-6-carboxylate ⑤;

[0026] Step (4): React ethyl 2-chloro-7H-pyrrolo[2,3-d]pyrimidine-6-carboxylate ⑤ with a halogenating reagent to obtain ethyl 2-chloro-7-cyanomethyl-7H-pyrrolo[2,3-d]pyrimidine-6-carboxylate ⑥.

[0027] In the said step (1), by molar ratio, 5-bromo-2,4-dichloropyrimidine ②: ammonia water = 1:(1 - 5), more preferably 1:(2 - 4);

[0028] In the said step (1), in the mixing reaction of 5-bromo-2,4-dichloropyrimidine ② and ammonia water, it is carried out in a solvent system, and the solvent used is one or several of methanol, ethanol, water, dichloromethane, tetrahydrofuran, acetonitrile, and ethyl acetate, preferably tetrahydrofuran. By mass-volume ratio, 5-bromo-2,4-dichloropyrimidine ②: solvent = 1:(3 - 10), preferably 1:(3 - 5);

[0029] In the said step (1), the reaction time is 4 - 10 hours, preferably 5 - 8 hours; the reaction temperature is 0 - 50 °C, preferably 20 - 40 °C.

[0030] In the said step (2), a catalyst is used to catalyze the reaction, and the catalyst is palladium metal, commonly Pd(OAc)2, Pd2(dba)3, Pd(dba)2, preferably Pd(OAc)2;

[0031] In the said step (2), in terms of molar ratio, 4-amino-5-bromo-2-chloropyrimidine ③: palladium metal = 1: (0.01 - 0.10), preferably 1: (0.03 - 0.05);

[0032] In the said step (2), for the coupling reaction between 4-amino-5-bromo-2-chloropyrimidine and pyruvic acid, DABCO also needs to be added. In terms of molar ratio, 4-amino-5-bromo-2-chloropyrimidine ③: pyruvic acid: DABCO = 1: (2 - 5): (2 - 5), preferably 1: (3 - 4): (3 - 4);

[0033] In the said step (2), the coupling of 4-amino-5-bromo-2-chloropyrimidine ③ and pyruvic acid is carried out in a solvent system. The solvents used are 1,4-dioxane, acetonitrile, toluene, N,N-dimethylformamide or N,N-dimethylacetamide, preferably N,N-dimethylformamide. In terms of mass-volume ratio, 4-amino-5-bromo-2-chloropyrimidine ③: solvent = 1: (3 - 10), preferably 1: (4 - 5);

[0034] In the said step (2), the reaction time is 13 - 18 hours, preferably 14 - 16 hours; the reaction temperature is 80 - 120 °C, preferably 100 - 110 °C.

[0035] In the said step (3), for the esterification reaction system, there are reactions of carboxylic acid and alcohol under acid catalysis, esterification of carboxylic acid and alcohol under the conditions of DCC and DMAP, reaction of carboxylic acid with thionyl chloride or oxalyl chloride to form acyl chloride and then esterification with alcohol. Preferably, the reaction of carboxylic acid with thionyl chloride to form acyl chloride and then esterification with alcohol;

[0036] In the said step (3), in terms of molar ratio, 2-chloro-7H-pyrrolo[2,3-d]pyrimidine-6-carboxylic acid ④: thionyl chloride or oxalyl chloride: ethanol = 1: (1.1 - 1.5): (3 - 10), preferably 1: (1.3 - 1.5): (5 - 8);

[0037] In the said step (3), the reaction time is 1 - 6 hours, preferably 3 - 5 hours; the reaction temperature is 0 - 80 °C, preferably 20 - 50 °C.

[0038] In the said step (4), ethyl 2-chloro-7H-pyrrolo[2,3-d]pyrimidine-6-carboxylate ⑤ reacts with a halogenating reagent under alkaline conditions. The bases used are one or a mixture of several of cesium carbonate, potassium carbonate, sodium carbonate, sodium hydride, triethylamine, N,N-diisopropylethylamine, preferably sodium hydride; the halogenating reagent is preferably bromoacetonitrile;

[0039] In the said step (4), by molar ratio, ethyl 2-chloro-7H-pyrrolo[2,3-d]pyrimidine-6-carboxylate ⑤: halogenating agent: base = 1:(1.1 - 1.5):(1.1 - 1.5), preferably 1:(1.1 - 1.3):(1.1 - 1.3);

[0040] In the said step (4), the reaction time is 1 - 5 hours, preferably 1 - 3 hours; the reaction temperature is 0 - 60 °C, preferably 20 - 40 °C.

[0041] Another intermediate of trilaciclib of the present invention is shown by the following formula, which is ethyl 2-chloro-7-(1-cyanocyclohexyl)-7H-pyrrolo[2,3-d]pyrimidine-6-carboxylate, and its structure is:

[0042]

[0043] On the preparation method of ethyl 2-chloro-7-(1-cyanocyclohexyl)-7H-pyrrolo[2,3-d]pyrimidine-6-carboxylate, the following steps are further included:

[0044] Step (5): React ethyl 2-chloro-7-cyanomethyl-7H-pyrrolo[2,3-d]pyrimidine-6-carboxylate ⑥ with a halide to form ethyl 2-chloro-7-(1-cyanocyclohexyl)-7H-pyrrolo[2,3-d]pyrimidine-6-carboxylate ⑦.

[0045] In the said step (5), the halide is selected from 1,5-dibromopentane, 1,5-dichloropentane, 1,5-diiodopentane, preferably 1,5-dibromopentane;

[0046] In the said step (5), ethyl 2-chloro-7-cyanomethyl-7H-pyrrolo[2,3-d]pyrimidine-6-carboxylate ⑥ reacts with the halide under alkaline conditions. Common bases include sodium hydroxide, sodium ethoxide, potassium tert-butoxide, sodium hydride, preferably sodium hydride;

[0047] In the said step (5), by molar ratio, ethyl 2-chloro-7-cyanomethyl-7H-pyrrolo[2,3-d]pyrimidine-6-carboxylate ⑥: halide: base = 1:(1.1 - 1.5):(2 - 5), preferably 1:(1.1 - 1.3):(3 - 4);

[0048] In the said step (5), the reaction time is 8 - 15 hours, preferably 10 - 13 hours; the reaction temperature is 90 - 120 °C, preferably 100 - 110 °C.

[0049] A preparation method of trilaciclib according to the present invention uses the above-mentioned trilaciclib intermediate ethyl 2-chloro-7-(cyanomethyl)-7H-pyrrolo[2,3-d]pyrimidine-6-carboxylate ⑥ and ethyl 2-chloro-7-(1-cyanocyclohexyl)-7H-pyrrolo[2,3-d]pyrimidine-6-carboxylate ⑦, and further includes the following steps:

[0050] Step (6): Ethyl 2-chloro-7-(1-cyanocyclohexyl)-7H-pyrrolo[2,3-d]pyrimidine-6-carboxylate ⑦ undergoes cyclization under a sodium borohydride and Lewis acid system to form 2'-chloro-7',8'-dihydro-6'H-spiro[cyclohexane-1,9'-pyrazinone[1',2':1,5]pyrrolo[2,3-d]pyrimidine]-6'-one ⑧;

[0051] Step (7): 2'-Chloro-7',8'-dihydro-6'H-spiro[cyclohexane-1,9'-pyrazinone[1',2':1,5]pyrrolo[2,3-d]pyrimidine]-6'-one ⑧ undergoes a coupling reaction with 5-(4-methylpiperazin-1-yl)pyridin-2-amine to form trilaciclib ①.

[0052] In the above-mentioned step (6): The Lewis acid is selected from ferric chloride, aluminum chloride, cobalt chloride, boron trifluoride, and preferably cobalt chloride;

[0053] In the above-mentioned step (6): By molar ratio, ethyl 2-chloro-7-(1-cyanocyclohexyl)-7H-pyrrolo[2,3-d]pyrimidine-6-carboxylate ⑦: Lewis acid: sodium borohydride = 1:(1-4):(5-12), and preferably 1:(2-3):(8-10);

[0054] In the above-mentioned step (6): The reaction time is 6 to 12 hours, preferably 8 to 10 hours; the reaction temperature is 30 to 70 °C, preferably 50 to 60 °C.

[0055] In the above-mentioned step (7): The coupling of 2'-chloro-7',8'-dihydro-6'H-spiro[cyclohexane-1,9'-pyrazinone[1',2':1,5]pyrrolo[2,3-d]pyrimidine]-6'-one ⑧ and 5-(4-methylpiperazin-1-yl)pyridin-2-amine is carried out under palladium catalysis. The catalyst and ligand do not actually have a fixed combination, but common combinations include Pd(OAc)2 / BINAP, Pd2(dba)3 / Xantphos, Pd(dba)2 / t-Bu3P, and preferably Pd(OAc)2 / BINAP;

[0056] In the above-mentioned step (7): In the coupling reaction, a strong base is crucial for the catalytic cycle. Commonly used bases include Cs2CO3, NaOH, t-BuONa, t-BuOK, and preferably t-BuONa;

[0057] In step (7): by molar ratio, 2'-chloro-7',8'-dihydro-6'H-spiro[cyclohexane-1,9'-pyrazino[1',2':1,5]pyrrolo[2,3-d]pyrimidin]-6'-one ⑧: 5-(4-methylpiperazin-1-yl)pyridin-2-amine: t-BuONa = 1:(1.1 - 1.5):(1.1 - 3.0), preferably 1:(1.1 - 1.3):(1.5 - 2.0);

[0058] In step (7): the coupling is carried out in a solvent system, and the solvent used is selected from 1,4-dioxane, acetonitrile, toluene, N,N-dimethylformamide, N,N-dimethylacetamide, more preferably 1,4-dioxane. By mass-volume ratio, 2'-chloro-7',8'-dihydro-6'H-spiro[cyclohexane-1,9'-pyrazino[1',2':1,5]pyrrolo[2,3-d]pyrimidin]-6'-one ⑧: solvent = 1:(2 - 8), preferably 1:(3 - 5);

[0059] In step (7): the reaction time is 7 - 15 hours, preferably 8 - 10 hours; the reaction temperature is 80 - 110 °C, preferably 100 - 110 °C.

[0060] The reaction equation involved in the above steps is:

[0061]

[0062] A preparation method of trilaciclib and its intermediate of the present invention has the beneficial effects that:

[0063] The present invention provides a preparation method of trilaciclib and its key intermediate compound. Trilaciclib is synthesized by a novel preparation method, which includes the following steps: using 5-bromo-2,4-dichloropyrimidine ② as the raw material, synthesizing 2'-chloro-7',8'-dihydro-6'H-spiro[cyclohexane-1,9'-pyrazino[1',2':1,5]pyrrolo[2,3-d]pyrimidin]-6'-one ⑧ through 6 steps of reactions, and then carrying out Buchwald-Hartwig coupling reaction with 5-(4-methylpiperazin-1-yl)pyridin-2-amine to generate trilaciclib ①. The synthetic route of the present invention selects commercially available 5-bromo-2,4-dichloropyrimidine as the starting material, which is inexpensive and easily available, reducing the synthesis cost. And the reaction conditions of each step are mild, and the intermediate and the final product are easy to purify and preserve. Moreover, the method of the present invention uses a new intermediate for synthetic research and has the potential for industrial production. Specific Embodiments

[0064] The following further illustrates the present invention with reference to embodiments, but is not limited thereto.

[0065] Example 1: Preparation of 4-Amino-5-bromo-2-chloropyrimidine ③

[0066] Dissolve 25.00 g (109.71 mmol) of 5-bromo-2,4-dichloropyrimidine in 50 mL of tetrahydrofuran and add it to a single-necked flask equipped with a magnetic stirrer. Then slowly add dropwise 11 mL (274.28 mmol) of 25% aqueous ammonia. After addition, react at room temperature for 6 hours and then stop the reaction. Concentrate the tetrahydrofuran under reduced pressure, extract with ethyl acetate (100 mL × 3), combine the organic phases, and concentrate under reduced pressure to obtain 18.78 g of a white solid with a yield of 82.3%. ESI-MS m / z: 209.88 [M+H] + 。

[0067] Example 2: Preparation of 2-Chloro-7H-pyrrolo[2,3-d]pyrimidine-6-carboxylic acid ④

[0068] Add 15.00 g (71.96 mmol) of 4-amino-5-bromo-2-chloropyrimidine and 280 mL of anhydrous DMF to a 500 mL three-necked flask equipped with a thermometer, a constant pressure dropping funnel, a magnetic stirrer, and a condenser. Stir to dissolve it, add 24.22 g (215.88 mmol) of DABCO in batches, stir for 20 min to dissolve it, and degas with argon for 10 min. Slowly add dropwise 15.2 mL (215.88 mmol) of pyruvic acid. After addition, degas with argon for 10 min. Add 0.81 g (3.60 mmol) of palladium acetate. Raise the temperature to 110 °C and continue the reaction for 15 hours. After the reaction is completed, concentrate the DMF under reduced pressure, extract with ethyl acetate (50 mL × 3), combine the organic phases, concentrate under reduced pressure, and adjust the pH to 2 - 3 with concentrated hydrochloric acid to obtain 9.35 g of a yellow solid with a yield of 65.8%. ESI-MS m / z: 198.69 [M+H] + 。

[0069] Example 3: Preparation of Ethyl 2-Chloro-7H-pyrrolo[2,3-d]pyrimidine-6-carboxylate ⑤

[0070] 10.00 g (50.76 mmol) of 2-chloro-7H-pyrrolo[2,3-d]pyrimidine-6-carboxylic acid and 100 mL of dichloromethane solvent were added to a single-necked flask equipped with a magnetic stirrer. 4.5 mL (60.99 mmol) of thionyl chloride and 0.1 mL of anhydrous DMF were added as catalysts respectively. After addition, the reaction was carried out at room temperature for 3 hours and then stopped. The reaction mixture was concentrated under reduced pressure. After adding a small amount of dichloromethane solution to form a suspension, it was dropped into 9 mL (152.28 mmol) of absolute ethanol. The reaction was significantly exothermic and was controlled below 0 °C in a low-temperature cold trap. After dropping, the reaction was carried out at room temperature for 2 hours. The reaction was completed and the reaction was stopped. The solvent was evaporated, and the residue was extracted with ethyl acetate (30 mL × 3). The organic phases were combined and washed with saturated brine. Dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain a pale yellow solid. After dissolving in 20 mL of dichloromethane, 10 mL of n-hexane was added to precipitate a white solid. The solid was filtered by suction, and the filter cake was dried in vacuo to obtain 12.42 g of a white solid, with a yield of 92.7%, ESI-MS m / z: 226.01 [M+H] + 。

[0071] Example 4: Preparation of ethyl 2-chloro-7-cyanomethyl-7H-pyrrolo[2,3-d]pyrimidine-6-carboxylate ⑥

[0072] 15.00 g (66.67 mmol) of ethyl 2-chloro-7H-pyrrolo[2,3-d]pyrimidine-6-carboxylate and 150 mL of anhydrous DMF were added to a single-necked flask equipped with a magnetic stirrer. 3.47 g (86.67 mmol) of 60% sodium hydride and 5 mL (73.33 mmol) of bromoacetonitrile were added at 0 °C. After addition, the reaction was carried out at room temperature for 1 hour and then stopped. The reaction mixture was concentrated under reduced pressure. Extracted with ethyl acetate (50 mL × 3). The organic phases were combined and washed with saturated brine. Dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain a yellow solid. After dissolving in 25 mL of dichloromethane, 10 mL of n-hexane was added to precipitate a solid. The solid was filtered by suction, and the filter cake was dried in vacuo to obtain 16.47 g of a pale yellow solid, with a yield of 93.6%, ESI-MS m / z: 265.02 [M+H] + 。

[0073] Example 5: Preparation of ethyl 2-chloro-7-(1-cyanocyclohexyl)-7H-pyrrolo[2,3-d]pyrimidine-6-carboxylate ⑦

[0074] 15.00 g (56.82 mmol) of ethyl 2-chloro-7-(cyanomethyl)-7H-pyrrolo[2,3-d]pyrimidine-6-carboxylate and 150 mL of anhydrous DMF were added to a single-necked flask equipped with magnetic stirring. 6.82 g (170.46 mmol) of 60% sodium hydride and 10 mL (73.87 mmol) of 1,5-dibromopentane were added respectively. After the addition was completed, the reaction was carried out at 110 °C for 13 hours. Then the reaction ended and the reaction was stopped. The reaction mixture was concentrated under reduced pressure, extracted with ethyl acetate (50 mL × 3), the organic phases were combined, and washed with saturated brine. Dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain a yellow solid. Recrystallized from 20 mL of ethyl acetate to obtain 12.77 g of a pale yellow solid, with a yield of 67.5%, ESI-MS m / z: 356.04 [M+Na] + 。

[0075] Example 6: Preparation of 2'-chloro-7',8'-dihydro-6'H-spiro[cyclohexane-1,9'-pyrazino[1',2':1,5]pyrrolo[2,3-d]pyrimidine]-6'-one ⑧

[0076] A mixed solution of 15.00 g (45.05 mmol) of ethyl 2-chloro-7-(1-cyanocyclohexyl)-7H-pyrrolo[2,3-d]pyrimidine-6-carboxylate, 100 mL of tetrahydrofuran and 50 mL of methanol was added to a single-necked flask equipped with magnetic stirring. 11.70 g (90.10 mmol) of anhydrous cobalt chloride and 17.04 g (450.50 mmol) of sodium borohydride were added respectively at 0 °C. After the addition was completed, the reaction was refluxed for 8 hours. Then the reaction ended and the reaction was stopped. The pH was adjusted to 5 with 1 mol / L hydrochloric acid, concentrated under reduced pressure, extracted with ethyl acetate (50 mL × 3), the organic layers were combined, and washed with saturated brine. Dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain a pale yellow solid. Recrystallized from 15 mL of ethyl acetate to obtain 8.20 g of a white solid, with a yield of 62.8%, ESI-MS m / z: 291.15 [M+H] + 。

[0077] Example 7: Preparation of Trilaciclib ①

[0078] 5.00 g (17.20 mmol) of 2'-chloro-7',8'-dihydro-6'H-spiro[cyclohexane-1,9'-pyridazino[1',2':1,5]pyrrolo[2,3-d]pyrimidin]-6'-one and 100 mL of 1,4-dioxane were added to a single-necked flask equipped with a magnetic stirrer. 4.30 g (22.36 mmol) of 5-(4-methylpiperazin-1-yl)pyridin-2-amine and 3.30 g (34.39 mmol) of sodium tert-butoxide were added. After degassing with argon for 10 min, 0.39 g (1.72 mmol) of palladium acetate and 1.07 g (1.72 mmol) of BINAP were added respectively. After the addition was completed, the reaction was carried out at 110 °C for 10 hours and then stopped. After cooling to room temperature, the reaction solution was poured into water to precipitate a pale yellow solid. It was slurried with 10 mL of ethyl acetate, filtered by suction, and the filter cake was dried in vacuo to obtain 5.90 g of a white solid, with a yield of 76.8%, mp: >300 °C, ESI-MS m / z: 447.53 [M+H] + 。 1 H NMR (600 MHz, DMSO-d6) δ 9.55 (s, 1H), 8.85 (s, 1H), 8.15 (s, 1H), 8.08 (d, J = 9.1 Hz, 1H), 8.02 (s, 1H), 7.43 - 7.41 (m, 1H), 7.06 (s, 1H), 3.64 (s, 2H), 3.13 (s, 4H), 2.93 - 2.88 (m, 2H), 2.47 (s, 4H), 2.23 (s, 3H), 1.88 (d, J = 12.8 Hz, 2H), 1.79 - 1.74 (m, 3H), 1.49 - 1.42 (m, 2H), 1.39 - 1.34 (m, 1H).

[0079] 13 C NMR (151 MHz, DMSO-d6) δ 160.34, 155.63, 154.05, 151.41, 151.06, 138.71, 136.80, 130.76, 113.51, 112.42, 110.99, 108.96, 59.40, 57.55 (2C), 55.31 (2C), 54.80, 45.58, 31.87 (2C), 23.96, 22.66 (2C).

[0080] A series of new intermediates ⑥ and ⑦ were used in the present invention to synthesize Trilaciclib ①. In this route, commercially available 5-bromo-2,4-dichloropyrimidine was selected as the starting material, which is inexpensive and easily available, reducing the synthesis cost. Moreover, the reaction conditions for each step are mild, and the intermediates and the final product are easy to purify and preserve.

[0081] The synthesis routes of Examples 1 - 7 above are as follows:

[0082]

[0083] Example 8:

[0084] A preparation method of trilaciclib and the key intermediates involved, including the following process steps:

[0085] (1) 5-Bromo-2,4-dichloropyrimidine reacts with ammonia water at room temperature for 6 hours to prepare 4-amino-5-bromo-2-chloropyrimidine ③; by molar ratio, 5-bromo-2,4-dichloropyrimidine ②: ammonia water = 1:3; compared with Example 1, the solvent used in this example is acetonitrile.

[0086] (2) Mix the 4-amino-5-bromo-2-chloropyrimidine ③ generated in step (1) with pyruvic acid and DABCO, and heat to 110 °C for reaction for 15 h to generate 2-chloro-7H-pyrrolo[2,3-d]pyrimidine-6-carboxylic acid ④; by molar ratio, 4-amino-5-bromo-2-chloropyrimidine ③: pyruvic acid: DABCO = 1:3:3, and the catalyst used in this example is Pd2(dba)3.

[0087] (3) After the 2-chloro-7H-pyrrolo[2,3-d]pyrimidine-6-carboxylic acid ④ generated in step (2) reacts with oxalyl chloride to form an acyl chloride, it reacts with ethanol at room temperature for 2 h to generate ethyl 2-chloro-7H-pyrrolo[2,3-d]pyrimidine-6-carboxylate ⑤; by molar ratio, 2-chloro-7H-pyrrolo[2,3-d]pyrimidine-6-carboxylic acid ④: oxalyl chloride: ethanol = 1:1.2:3, and the oxalyl chloride is used in this example.

[0088] (4) The ethyl 2-chloro-7H-pyrrolo[2,3-d]pyrimidine-6-carboxylate ⑤ generated in step (3) reacts with bromoacetonitrile at room temperature for 1 hour to generate ethyl 2-chloro-7-cyanomethyl-7H-pyrrolo[2,3-d]pyrimidine-6-carboxylate ⑥; by molar ratio, ethyl 2-chloro-7H-pyrrolo[2,3-d]pyrimidine-6-carboxylate ⑤: bromoacetonitrile: potassium carbonate = 1:1.1:1.5, and the base used in this example is potassium carbonate.

[0089] (5) The ethyl 2-chloro-7-cyanomethyl-7H-pyrrolo[2,3-d]pyrimidine-6-carboxylate ⑥ generated in step (4) reacts with 1,5-dichloropentane at 110 °C for 13 hours to generate ethyl 2-chloro-7-(1-cyanocyclohexyl)-7H-pyrrolo[2,3-d]pyrimidine-6-carboxylate ⑦; by molar ratio, ethyl 2-chloro-7-cyanomethyl-7H-pyrrolo[2,3-d]pyrimidine-6-carboxylate ⑥: 1,5-dichloropentane: sodium hydride = 1:1.3:3, and the solvent used in this example is N,N-dimethylacetamide.

[0090] (6) React the ethyl 2-chloro-7-(1-cyanocyclohexyl)-7H-pyrrolo[2,3-d]pyrimidine-6-carboxylate ⑦ generated in step (5) with sodium borohydride and cobalt chloride hexahydrate at 70 °C for 8 hours to form 2'-chloro-7',8'-dihydro-6'H-spiro[cyclohexane-1,9'-pyrazino[1',2':1,5]pyrrolo[2,3-d]pyrimidine]-6'-one ⑧. By molar ratio, ethyl 2-chloro-7-(1-cyanocyclohexyl)-7H-pyrrolo[2,3-d]pyrimidine-6-carboxylate ⑦ : cobalt chloride hexahydrate : sodium borohydride = 1 : 2 : 10. The solvent used in this example is tetrahydrofuran-methanol.

[0091] (7) React 2'-chloro-7',8'-dihydro-6'H-spiro[cyclohexane-1,9'-pyrazino[1',2':1,5]pyrrolo[2,3-d]pyrimidine]-6'-one ⑧ generated in step (6) with 5-(4-methylpiperazin-1-yl)pyridin-2-amine and potassium tert-butoxide at 110 °C for 10 h to form trilaciclib ①. The solvent used in this example is toluene.

[0092] Example 9:

[0093] The preparation of trilaciclib comprises the following process steps:

[0094] (1) React 5-bromo-2,4-dichloropyrimidine ② with ammonia water to prepare 4-amino-5-bromo-2-chloropyrimidine ③;

[0095] In this example, the molar ratio of 5-bromo-2,4-dichloropyrimidine ② to ammonia water is 1:4;

[0096] The reaction is carried out in a solvent system, and the solvent is ethyl acetate;

[0097] The reaction time is 5 hours, and the reaction temperature is 40 °C.

[0098] (2) Carry out a coupling reaction on 4-amino-5-bromo-2-chloropyrimidine ③ generated in step (1) with pyruvic acid and DABCO to form 2-chloro-7H-pyrrolo[2,3-d]pyrimidine-6-carboxylic acid ④;

[0099] In this example, the molar ratio of 4-amino-5-bromo-2-chloropyrimidine ③ to pyruvic acid and DABCO is 1:4:4;

[0100] The reaction is carried out in a solvent system, and the solvent is 1,4-dioxane;

[0101] The reaction time is 14 hours, and the reaction temperature is 100 °C.

[0102] (3) React the 2-chloro-7H-pyrrolo[2,3-d]pyrimidine-6-carboxylic acid ④ generated in step (2) with thionyl chloride to form an acyl chloride, and then react with ethanol to form ethyl 2-chloro-7H-pyrrolo[2,3-d]pyrimidine-6-carboxylate ⑤;

[0103] In this example, the molar ratio of 2-chloro-7H-pyrrolo[2,3-d]pyrimidine-6-carboxylic acid ④, thionyl chloride and ethanol is 1:1.5:8;

[0104] The reaction is carried out in a solvent system, and the solvent is dichloromethane;

[0105] The reaction time is 1 hour and the reaction temperature is 40 °C.

[0106] (4) React ethyl 2-chloro-7H-pyrrolo[2,3-d]pyrimidine-6-carboxylate ⑤ generated in step (3) with bromoacetonitrile to form ethyl 2-chloro-7-cyanomethyl-7H-pyrrolo[2,3-d]pyrimidine-6-carboxylate ⑥;

[0107] In this example, the molar ratio of ethyl 2-chloro-7H-pyrrolo[2,3-d]pyrimidine-6-carboxylate ⑤, bromoacetonitrile and sodium hydride is 1:1.2:1.3;

[0108] The reaction is carried out in a solvent system, and the solvent is N,N-dimethylacetamide;

[0109] The reaction time is 1 hour and the reaction temperature is room temperature.

[0110] (5) React ethyl 2-chloro-7-cyanomethyl-7H-pyrrolo[2,3-d]pyrimidine-6-carboxylate ⑥ generated in step (4) with 1,5-dibromopentane to form ethyl 2-chloro-7-(1-cyanocyclohexyl)-7H-pyrrolo[2,3-d]pyrimidine-6-carboxylate ⑦;

[0111] In this example, the molar ratio of ethyl 2-chloro-7-cyanomethyl-7H-pyrrolo[2,3-d]pyrimidine-6-carboxylate ⑥, 1,5-dibromopentane and sodium hydride is 1:1.3:4;

[0112] The reaction is carried out in a solvent system, and the solvent is N,N-dimethylacetamide;

[0113] The reaction time is 10 hours and the reaction temperature is 110 °C.

[0114] (6) React the ethyl 2-chloro-7-(1-cyanocyclohexyl)-7H-pyrrolo[2,3-d]pyrimidine-6-carboxylate ⑦ generated in step (5) with sodium borohydride and cobalt chloride hexahydrate to form 2'-chloro-7',8'-dihydro-6'H-spiro[cyclohexane-1,9'-pyrazino[1',2':1,5]pyrrolo[2,3-d]pyrimidine]-6'-one ⑧;

[0115] In this example, the molar ratio of ethyl 2-chloro-7-(1-cyanocyclohexyl)-7H-pyrrolo[2,3-d]pyrimidine-6-carboxylate ⑦ to cobalt chloride and sodium borohydride is 1:3:10;

[0116] The reaction is carried out in a solvent system, and the solvent is tetrahydrofuran-methanol (v / v = 1:1);

[0117] The reaction time is 9 hours, and the reaction temperature is 60 °C.

[0118] (7) React 2'-chloro-7',8'-dihydro-6'H-spiro[cyclohexane-1,9'-pyrazino[1',2':1,5]pyrrolo[2,3-d]pyrimidine]-6'-one ⑧ generated in step (6) with 5-(4-methylpiperazin-1-yl)pyridin-2-amine and sodium tert-butoxide to prepare trilaciclib ①;

[0119] In this example, the molar ratio of 2'-chloro-7',8'-dihydro-6'H-spiro[cyclohexane-1,9'-pyrazino[1',2':1,5]pyrrolo[2,3-d]pyrimidine]-6'-one ⑧ to 5-(4-methylpiperazin-1-yl)pyridin-2-amine and sodium tert-butoxide is 1:1.3:2;

[0120] The reaction is carried out in a solvent system, and the solvent is N,N-dimethylformamide;

[0121] The reaction time is 10 hours, and the reaction temperature is 110 °C.

[0122] The above embodiments are only for illustrating the technical concept and characteristics of the present invention, and the purpose is to enable those skilled in the art to understand the content of the present invention and implement it. It is not intended to limit the protection scope of the present invention. Any equivalent changes or modifications made according to the spirit of the present invention should be covered within the protection scope of the present invention.

Claims

1. A preparation method of trilaciclib, characterized in that, Comprising the following steps: Step (1): Ethyl 2-chloro-7-(1-cyanocyclohexyl)-7H-pyrrolo[2,3-d]pyrimidine-6-carboxylate (7) undergoes cyclization under a sodium borohydride and Lewis acid system to form 2'-chloro-7',8'-dihydro-6'H-spiro[cyclohexane-1,9'-pyrazino[1',2':1,5]pyrrolo[2,3-d]pyrimidin]-6'-one (8); Step (2): 2'-Chloro-7',8'-dihydro-6'H-spiro[cyclohexane-1,9'-pyrazino[1',2':1,5]pyrrolo[2,3-d]pyrimidin]-6'-one (8) undergoes a coupling reaction with 5-(4-methylpiperazin-1-yl)pyridin-2-amine to form Trilaciclib (1); 2. The preparation method of Trilaciclib according to claim 1, characterized in that, In the said step (1): The Lewis acid is selected from ferric chloride, aluminum chloride, cobalt chloride, boron trifluoride; by molar ratio, ethyl 2-chloro-7-(1-cyanocyclohexyl)-7H-pyrrolo[2,3-d]pyrimidine-6-carboxylate (7): Lewis acid: sodium borohydride = 1:(1 - 4):(5 - 12).

3. The preparation method of Trilaciclib according to claim 1, characterized in that, In the said step (1): The reaction time is 6 to 12 hours, and the reaction temperature is 30 to 70 °C.

4. The preparation method of Trilaciclib according to claim 1, characterized in that In the said step (2): The coupling reaction of 2'-chloro-7',8'-dihydro-6'H-spiro[cyclohexane-1,9'-pyrazino[1',2':1,5]pyrrolo[2,3-d]pyrimidin]-6'-one (8) with 5-(4-methylpiperazin-1-yl)pyridin-2-amine is carried out under palladium catalysis, and the catalyst and ligand combination is selected from Pd(OAc)2 / BINAP, Pd2(dba)3 / Xantphos, Pd(dba)2 / t-Bu3P; a base also needs to be added in the coupling reaction, and the base is selected from Cs2CO3, NaOH, t-BuONa, t-BuOK; by molar ratio, 2'-chloro-7',8'-dihydro-6'H-spiro[cyclohexane-1,9'-pyrazino[1',2':1,5]pyrrolo[2,3-d]pyrimidin]-6'-one (8): 5-(4-methylpiperazin-1-yl)pyridin-2-amine: base = 1:(1.1 - 1.5):(1.1 - 3.0).

5. The preparation method of Trilaciclib according to claim 1, characterized in that, The preparation method of the said ethyl 2-chloro-7-(1-cyanocyclohexyl)-7H-pyrrolo[2,3-d]pyrimidine-6-carboxylate (7) comprises the following steps: Ethyl 2-chloro-7-cyanomethyl-7H-pyrrolo[2,3-d]pyrimidine-6-carboxylate (6) reacts with a halide to form ethyl 2-chloro-7-(1-cyanocyclohexyl)-7H-pyrrolo[2,3-d]pyrimidine-6-carboxylate (7); the halide is selected from 1,5-dibromopentane, 1,5-dichloropentane, 1,5-diiodopentane; 6. The preparation method of Trilaciclib according to claim 5, characterized in that, Ethyl 2-chloro-7-(cyanomethyl)-7H-pyrrolo[2,3-d]pyrimidine-6-carboxylate (6) reacts with a halide under basic conditions, and the base is selected from sodium hydroxide, sodium ethoxide, potassium tert-butoxide, and sodium hydride; in terms of molar ratio, ethyl 2-chloro-7-(cyanomethyl)-7H-pyrrolo[2,3-d]pyrimidine-6-carboxylate (6):halide:base = 1:(1.1 - 1.5):(2 - 5); the reaction time is 8 to 15 hours, and the reaction temperature is 90 to 120 °C.

7. The preparation method of trilaciclib according to claim 5, wherein, The preparation method of the described ethyl 2-chloro-7-(cyanomethyl)-7H-pyrrolo[2,3-d]pyrimidine-6-carboxylate (6) specifically includes the following steps: Step (1): Mix 5-bromo-2,4-dichloropyrimidine (2) with ammonia water for reaction to prepare 4-amino-5-bromo-2-chloropyrimidine (3); Step (2): Perform a coupling reaction on 4-amino-5-bromo-2-chloropyrimidine (3) and pyruvic acid to obtain 2-chloro-7H-pyrrolo[2,3-d]pyrimidine-6-carboxylic acid (4); Step (3): Perform an esterification reaction on 2-chloro-7H-pyrrolo[2,3-d]pyrimidine-6-carboxylic acid (4) and ethanol to obtain ethyl 2-chloro-7H-pyrrolo[2,3-d]pyrimidine-6-carboxylate (5); Step (4): React ethyl 2-chloro-7H-pyrrolo[2,3-d]pyrimidine-6-carboxylate (5) with bromoacetonitrile to obtain ethyl 2-chloro-7-(cyanomethyl)-7H-pyrrolo[2,3-d]pyrimidine-6-carboxylate (6); 8. The preparation method of trilaciclib according to claim 7, wherein, In the described step (1), in terms of molar ratio, 5-bromo-2,4-dichloropyrimidine (2):ammonia water = 1:(1 - 5); the reaction time is 4 to 10 hours, and the reaction temperature is 0 to 50 °C; in the described step (2), the catalyst is selected from Pd(OAc)2, Pd2(dba)3, Pd(dba)2; in terms of molar ratio, 4-amino-5-bromo-2-chloropyrimidine (3):catalyst = 1:(0.01 - 0.10); when 4-amino-5-bromo-2-chloropyrimidine (3) and pyruvic acid perform a coupling reaction, DABCO also needs to be added, and in terms of molar ratio, 4-amino-5-bromo-2-chloropyrimidine (3):pyruvic acid:DABCO = 1:(2 - 5):(2 - 5); the reaction time is 13 to 18 hours, and the reaction temperature is 80 to 120 °C.

9. The preparation method of Trilaciclib according to claim 7, wherein, In the described step (3), the carboxylic acid reacts with thionyl chloride or oxalyl chloride to form an acyl chloride and then performs an esterification reaction with an alcohol; in terms of molar ratio, 2-chloro-7H-pyrrolo[2,3-d]pyrimidine-6-carboxylic acid (4):thionyl chloride or oxalyl chloride:ethanol = 1:(1.1 - 1.5):(3 - 10); the reaction time is 1 to 6 hours, and the reaction temperature is 0 to 80 °C.

10. The preparation method of trilaciclib according to claim 7, characterized in that, In the step (4), ethyl 2-chloro-7H-pyrrolo[2,3-d]pyrimidine-6-carboxylate (5) reacts with bromoacetonitrile under alkaline conditions, and the base used is selected from cesium carbonate, potassium carbonate, sodium carbonate, sodium hydride, triethylamine, N,N-diisopropylethylamine; by molar ratio, ethyl 2-chloro-7H-pyrrolo[2,3-d]pyrimidine-6-carboxylate (5): bromoacetonitrile: base = 1:(1.1-1.5):(1.1-1.5); the reaction time is 1 to 5 hours, and the reaction temperature is 0 to 60 °C.

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