CN112500356A - 3, 4-dihydropyrimidine benzonitrile derivative, and preparation method and application thereof - Google Patents

3, 4-dihydropyrimidine benzonitrile derivative, and preparation method and application thereof Download PDF

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CN112500356A
CN112500356A CN202011444196.5A CN202011444196A CN112500356A CN 112500356 A CN112500356 A CN 112500356A CN 202011444196 A CN202011444196 A CN 202011444196A CN 112500356 A CN112500356 A CN 112500356A
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compound
concentrating
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formula
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龙承基
徐彬滨
金秉德
朱静
邹斌彬
朱维君
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Hunan Qianjin Xiangjiang Pharmaceutical Co ltd
Zhuzhou Qianjin Pharmaceutical Co Ltd
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Zhuzhou Qianjin Pharmaceutical Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D239/00—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings
    • C07D239/02—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings
    • C07D239/24—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members
    • C07D239/28—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, directly attached to ring carbon atoms
    • C07D239/46—Two or more oxygen, sulphur or nitrogen atoms
    • C07D239/52—Two oxygen atoms
    • C07D239/54—Two oxygen atoms as doubly bound oxygen atoms or as unsubstituted hydroxy radicals
    • C07D239/545—Two oxygen atoms as doubly bound oxygen atoms or as unsubstituted hydroxy radicals with other hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, directly attached to ring carbon atoms
    • C07D239/553—Two oxygen atoms as doubly bound oxygen atoms or as unsubstituted hydroxy radicals with other hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, directly attached to ring carbon atoms with halogen atoms or nitro radicals directly attached to ring carbon atoms, e.g. fluorouracil
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D239/00—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings
    • C07D239/02—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings
    • C07D239/24—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members
    • C07D239/28—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, directly attached to ring carbon atoms
    • C07D239/46—Two or more oxygen, sulphur or nitrogen atoms
    • C07D239/60—Three or more oxygen or sulfur atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
    • C07D401/04—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings directly linked by a ring-member-to-ring-member bond
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation

Abstract

The invention provides a 3, 4-dihydropyrimidine benzonitrile derivative, and a preparation method and application thereof. The invention discloses a structure of a specific impurity (RRT is 1.22 impurity) generated in the process of preparing alogliptin benzoate by adopting a route described in the original patent. The series of impurities have warning structures, and the content of the impurities in the alogliptin benzoate finished product needs to be detected according to the related limit of genotoxic impurities. The invention also provides a preparation and purification method of the compound with high purity as shown in the formula (A) and a detection method of the content of the impurity compound in the alogliptin benzoate medicine, thereby realizing the quality control of the alogliptin benzoate medicine.

Description

3, 4-dihydropyrimidine benzonitrile derivative, and preparation method and application thereof
Technical Field
The invention relates to the field of chemical drugs, in particular to a 3, 4-dihydropyrimidine benzonitrile derivative, and a preparation method and application thereof.
Background
Alogliptin benzoate is a drug for treating type II diabetes based on a DPP-4 inhibitor mechanism. The synthetic route protected in the original patent WO2007035629A2 (figure 1) has the characteristics of cheap and easily obtained raw materials, few reaction steps, simple post-treatment, high product purity and the like. Therefore, the route is generally adopted by domestic industrial mass production.
Multiple batches of tests show that an impurity with an RRT of 1.22 is inevitably generated in the process of preparing the compound 2a by adopting the route, and the content of the impurity is between 0.8 and 1.2 percent. At present, no research report on the impurities exists, and comprehensive and systematic research on the impurities is urgently needed to reduce the content of the impurities in the alogliptin benzoate medicine.
Disclosure of Invention
The invention aims to provide a 3, 4-dihydropyrimidine benzonitrile derivative, and a preparation method and application thereof.
The invention also aims to provide a method for detecting the content of the 3, 4-dihydropyrimidine benzonitrile derivative in the alogliptin benzoate medicine.
In order to achieve the object, the invention provides a 3, 4-dihydropyrimidine benzonitrile derivative, which has a structure shown in formula (A):
Figure BDA0002823658830000011
wherein: r1Selected from the group consisting of-F, -Cl, -Br, -I, -OH, -O- (CH)2)n-CH3A piperidine ring or a 3-aminopiperidine, wherein n is an integer between 0 and 5; preferably-Cl, -OCH3Or 3-aminopiperidine;
R2selected from-H, -F, -Cl, -Br or-I, preferably-Br.
Preferably, the structure of the 3, 4-dihydropyrimidine benzonitrile derivative is shown as the formula (1A):
Figure BDA0002823658830000012
preferably, the structure of the 3, 4-dihydropyrimidine benzonitrile derivative is shown as the formula (2A):
Figure BDA0002823658830000021
preferably, the structure of the 3, 4-dihydropyrimidine benzonitrile derivative is shown as the formula (3A):
Figure BDA0002823658830000022
in a second aspect, the present invention provides a process for the preparation of a compound of formula (A), the synthetic route being shown in FIG. 2. The method comprises the following steps:
(1) reacting a compound of formula (i) with a compound of formula (ii) in the presence of a base to produce a compound of formula (iii);
(2) reacting the compound of formula (iii) with a halogenating agent to give a compound of formula (A).
Wherein the structures of the compounds of formula (i), (ii) and (iii) are respectively as follows:
Figure BDA0002823658830000023
the reaction in the step (1) is carried out in an organic solvent; the organic solvent may be selected from at least one of dichloromethane, toluene, acetonitrile, xylene, N' N-dimethylformamide, dimethyl sulfoxide, etc., preferably toluene or acetonitrile.
The base may be an inorganic base or an organic base; the inorganic base can be at least one selected from sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate and the like, and sodium carbonate is preferred; the organic base can be at least one selected from methylamine, triethylamine, tri-N-butylamine, diisopropylethyl, N-dimethylaminopyridine, N-dimethylaminoaniline and the like, and triethylamine is preferred.
The reaction temperature of the step (1) is 30-110 ℃, and preferably 80 ℃.
The conversion (yield) of the reaction under different conditions in step (1) in the actual development process is shown in table 1.
TABLE 1
Figure BDA0002823658830000024
Figure BDA0002823658830000031
Note: a yield of 0 indicates no chemical reaction under these conditions.
The reaction in the step (2) is carried out in an organic solvent; the organic solvent is at least one selected from solvents such as dichloromethane, dichloroethane, toluene, acetonitrile, xylene, N' N-dimethylformamide, dimethyl sulfoxide, methanol, ethanol, isopropanol, N-butanol, etc., preferably dichloromethane or methanol.
The reaction temperature in the step (2) is 40-100 ℃, and preferably 80 ℃.
The conversion (yield) of the reaction under different conditions in step (2) in the actual development process is shown in table 2.
TABLE 2
Figure BDA0002823658830000032
Note: a yield of 0 indicates no chemical reaction under these conditions.
The molar ratio of the compound of formula (i) to the compound of formula (ii) in step (1) is 1: 1-3.
The molar ratio of the compound in the formula (iii) in the step (2) to the halogenating agent is 1: 1-3.
A process for the preparation of a compound of formula (1A) comprising: 45ml of dichloroethane and 10.9mmol of the compound M1 are added to the reactor, and 13.1mmol of N are added with stirringBS and 0.5mmol AIBN, heating the reaction solution to 40-80 ℃, preserving heat for 2-6h, then cooling to 20-30 ℃ in an ice water bath (preferably, heating the reaction solution to 80 ℃, preserving heat for 3h, then cooling to 20 ℃ in the ice water bath); 30ml of water was added to the reaction system, and after washing with stirring, the lower organic phase was collected and then washed with 15ml of 5% NaHCO3Washing the solution twice, washing the solution once by 15ml of saturated saline solution, collecting a lower organic phase, and drying the lower organic phase by anhydrous sodium sulfate; filtering, concentrating the filtrate under reduced pressure to obtain white solid powder, purifying by column chromatography, gradient eluting with PE and EA mixed solution of different concentrations, collecting eluate, and concentrating to obtain compound of formula (1A).
Wherein, the structure of the compound M1 is as follows:
Figure BDA0002823658830000041
the PE and EA mixed liquids with different concentrations are respectively as follows: PE, EA is 3:1, PE, EA is 2:1, PE, EA is 1: 1.
A process for the preparation of a compound of formula (2A) comprising: 14.1mmol of compound 1A and 40ml of methanol are added into a reactor, stirred to dissolve and clear, and 28.2mmol of K is added2CO3Heating to 40-65 ℃, and reacting for 3-7h under heat preservation (preferably heating to 65 ℃, and reacting for 6h under heat preservation); concentrating the reaction solution under reduced pressure to dryness, adding 40ml of water and 30ml of dichloromethane, stirring, collecting an organic phase, continuously extracting the water phase with 20ml of dichloromethane twice, combining the organic phases, adding anhydrous sodium sulfate, and drying; filtering, concentrating the filtrate under reduced pressure to dryness to obtain off-white solid powder, and pulping with 20ml MTBE at room temperature for 1-3h (preferably 2 h); filtration afforded the compound of formula (2A) as a white solid powder.
A process for the preparation of a compound of formula (3A) comprising: adding 45ml of dichloroethane and 14.7mmol of compound M2 (namely alogliptin) into a reactor, adding 20.8mmol of NBS and 0.7mmol of AIBN under stirring, heating to 40-80 ℃, carrying out heat preservation reaction for 2-6h, then cooling to 20-30 ℃ under ice-water bath (preferably, heating to 80 ℃, carrying out heat preservation reaction for 4h, and then cooling to 20 ℃ under ice-water bath); adding 30ml of water into the reaction solution, stirring and washing, and collecting the lower organic layerThe phases were continued with 15ml of 5% NaHCO3Washing the solution twice, washing the solution once by using 15ml of saturated saline solution, collecting a lower organic phase, and adding anhydrous sodium sulfate for drying; filtering, and concentrating the filtrate under reduced pressure to obtain white solid powder; purifying by column chromatography, gradient eluting with PE and EA mixed solution of different concentrations, collecting eluate, and concentrating to obtain white solid powder of formula (3A) compound.
Wherein, the structure of the compound M2 is as follows:
Figure BDA0002823658830000042
in a third aspect, the invention provides an application of the 3, 4-dihydropyrimidine benzonitrile derivative in the production quality control of alogliptin benzoate medicines.
In a fourth aspect, the present invention provides a method for detecting the content of the compound of formula (a) in alogliptin benzoate, comprising:
1) preparing a sample to be detected into a solution, and adding the solution into a reversed phase high performance liquid chromatography (RP-HPLC);
2) eluting the sample with a mobile phase gradient;
3) detecting the effluent of the chromatographic column by using an ultraviolet detector to detect:
signal peaks corresponding to alogliptin benzoate;
signal peaks corresponding to one or more impurities in the sample to be measured;
4) and calculating the content of the impurities in the sample to be detected according to the peak area of the detected signal peak.
In the step 1), a sample to be detected is prepared into a solution by using a diluent solvent, wherein the diluent solvent is as follows: 0.02-0.03mol/L potassium dihydrogen phosphate solution (pH adjusted to 3.2-4.1 with phosphoric acid) and acetonitrile according to (80-90): (10-20) mixing liquid in volume ratio; preferably, the dilution solvent is: 0.025mol/L potassium dihydrogen phosphate solution (pH adjusted to 4.0 with phosphoric acid) and acetonitrile at a volume ratio of 86: 14.
Chromatographic conditions are as follows:
detection wavelength: 277 nm; flow rate: 1.0 ml/min; column temperature: at 30 ℃. Sample introduction amount: 20 μ l.
Mobile phase used for gradient elution in step 2):
phase A: 0.02-0.03mol/L potassium dihydrogen phosphate solution (pH adjusted to 3.2-4.1 with phosphoric acid), preferably 0.025mol/L potassium dihydrogen phosphate solution (pH adjusted to 4.0 with phosphoric acid);
phase B: and (3) acetonitrile.
The gradient elution procedure is shown in table 3.
TABLE 3
Time (min) A(％) B(％)
0 86 14
30 70 30
50 40 60
60 40 60
By the technical scheme, the invention at least has the following advantages and beneficial effects:
the invention discloses a structure of a specific impurity (RRT is 1.22 impurity) generated in the process of preparing alogliptin benzoate by adopting a route described in the original patent. The series of impurities have warning structures, and the content of the impurities in the alogliptin benzoate finished product needs to be detected according to the related limit of genotoxic impurities. The invention also provides a preparation and purification method of the compound with high purity as shown in the formula (A) and a detection method of the content of the impurity compound in the alogliptin benzoate medicine, thereby realizing the quality control of the alogliptin benzoate medicine.
Drawings
FIG. 1 shows the synthesis of alogliptin benzoate in the original patent in the preferred embodiment of the present invention. RRT1.22 is the relative retention time of the impurity in intermediate product 1.
FIG. 2 shows a scheme for synthesizing the compound of formula (A) according to the present invention.
FIG. 3 is a scheme showing the synthesis of the compound of formula (1A) in a preferred embodiment of the present invention.
FIG. 4 is a scheme showing the synthesis of the compound of formula (2A) in a preferred embodiment of the present invention.
FIG. 5 is a scheme showing the synthesis of the compound of formula (3A) in a preferred embodiment of the present invention.
FIG. 6 shows the HPLC analysis result of the compound of formula (1A) in the preferred embodiment of the present invention. The peak at 41.889min was compound (1A).
FIGS. 7A to 7D are MS analysis results of the compound of formula (1A) in the preferred embodiment of the present invention.
FIG. 8 shows the HNMR analysis of the compound of formula (1A) in a preferred embodiment of the present invention.
FIG. 9 shows the HPLC analysis result of the compound of formula (2A) in the preferred embodiment of the present invention. The peak at 37.634min was compound (2A).
FIGS. 10A and 10B are MS analysis results of the compound of formula (2A) in the preferred embodiment of the present invention.
FIG. 11 shows the results of HNMR analysis of a compound of formula (2A) in a preferred embodiment of the present invention.
FIGS. 12A and 12B are HPLC analysis results of the compound of formula (3A) in a preferred embodiment of the present invention. The peak at 15.426min was compound (3A).
FIGS. 13A to 13D are MS analysis results of the compound of formula (3A) in the preferred embodiment of the present invention.
FIG. 14 shows the HNMR analysis of the compound of formula (3A) in a preferred embodiment of the present invention.
Detailed Description
The terms of the invention:
rrt (relative Retention time), the relative Retention time, is the ratio of the corrected Retention time of a component to the corrected Retention time of the corresponding standard.
NBS: n-bromosuccinimide;
AIBN: azobisisobutyronitrile;
PE: petroleum ether;
EA: ethyl acetate;
MTBE: methyl tert-butyl ether.
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are commercially available products.
The column chromatography is silica gel column, available from Qingdao oceanic plant, and is model No. GF 254.
The compounds of formula (a) prepared in the following examples are shown in table 4:
TABLE 4
Figure BDA0002823658830000061
Example 1 preparation of a compound of formula (1A): 2- ((5-bromo-6-chloro-3-methyl-2, 4-dioxo-3, 4-dihydropyrimidin-1 (2H) -yl) methyl) benzonitrile
The synthetic route for the compound of formula (1A) is shown in FIG. 3. The specific method comprises the following steps:
dichloroethane (45ml) and compound M1(3.00,10.9mmol) were put into a three-necked flask, NBS (2.33g,13.1mmol) and AIBN (0.09g,0.5mmol) were added with stirring, heating was turned on, the reaction solution was heated to 80 ℃ and kept warm for 3h, heating was turned off, and cooling was carried out to 20 ℃ in an ice-water bath. Adding 30ml of water into the reaction solution, stirring and washing, collecting a lower organic phase, and continuing to use 15ml of 5% NaHCO3The solution is washed for 2 times, and then,the organic layer was washed once with 15ml of saturated brine, and the lower organic phase was collected and dried by adding 3g of anhydrous sodium sulfate. Filtering, and concentrating the filtrate under reduced pressure to obtain white solid powder. Purifying by column chromatography, eluting with gradient eluent PE: EA ═ 3:1 → 1:1 (specifically: PE: EA ═ 3:1, PE: EA ═ 2:1 and PE: EA ═ 1:1), collecting the target fraction eluent, and concentrating to obtain 2.97g of the high-purity compound of formula (1A), with a yield of 76.9% and a purity of 92.13%.
After the impurities are separated by preparative liquid chromatography (the HPLC analysis result is shown in figure 6), MS analysis (the MS analysis result is shown in figures 7A-7D) is carried out, the molecular weight and the characteristic functional groups of the impurities are determined, the structure of the impurities is preliminarily deduced, and then the accurate structure of the impurities is analyzed by HPLC positioning and HNMR information (the HNMR analysis result is shown in figure 8).
Example 2 preparation of a compound of formula (2A): 2- ((5-bromo-6-methoxy-3-methyl-2, 4-dioxo-3, 4-dihydropyrimidin-1 (2H) -yl) methyl) benzonitrile
The synthetic route for the compound of formula (2A) is shown in FIG. 4. The specific method comprises the following steps:
adding compound 1A (5g,14.1mmol) and 40ml methanol into a three-neck flask, stirring to dissolve, adding K2CO3(3.90g,28.2 mmol). The heating is started and the reaction is incubated for 6h at 65 ℃. The reaction mixture is concentrated to dryness under reduced pressure, 40ml of water and 30ml of dichloromethane are added, the organic phase is collected after stirring, the aqueous phase is extracted twice with 20ml of dichloromethane, the organic phases are combined, and 5g of anhydrous sodium sulfate is added for drying. Filtration and concentration of the filtrate to dryness under reduced pressure gave an off-white solid powder which was slurried with 20ml of MTBE for 2h at room temperature. Filtration gave 3.73g of a white solid powder of the compound of formula (2A) in 60.6% yield and 95.28% purity. The structure of the compound of formula (2A) is as follows:
Figure BDA0002823658830000071
after the impurities are separated by preparative liquid chromatography (the HPLC analysis result is shown in figure 9), MS analysis (the MS analysis result is shown in figures 10A and 10B) is carried out, the molecular weight and the characteristic functional groups of the impurities are determined, the structures of the impurities are preliminarily deduced, and then the accurate structures of the impurities are analyzed through HPLC positioning and HNMR information by preparing the impurities (the HNMR analysis result is shown in figure 11).
Example 3 preparation of a compound of formula (3A): (R) -2- ((6- (3-aminopiperidin-1-yl) -5-bromo-3-methyl-2, 4-dioxo-3, 4-dihydropyrimidin-1 (2H) -yl) methyl) benzonitrile
The synthetic route for the compound of formula (3A) is shown in FIG. 5. The specific method comprises the following steps:
putting dichloroethane (45ml) and a compound M2(5.00,14.7mmol) into a three-necked bottle, adding NBS (3.70g,20.8mmol) and AIBN (0.09g,0.7mmol) under stirring, starting heating, heating the reaction solution to 80 ℃, keeping the temperature for 4 hours, stopping heating, and cooling to 20 ℃ under ice-water bath. Adding 30ml of water into the reaction solution, stirring and washing, collecting a lower organic phase, and continuing to use 15ml of 5% NaHCO3The solution was washed 2 times, once with 15ml of saturated brine, and the lower organic phase was collected and dried by adding 3g of anhydrous sodium sulfate. Filtering, and concentrating the filtrate under reduced pressure to obtain off-white solid powder 3A. Purification was performed by column chromatography using gradient eluents PE: EA ═ 3:1 → 1:1 (specifically: PE: EA ═ 3:1, PE: EA ═ 2:1 and PE: EA ═ 1:1), and the target fraction eluates were collected and concentrated to give 3.56 g of the compound of formula (3A) as a high-purity white solid powder with a yield of 57.7% and a purity of 93.61%. The structure of the compound of formula (3A) is as follows:
Figure BDA0002823658830000081
the structure of compound M2 (i.e. alogliptin) is as follows:
Figure BDA0002823658830000082
after the impurities are separated by preparative liquid chromatography (HPLC analysis results are shown in fig. 12A and fig. 12B), MS analysis (MS analysis results are shown in fig. 13A-fig. 13D) is carried out, the molecular weight and characteristic functional groups of the impurities are determined, the structures of the impurities are preliminarily deduced, and then the accurate structures of the impurities are analyzed through HPLC positioning and HNMR information by preparing the impurities (HNMR analysis results are shown in fig. 14).
EXAMPLE 4 quantitative detection of impurity 2- ((5-bromo-6-chloro-3-methyl-2, 4-dioxo-3, 4-dihydropyrimidin-1 (2H) -yl) methyl) benzonitrile (1A) in alogliptin benzoate
1. Reagent and reagent: acetonitrile, purified water, monopotassium phosphate and phosphoric acid.
2. Instruments and equipment: an electronic analytical balance, Thermo Hypersil GOLD C18(4.6 × 250mm, 5 μm), high performance liquid chromatography (UV detector), and the like.
3. Chromatographic conditions
1. And calculating results by an HPLC method and an external standard method.
2. A chromatographic column: thermo Hypersil GOLD C18(4.6mm × 250mm, 5 μm), octadecylsilane chemically bonded silica was used as a filler.
The chromatographic conditions were as follows:
detection wavelength: 277 nm; flow rate: 1.0 ml/min; sample introduction amount: 20 mu l of the mixture; column temperature: at 30 ℃.
3. Mobile phase: phase A: 0.025mol/L potassium dihydrogen phosphate solution (pH adjusted to 4.0 with phosphoric acid); phase B: and (3) acetonitrile. The gradient elution procedure is shown in table 3.
4. Solution preparation
An appropriate amount of the pure compound of formula (1A) prepared in example 1 was weighed out precisely, dissolved in an appropriate amount of acetonitrile, and diluted with a diluent solvent to give a solution containing about 0.2. mu.g of the impurity of formula (1A) per 1ml, as a control solution. And taking a proper amount of alogliptin benzoate, precisely weighing, adding a diluting solvent to dissolve, and quantitatively diluting to prepare a solution containing about 5mg of alogliptin benzoate in each 1ml of solution to be used as a test solution.
Diluting the solvent: 0.025mol/L potassium dihydrogen phosphate solution (pH adjusted to 4.0 with phosphoric acid) and acetonitrile at a volume ratio of 86: 14.
5. Measuring method (external standard method)
Precisely measuring 20 μ l of each of the sample solution and the reference solution, respectively injecting into a liquid chromatograph, and recording chromatogram. If the sample solution chromatogram has an impurity 1A peak, the impurity content of the formula (1A) should not exceed 40ppm calculated by the peak area according to an external standard method.
6. Formula for calculation
Figure BDA0002823658830000091
Wherein S isHetero compoundThe area of impurity 1A peak, M, of the test solutionTo pairAs impurity 1A control weight, omega To pair1A reference content, STo pairAs the peak area of 1A in the control solution, MSample (A)Is the sample weight.
The results are put into the formula, with impurity 1A being detected in alogliptin benzoate at maximum 6.2ppm, below 30% (12ppm) of the threshold limit.
EXAMPLE 5 quantitative detection of impurity 2- ((5-bromo-6-methoxy-3-methyl-2, 4-dioxo-3, 4-dihydropyrimidin-1 (2H) -yl) methyl) benzonitrile (2A) in alogliptin benzoate
1. Reagent and reagent: acetonitrile, purified water, monopotassium phosphate and phosphoric acid.
2. Instruments and equipment: an electronic analytical balance, Thermo Hypersil GOLD C18(4.6 × 250mm, 5 μm), high performance liquid chromatography (UV detector), and the like.
3. Chromatographic conditions
1. HPLC method, main component comparison method calculation results.
2. A chromatographic column: thermo Hypersil GOLD C18(4.6mm × 250mm, 5 μm), octadecylsilane chemically bonded silica was used as a filler.
The chromatographic conditions were as follows:
detection wavelength: 277 nm; flow rate: 1.0 ml/min; sample introduction amount: 20 mu l of the mixture; column temperature: at 30 ℃.
3. Mobile phase: phase A: 0.025mol/L potassium dihydrogen phosphate solution (pH adjusted to 4.0 with phosphoric acid); phase B: and (3) acetonitrile. The gradient elution procedure is shown in table 3.
4. Solution preparation
Taking a proper amount of alogliptin benzoate, precisely weighing, adding a diluting solvent to dissolve, and quantitatively diluting to prepare a solution containing about 0.4mg of alogliptin benzoate in every 1ml, wherein the solution is used as a test solution; and taking a proper amount of alogliptin benzoate as a reference substance, precisely weighing, adding a diluting solvent to dissolve, and quantitatively diluting to prepare a solution containing about 0.4 mu g of alogliptin benzoate in each 1ml of the solution as a reference substance solution.
Diluting the solvent: 0.025mol/L potassium dihydrogen phosphate solution (pH adjusted to 4.0 with phosphoric acid) and acetonitrile at a volume ratio of 86: 14.
5. Measuring method (external standard method)
Precisely measuring 20 μ l of each of the sample solution and the reference solution, respectively injecting into a liquid chromatograph, and recording chromatogram. If an impurity 2A peak exists in a chromatogram of a test solution, the content of the impurity in the formula (2A) should not exceed 0.10 percent according to the peak area calculation of an external standard method of a main component (alogliptin benzoate).
6. Formula for calculation
Figure BDA0002823658830000101
Wherein SHetero compoundThe area of impurity 2A peak, M, of the test solutionTo pairAs control weight, omegaTo pairAs a control content, STo pairThe peak area of the main peak in the control, MSample (A)Is the sample weight.
The results are substituted into a formula, and the content of the impurity 2A in alogliptin benzoate is not detected.
EXAMPLE 6 quantitative determination of the impurity (R) -2- ((6- (3-aminopiperidin-1-yl) -5-bromo-3-methyl-2, 4-dioxo-3, 4-dihydropyrimidin-1 (2H) -yl) methyl) benzonitrile (3A) in alogliptin benzoate
1. Reagent and reagent: acetonitrile, purified water, monopotassium phosphate and phosphoric acid.
2. Instruments and equipment: an electronic analytical balance, Thermo Hypersil GOLD C18(4.6 × 250mm, 5 μm), high performance liquid chromatography (UV detector), and the like.
3. Chromatographic conditions
1. HPLC method, main component comparison method calculation results.
2. A chromatographic column: thermo Hypersil GOLD C18(4.6mm × 250mm, 5 μm), octadecylsilane chemically bonded silica was used as a filler.
The chromatographic conditions were as follows:
detection wavelength: 277 nm; flow rate: 1.0 ml/min; sample introduction amount: 20 mu l of the mixture; column temperature: at 30 ℃.
3. Mobile phase: phase A: 0.025mol/L potassium dihydrogen phosphate solution (pH adjusted to 4.0 with phosphoric acid); phase B: and (3) acetonitrile. The gradient elution procedure is shown in table 3.
4. Solution preparation
Taking a proper amount of alogliptin benzoate, precisely weighing, adding a diluting solvent to dissolve, and quantitatively diluting to prepare a solution containing about 0.4mg of alogliptin benzoate in every 1ml, wherein the solution is used as a test solution; and taking a proper amount of alogliptin benzoate as a reference substance, precisely weighing, adding a diluting solvent to dissolve, and quantitatively diluting to prepare a solution containing about 0.4 mu g of alogliptin benzoate in each 1ml of the solution as a reference substance solution.
Diluting the solvent: 0.025mol/L potassium dihydrogen phosphate solution (pH adjusted to 4.0 with phosphoric acid) and acetonitrile at a volume ratio of 86: 14.
5. Measuring method (external standard method)
Precisely measuring 20 μ l of each of the sample solution and the reference solution, respectively injecting into a liquid chromatograph, and recording chromatogram. If an impurity 3A peak exists in a chromatogram of a test solution, the content of the impurity in the formula (3A) should not exceed 0.10 percent according to the peak area calculation of an external standard method of a main component (alogliptin benzoate).
6. Formula for calculation
Figure BDA0002823658830000111
Wherein SHetero compoundThe area of impurity 3A peak, M, of the test solutionTo pairAs control weight, omegaTo pairAs a control content, STo pairThe peak area of the main peak in the control, MSample (A)Is the sample weight.
The results are substituted into a formula, and the content of the impurity 3A in alogliptin benzoate is not detected.
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Claims (7)

  1. The 3, 4-dihydropyrimidine benzonitrile derivative is characterized in that the structure is shown as the formula (2A):
    Figure FDA0002823658820000011
  2. 2. a process for preparing a 3, 4-dihydropyrimidine benzonitrile derivative as claimed in claim 1, which comprises:
    14.1mmol of compound 1A and 40ml of methanol are added into a reactor, stirred to dissolve and clear, and 28.2mmol of K is added2CO3Heating to 40-65 ℃, and reacting for 3-7h under heat preservation; concentrating the reaction solution under reduced pressure to dryness, adding 40ml of water and 30ml of dichloromethane, stirring, collecting an organic phase, continuously extracting the water phase with 20ml of dichloromethane twice, combining the organic phases, adding anhydrous sodium sulfate, and drying; filtering, concentrating the filtrate under reduced pressure to dryness to obtain white solid powder, and pulping with 20ml of MTBE at room temperature for 1-3 h; filtering to obtain white solid powder of the compound of the formula (2A);
    wherein, the structure of compound 1A is as follows:
    Figure FDA0002823658820000012
  3. 3. the method of claim 2, comprising: 14.1mmol of compound 1A and 40ml of methanol are added into a reactor, stirred to dissolve and clear, and 28.2mmol of K is added2CO3Heating to 65 ℃, and reacting for 6 hours in a heat preservation way; concentrating the reaction solution under reduced pressure to dryness, adding 40ml of water and 30ml of dichloromethane, stirring, collecting an organic phase, continuously extracting the water phase with 20ml of dichloromethane twice, combining the organic phases, adding anhydrous sodium sulfate, and drying; filtering, concentrating the filtrate under reduced pressure to dryness to obtain white solid powder, and pulping with 20ml of MTBE at room temperature for 2 h; filtering to obtain the product.
  4. 4. The method of claim 2 or 3, wherein compound 1A is prepared by a process comprising: adding 45ml of dichloroethane and 10.9mmol of compound M1 into a reactor, adding 13.1mmol of NBS and 0.5mmol of AIBN under stirring, heating the reaction solution to 40-80 ℃, preserving heat for 2-6h, and then cooling to 20-30 ℃ in an ice-water bath; 30ml of water was added to the reaction system, and after washing with stirring, the lower organic phase was collected and then washed with 15ml of 5% NaHCO3Washing the solution twice, washing the solution once by 15ml of saturated saline solution, collecting a lower organic phase, and drying the lower organic phase by anhydrous sodium sulfate; filtering, concentrating the filtrate under reduced pressure to obtain white solid powder, purifying by column chromatography, gradient eluting with PE and EA mixed solution of different concentrations, collecting eluate, and concentrating;
    wherein, the structure of the compound M1 is as follows:
    Figure FDA0002823658820000021
  5. 5. the method of claim 4, wherein compound 1A is prepared by a process comprising: adding 45ml of dichloroethane and 10.9mmol of compound M1 into a reactor, adding 13.1mmol of NBS and 0.5mmol of AIBN while stirring, heating the reaction solution to 80 ℃, preserving heat for 3 hours, and then cooling to 20 ℃ in an ice-water bath; 30ml of water was added to the reaction system, and after washing with stirring, the lower organic phase was collected and then washed with 15ml of 5% NaHCO3Washing the solution twice, washing the solution once by 15ml of saturated saline solution, collecting a lower organic phase, and drying the lower organic phase by anhydrous sodium sulfate; filtering, concentrating the filtrate under reduced pressure to obtain white solid powder, purifying by column chromatography, gradient eluting with PE and EA mixed solution of different concentrations, collecting eluate, and concentrating.
  6. 6. The use of the 3, 4-dihydropyrimidine benzonitrile derivative as in claim 1 in the quality control of alogliptin benzoate production.
  7. 7. The method for detecting the content of the 3, 4-dihydropyrimidine benzonitrile derivative in alogliptin benzoate according to claim 1, which is characterized by comprising the following steps:
    1) preparing a sample to be detected into a solution and adding the solution into a reversed-phase high performance liquid chromatography column;
    2) eluting the sample with a mobile phase gradient;
    3) detecting the effluent of the chromatographic column by using an ultraviolet detector to detect:
    signal peaks corresponding to alogliptin benzoate;
    signal peaks corresponding to impurities in the sample to be detected;
    4) calculating the impurity content in the sample to be detected according to the peak area of the detected signal peak;
    wherein the impurities refer to 3, 4-dihydropyrimidine benzonitrile derivatives;
    in the step 1), a sample to be detected is prepared into a solution by using a diluent solvent, wherein the diluent solvent is as follows: 0.02-0.03mol/L potassium dihydrogen phosphate solution with pH of 3.2-4.1 and acetonitrile according to (80-90): (10-20) mixing liquid in volume ratio; preferably, the dilution solvent is: 0.025mol/L potassium dihydrogen phosphate solution with pH of 4.0 and acetonitrile according to 86:14 volume ratio;
    the chromatographic conditions were as follows: detection wavelength: 277 nm; flow rate: 1.0 ml/min; column temperature: 30 ℃;
    the mobile phase used for gradient elution in step 2) is as follows: phase A: 0.02-0.03mol/L potassium dihydrogen phosphate solution with pH of 3.2-4.1, preferably 0.025mol/L potassium dihydrogen phosphate solution with pH of 4.0; phase B: acetonitrile;
    the gradient elution procedure was as follows:
    Figure FDA0002823658820000022
    Figure FDA0002823658820000031
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