阅读说明：本技术 一种吡啶喹唑啉中未知杂质的分离纯化与结构鉴定方法 (Separation and purification and structure identification method for unknown impurities in pyridine quinazoline ) 是由 吴宏霞 杨彬 刘鹏 王宇 于 2020-12-03 设计创作，主要内容包括：本发明公开了一种吡啶喹唑啉中未知杂质的分离纯化与结构鉴定方法,先将吡啶喹唑啉原药结晶母液经硅胶柱初步分离,采用制备级制备液相色谱梯度分离得到含量大于98%未知杂质,并通过LC-MS、～1H NMR等分析手段对其进行结构鉴定,确定了吡啶喹唑啉原药中该未知杂质的结构为1-(5-(全氟丙烷-2-基)苯并异噁唑-1(3H)-基)乙烷-1-酮并分析了其产生原因。该杂质为首次发现的新杂质,为吡啶喹唑啉的合成工艺研究提供了杂质对照品。(The invention discloses a method for separating, purifying and identifying the structure of unknown impurities in pyridine quinazoline, which comprises the steps of firstly, preliminarily separating the mother liquor of pyridine quinazoline original drug crystallization by a silica gel column, adopting preparative liquid chromatography gradient separation to obtain the unknown impurities with the content of more than 98%, and performing LC-MS, 1 The unknown impurity in the pyridine quinazoline original drug is determined to be 1- (5- (perfluoropropane-2-yl) benzisoxazol-1 (3H) -yl) ethane-1-ketone by analyzing means such as H NMR and the like to carry out structural identification, and the generation reason is analyzed. The impurity is a new impurity discovered for the first time, and provides an impurity reference substance for the research of the synthetic process of the pyridine quinazoline.)

1. a separation and purification and structure identification method of unknown impurities in pyridine quinazoline is characterized in that: recrystallizing pyridine quinazoline raw drug to obtain crystallization mother liquor containing 0.8% of unknown impurity, preliminarily separating by silica gel column, performing gradient preparation separation by adopting full-automatic preparation liquid chromatograph to obtain the unknown impurity with the content of more than 98%, and performing ultraviolet absorption spectrum, LC-MS and LC-MS¹H NMR instrumental analysis confirmed that its structure was 1- (5- (perfluoropropan-2-yl) benzisoxazol-1 (3H) -yl) ethan-1-one and analyzed for its cause of occurrence, which is of the formula:

。

2. the method for separating, purifying and structurally identifying the unknown impurities in the pyridine quinazoline according to claim 1, characterized in that the step of performing primary separation on the unknown impurities by using a pyridine quinazoline technical crystal mother liquor comprises: dissolving 20g of raw pyridine quinazoline with the unknown impurity content of 0.2% in acetonitrile under the conditions of heating and stirring until saturation, cooling, crystallizing, filtering, concentrating the obtained filtrate to obtain the unknown impurity with the content of 0.8%, separating by using a silica gel column, and concentrating by using a rotary evaporator to obtain a concentrated solution enriched with the unknown impurity.

3. The method for separating, purifying and structurally identifying the unknown impurities in the pyridine quinazoline according to claim 1, characterized in that the concentrated solution which is subjected to primary separation and is enriched in the unknown impurities is subjected to gradient separation again by using a full-automatic preparative liquid chromatograph, and the preparative liquid chromatography separation conditions are as follows: shimadzu Shim pack GIST, 5 μm, 10X 250mm C18 column; mobile phase: gradient elution is carried out by taking acetonitrile as a mobile phase A and pure water as a mobile phase B (0-40 min, 10% A → 50% A; 40-60 min, 50% A → 10% A); flow rate: 5 mL/min; detection wavelength: 254nm, the fraction of unknown impurity is desolventized and dried, and the HPLC content is 98%.

4. The method for separating, purifying and structurally identifying unknown impurities in pyridine quinazoline according to claims 1 and 3, characterized in that a mobile phase gradient for liquid chromatographic separation is prepared (see table 1):

TABLE 1 gradient of mobile phase

。

Technical Field

The invention relates to a method for separating and purifying unknown impurities in pesticide raw materials and carrying out structural identification on the unknown impurities.

Background

The pyridine quinazoline is a novel quinazoline insecticide which is effective on stinkbug pests and is more effective on aphids and whitefliesThe effects of mealybugs, leafhoppers and thrips are excellent. The molecular formula is as follows: c₁₉H₁₅F₇N₄O₂The synthetic process route is as follows:

because the synthesis process is complex, some side reactions are inevitably generated, impurities of the side reactions directly affect the quality of the original medicine, and in order to better control the content of the impurities and improve the quality of the original medicine, the structures of the impurities need to be analyzed and the generation reasons of the impurities need to be analyzed, however, the separation and purification and the structural identification of the impurities have certain difficulty, and the research reports in the aspect are few at present.

Disclosure of Invention

The invention aims to provide a method for separating and purifying trace unknown impurities in a pyridine quinazoline raw drug, which utilizes a silica gel column for preliminary separation and then uses a full-automatic preparative liquid chromatograph for further gradient elution to realize separation and purification, and can obtain the impurities with the content of 98%.

It is another object of the invention to identify the chemical structure of the unknown impurity using polychromatic spectroscopy.

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

the unknown impurity with the content of 0.2% exists in the pyridine quinazoline crude drug, the unknown impurity is firstly recrystallized to obtain a crystallization mother liquor, the crystallization mother liquor is initially separated by a silica gel column, and the separation and the concentration are carried out by adopting a liquid chromatograph to realize the separation, so that the unknown impurity with the content of 98% is obtained.

Wherein the mother liquor extraction step comprises: weighing 20g of pyridine quinazoline raw drug, dissolving in acetonitrile under the condition of heating and stirring until saturation, cooling, carrying out suction filtration for solid-liquid separation, and concentrating the filtrate under reduced pressure until the filtrate is dry.

The primary separation by silica gel column comprises: taking a glass chromatographic column with the diameter of 3.3cm, filling the column with the height of about 20cm, adding 10mL of dichloromethane into the concentrated solid sample of the recrystallization mother liquor for dissolving, then adding 8g of 200-mesh silica gel, concentrating under reduced pressure until the mixture is dry, then adopting a dry method for preparing the sample, loading the sample on the column, flushing the column with petroleum ether-ethyl acetate (20: 1-0: 1), collecting the component with the Rf of 0.5, and concentrating under reduced pressure until the component is dry to obtain 2g of 10% crude unknown impurities.

The preparative liquid chromatography separation step comprises: dissolving the crude product containing 10% of the unknown impurity in 10mL of acetonitrile to prepare a solution of 200mg/mL, separating by using a preparative liquid chromatography, injecting samples 10 times, wherein the sample injection amount is 1mL, and the column temperature is as follows: 40 ℃, mobile phase: gradient elution is carried out by taking acetonitrile as a mobile phase A and pure water as a mobile phase B (0-40 min, 10% A → 50% A; 40-60 min, 50% A → 10% A); collecting 19.5min chromatographic peak fraction under the condition of preparation and separation with flow rate of 5mL/min and detection wavelength of 254nm, and concentrating under reduced pressure to dryness to obtain 100mg of 98% unknown impurity.

The chemical structure of the unknown impurity is identified by the following method:

firstly, determining that the impurity is the impurity in the preparation process of the pyridine quinazoline by using a high performance liquid chromatograph according to retention time and an ultraviolet absorption spectrum, and determining the impurity by using a liquid chromatogram and an ultraviolet absorption spectrum chart.

The molecular weight of the unknown impurity was then determined by LC-MS: obtaining the excimer ion peak M/z 330[ M-H ] from LC-MS spectrogram]If the molecular weight of the unknown impurity is 331, the molecule should contain an odd number of N atoms, and the molecular formula is deduced according to the synthesis process: c₁₁H₈F₇NO₂。

Elemental analysis results: experimental values C43.46%, H2.41%, F40.06%, N4.33%, O9.74%, theoretical values C43.52%, H2.43%, F40.15%, N4.23%, O9.66%

Finally pass through¹H NMR analysis confirms the chemical structure of the compound,¹the H NMR spectrum structure is analyzed as follows: delta 7.933 (d, 1H, H-3), delta 7.589 (d, 1H, H-2), delta 7.442 (s, 1H, H-1), delta 4.638 (s, 2H, H-4), delta 2.606 (s, 3H, H-5).

Analyzing the generation reason: in the presence of acid and palladium-carbon catalyst, a small amount of dihydroquinolinone ring in the pyridine quinazoline generates intramolecular nucleophilic substitution reaction, and is rearranged into dihydrooxazole ring in molecule to finally form the impurity.

Drawings

FIG. 1 is a sample HPLC chromatogram.

FIG. 2 is a graph of the ultraviolet absorption spectrum of a sample.

FIG. 3 is a sample LC-MS ion flow spectrum.

FIG. 4 is a sample LC-MS spectrum.

FIG. 5 is a sample¹H NMR spectrum.

Detailed Description

The invention will be further described with reference to specific embodiments and associated drawings, the advantages and features of which will become apparent as the description proceeds. The examples are illustrative only and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be made without departing from the spirit and scope of the invention.

Example 1: separating and purifying unknown impurities in the pyridine quinazoline by the following steps: weighing 20g of pyridine quinazoline original drug (95%), dissolving the pyridine quinazoline original drug in 40mL of acetonitrile under the condition of heating and stirring until saturation, cooling and crystallizing, then carrying out solid-liquid separation by suction filtration, taking the obtained filtrate as a crystallization mother liquor, carrying out reduced pressure concentration until the filtrate is dry, obtaining a solid sample containing about 1% of the unknown impurity, adding 10mL of dichloromethane for dissolution, adding 8g of 200-mesh silica gel, carrying out reduced pressure concentration until the filtrate is dry, then adopting a dry method for sample preparation, loading the sample on the silica gel column, washing the column by petroleum ether-ethyl acetate (20: 1-0: 1), collecting the component with Rf of 0.5, carrying out reduced pressure concentration until the filtrate is dry, obtaining 2g of 10% of the unknown impurity crude product, adding 10mL of acetonitrile for dissolution, preparing a 200mg/mL solution, carrying out gradient elution by using a prepared liquid chromatography to realize complete. Respectively collecting eluates containing different chromatographic peaks, analyzing by HPLC to ensure that the collected chromatographic peaks are the unknown impurities, and concentrating under reduced pressure to dryness to obtain 100mg of unknown impurity sample with content of more than 98%.

Example 2: the unknown impurity in the pyridine quinazoline is separated and purified through the following steps: weighing 15g of pyridine quinazoline original drug (90%) and dissolving the pyridine quinazoline original drug in 30mL of acetonitrile under the condition of heating and stirring until saturation, cooling and crystallizing, then carrying out suction filtration for solid-liquid separation, taking the obtained filtrate as a crystallization mother liquor, carrying out reduced pressure concentration until the filtrate is dry to obtain a solid sample containing about 2% of the unknown impurity, then adding 10mL of dichloromethane for dissolution, adding 8g of 200-mesh silica gel, carrying out reduced pressure concentration until the filtrate is dry, then adopting a dry method for sample preparation, loading the sample into the silica gel column, washing the column with petroleum ether-ethyl acetate (20: 1-0: 1), collecting the component with Rf of 0.5, carrying out reduced pressure concentration until the filtrate is dry to obtain 1.5g of 18% of the unknown impurity crude product, adding 10mL of acetonitrile for dissolution, preparing a 150mg/mL solution, carrying out re-separation by using a. And respectively collecting eluates containing different chromatographic peaks, analyzing by HPLC to ensure that the collected chromatographic peaks are the unknown impurities, combining, concentrating under reduced pressure to dryness to obtain an unknown impurity sample with the content of more than 98% of 150 mg.

Example 3: carrying out structural analysis on the obtained unknown impurities by the following steps:

and (3) measuring the molecular weight: weighing about 13.00 mg of unknown impurities, placing the unknown impurities into a 25 mL volumetric flask, dissolving the unknown impurities with acetonitrile, diluting the unknown impurities to a scale, and preparing mother liquor of the unknown impurities. And (3) transferring 0.6 mL of unknown impurity mother liquor, placing the mother liquor into a 10mL volumetric flask, diluting the mother liquor to a scale with acetonitrile, and preparing an unknown impurity solution. The LC-MS analysis conditions preferably adopt Agilent 1260/6120 LC-MS, chromatographic column: eclipse Plus C18, 250mm × 4.6 mm, 5 μm, mobile phase: acetonitrile: water = 45: 55, column temperature: 35 ℃, ultraviolet detection wavelength: 210 nm, column flow: 1.0 mL/min, injection volume: 10 μ L, run time: 30.0 min, ion source: APCI, negative ion detection mode, fragmentation voltage: 70 Ev, nebulizer pressure: 60 psi, dry gas (N2) flow: 5.0 mL/min, evaporator temperature: 350 ℃, drying gas temperature: 350 ℃, capillary transport voltage: 3000V, mass scan range: 50-1000 amu. Obtaining the excimer ion peak M/z 330[ M-H ] from LC-MS spectrogram]-, then the unknown heteroThe mass molecular weight is 331, which is inferred to contain an odd number of N atoms and has the formula: c₁₁H₈F₇NO₂。

¹H NMR analysis: placing about 5 mg of the pure product of the unknown impurity in a nuclear magnetic tube, adding 0.5 mL of deuterated chloroform, dissolving sufficiently to prepare the solution of the unknown impurity, and performing¹H NMR analysis, preferably with a Varian INOVA-300 NMR spectrometer, operating frequency: 300 MHz, scan number: deuterated chloroform (CDCl 3) with a purity of 99.99%: Sigma-Aldrich. The 1H NMR spectrum was resolved as follows: delta 7.933 (d, 1H, H-3), delta 7.589 (d, 1H, H-2), delta 7.442 (s, 1H, H-1), delta 4.638 (s, 2H, H-4), delta 2.606 (s, 3H, H-5). The chemical formula of the unknown impurity can be determined as follows:

。