CN115894377A

CN115894377A - Isotopic psychoactive substance labeled compound, preparation method and application thereof

Info

Publication number: CN115894377A
Application number: CN202211506877.9A
Authority: CN
Inventors: 孙喆明; 林何; 石寒天; 疏天民; 洪利军
Original assignee: Third Research Institute of the Ministry of Public Security
Current assignee: Third Research Institute of the Ministry of Public Security
Priority date: 2022-11-29
Filing date: 2022-11-29
Publication date: 2023-04-04

Abstract

The isotopic psychoactive substance labeled compound provided by the invention can be used for an internal standard for measuring the content of a psychoactive substance 1- (4-nitrile) -N- (2-phenylpropyl-2-yl) -1H-indazole-3-amide in a hair inspection material, and qualitatively detecting the 1- (4-nitrile) -N- (2-phenylpropyl-2-yl) -1H-indazole-3-amide according to the retention time and ion pair matching, and specifically, quantitatively detecting the corresponding compound according to the chromatographic peak area. The isotope labeled compound is used as an internal standard substance, can reduce the matrix effect of a detection material, and has good application prospect in aspects such as judicial identification and the like.

Description

Isotopic psychoactive substance labeled compound, preparation method and application thereof

Technical Field

The invention belongs to the technical field of preparation and application of standard substances of mental active substances, and particularly relates to an isotopic mental active substance labeled compound, and a preparation method and application thereof.

Background

Aiming at the detection of trace and trace substances in a detected material, an isotope dilution mass spectrometry method of a liquid chromatogram-mass spectrometer combined with an isotope labeling standard substance internal standard is the most common and efficient detection means. The isotope labeled standard substance is used as the internal standard, and the chemical properties of the isotope labeled internal standard and the compound to be detected are completely consistent except for the molecular weight, so that the isotope dilution mass spectrometry can achieve high detection precision and sensitivity, and is an indispensable tool for accurately detecting trace psychoactive substances.

The detection of the psychoactive substances and related metabolites in the hair can reflect the condition that a detected object is contacted with the psychoactive substances for a long time, and is an essential detection means for judging drug addiction, community drug rehabilitation and the like.

At present, the method for trace 1- (4-nitrile) -N- (2-phenylpropyl-2-yl) -1H-indazole-3-amide (4 CN-CUMYL-BUTINACA) in a biological test material is generally liquid phase mass spectrometry, and the content of 4CN-CUMYL-BUTINACA in the test material is determined by a standard curve method by selecting a compound with chemical properties similar to that of 4CN-CUMYL-BUTINACA as an internal standard. The closer the internal standard is to the target, the lower its matrix effect. The currently selected internal standard is generally the more common methoxamine, D5-diazepam or D5-APINACA.

Therefore, it is an urgent problem to be solved by those skilled in the art to provide an isotopically labeled compound of a psychotropic substance for improving the accuracy and sensitivity of detection of the psychotropic substance 4CN-CUMYL-BUTINACA in a test material.

Disclosure of Invention

Aiming at the problems of low detection precision and low sensitivity of psychoactive substances in the prior art, the invention provides an isotopically active substance labeled compound, which has the following structural formula:

wherein R is ₁ 、R ₂ 、R ₃ 、R ₄ At least one of which is D, said psychoactive substance comprising 1- (4-nitrile) -N- (2-phenylpropyl-2-yl) -1H-indazol-3-amide (4 CN-CUMYL-BUTINACA).

Preferably, the isotopically active substance labeled compound has the structural formula:

another aspect of the present invention provides a method for preparing the isotopically active substance-labeled compound, comprising the steps of:

(1) Performing iodophoration on D6-indazole at the 3-position under the alkaline condition, and performing carbonyl insertion and saponification under the catalysis of palladium to synthesize D4-indazole-3-carboxylic acid;

(2) Generating D4-N- (4-cyanobutyl) indazole-3-carboxylic acid from 5-bromo-valeronitrile and D4-indazole-3-carboxylic acid under alkaline conditions;

(3) 2-phenyl-2 propylamine and D4-N- (4-cyanobutyl) indole-3-carboxylic acid are subjected to condensation reaction under the catalysis of DMAP to generate D4-4CN-CUMYL-BUTINACA.

In another aspect, the invention provides the use of the isotopically active substance labeled compound in detecting the content of a psychoactive substance 1- (4-nitrile) -N- (2-phenylpropyl-2-yl) -1H-indazole-3-amide (4 CN-CUMYL-butonac) in biological test materials or sewage.

The invention provides a method for detecting the content of a drug 1- (4-nitrile) -N- (2-phenylpropyl-2-yl) -1H-indazole-3-amide in biological detection materials or sewage, which comprises the following steps: detecting the drug 1- (4-nitrile) -N- (2-phenylpropyl-2-yl) -1H-indazole-3-amide using the isotopically labeled drug compound of

claim

1 or 2 as an internal standard.

Preferably, the method comprises the following steps:

(1) Setting the detection conditions of liquid chromatography-mass spectrometry;

(2) Drawing a standard curve: adding 4CN-CUMYL-BUTINACA and D4-4CN-CUMYL-BUTINACA standard solutions with different proportions into a negative test material corresponding to a sample to be detected, performing LC-MS/MS detection after the same pretreatment step as the sample to be detected, and making a standard curve for the peak area ratio and the concentration of the 4CN-CUMYL-BUTINACA and the D4-4 CN-CUMYL-BUTINACA; wherein said internal standard is said isotopically active substance-labeled compound;

(3) Pretreatment and determination of a sample to be detected: and adding the isotopic psychoactive substance labeled compound into the sample to be detected as an internal standard, performing LC-MS/MS detection after the same pretreatment step as that used in the drawing of the standard curve to obtain quantitative parameters of the sample to be detected and the internal standard, and calculating by using a formula of the standard curve of an internal standard method to obtain the content of the sample to be detected.

Preferably, in step (1), the mobile phase for liquid chromatography-mass spectrometry detection is: acetonitrile (0.01% formic acid), B: water (0.01% formic acid, 5% ammonium formate), gradient: 1min 10, A,2min 50%, A,4min 90%, A,6min 95% by weight; and (3) chromatographic column: poroshell120 PFP 3.0x100mm 1.9um; column temperature: 30 ℃; flow rate: 0.5mL/min; sample introduction amount: 5 mu L of the solution; an ion source: electrospray ion source, positive mode (ESI +); the spraying voltage is 3500V; ion source temperature: 340 ℃; collision gas: and (4) nitrogen.

Preferably, in the step (2), the mass concentration of the internal standard substance in the mixed standard solution is 0.1-99.9%.

Preferably, in the step (3), the mass concentration of the internal standard substance in the sample to be detected is 0.1-99.9%.

Preferably, in the step (3), the formula of the internal standard method is calculated as,

X＝(Y-b ₀ )/b ₁

wherein X is the concentration of the sample to be measured, and Y is the liquid chromatography-mass spectrometryDetecting the resulting peak area, b ₀ Is the intercept of a standard curve, b ₁ Is the slope of the standard curve.

Preferably, the quality content of the psychoactive substance 4CN-CUMYL-butina in the biological detection material or the sewage is 10 ^-7 ％～10％。

The isotopic psychoactive substance labeled compound D4-1- (4-nitrile) -N- (2-phenylpropyl-2-yl) -1H-indazole-3-amide provided by the invention can be used as an internal standard for content determination of a psychoactive substance 1- (4-nitrile) -N- (2-phenylpropyl-2-yl) -1H-indazole-3-amide in sewage or biological detection materials, can reduce the matrix effect of the detection materials, and has good application prospects in aspects such as judicial identification.

Drawings

The invention is further described below in conjunction with the appended drawings and the detailed description.

FIG. 1 high resolution mass spectrum of D4-4CN-CUMYL-BUTINACA in example 1.

FIG. 2 preparation of D4-4CN-CUMYL-BUTINACA in example 1 ¹ H-NMR spectrum.

FIG. 3 preparation of D4-4CN-CUMYL-BUTINACA in example 1 ¹³ C-NMR spectrum.

FIG. 4 preparation of D4-4CN-CUMYL-BUTINACA in example 1 ¹⁹ F-NMR spectrum.

FIG. 5 chromatogram 1 for the detection of a 4CN-CUMYL-BUTINACA sample from example 2.

FIG. 6 chromatogram of the detection of a 4CN-CUMYL-BUTINACA sample from example 2.

FIG. 7 Hair 4CN-CUMYL-BUTINACA standard curve in example 2.

Detailed Description

In order to make the technical means, the creation characteristics, the achievement purposes and the effects of the invention easy to understand, the invention is further explained below by combining the specific drawings.

In the present description, the term "matrix effect" refers to the influence and interference of substances other than the test substance 4 CN-CUMYL-butilaca on the analysis process and the test result.

In the present description, the concentration by mass of 4CN-CUMYL-BUTINACA in the hair is 10 ^-7 ％～10％。

The isotopically active substance labeled compound D4-1- (4-nitrile) -N- (2-phenylpropyl-2-yl) -1H-indazole-3-amide provided by the invention has the following structural formula:

the invention is further illustrated by the following specific examples.

Example 1

The preparation method of D4-4CN-CUMYL-BUTINACA comprises the following steps:

0.74g (5.94 mmol) of D6-indazole (CAS: 2645412-96-8) and 20mL of dry DMF are added into a flask, stirred and cooled to 0 ℃ under the protection of nitrogen, 3.01g (11.88 mmol) of iodine and 1.34g (23.76 mmol) of potassium hydroxide are added, stirring is completed for half an hour, the mixture is heated to room temperature and stirred for reaction for 3 hours, poured into a sodium thiosulfate solution, extracted by ethyl acetate, combined with ethyl acetate, washed by water, dried by anhydrous magnesium sulfate, filtered, concentrated, and subjected to silica gel column chromatography to obtain 1.2g of D4-iodoindazole. 1.2g of D4-iodoindazole (4.4 mmol, 96.0 mg) and (0.13 mmol) Pd (dppf) Cl2 (CAS number 95464-05-4) were added to a flask, toluene (15 mL) and methanol (15 mL) were added, 750mg triethylamine (7.41 mmol) was added, the mixture was heated to 70 ℃ with carbon monoxide and stirred for reaction for 6 hours, the reaction mixture was cooled to room temperature, the solvent was concentrated and purified, and silica gel column chromatography was performed to give 0.69g of methyl D4-indazole-3-carboxylate. 0.54g (3.0 mmol) of D4-indazole-3-carboxylic acid methyl ester, 600mg (15.0 mmol) of sodium hydroxide, THF (18.0 mL), meOH (12.0 mL), and water (4.5 mL) were added to a reaction flask, heated to 60 ℃ and stirred for reaction for 4 hours, the solvent was concentrated, diluted with water, washed with DCM, the aqueous layer was acidified, the solid was precipitated by filtration, and dried to give 495mg of D4-indazole-3-carboxylic acid. At-20 ℃) D4-indole-3-carboxylic acid (495mg, 3mmol, compound 4.) is dissolved in 50mL of anhydrous oxygen-free dichloromethane, triethylamine (0.3 mL) is added, followed by dropwise addition of a solution of 5-bromo-valeronitrile (557mg, 3.3mmol) in anhydrous oxygen-free dichloromethane (25 mL), stirring andslowly warmed to room temperature, held at room temperature for 12 hours, heated to 30 ℃ and heated for 2 hours. After cooling, the solvent is removed by rotary evaporation to obtain an intermediate, and the N- (4-cyanobutyl) indazole-3-carboxylic acid is generated. The intermediate was dissolved in 50mL of DMF, and L-tert-leucine methyl ester (CAS No. 63038-27-7) (543mg, 3mmol), 1-ethyl-3 (3-dimethylpropylamine) carbodiimide (EDCI) (CAS No. 25952-53-8) (960mg, 5mmol) and 4-Dimethylaminopyridine (DMAP) (CAS No. 1122-58-3) (61mg, 0.5 mmol) were added, and after reacting at room temperature for 48 hours, the solvent was evaporated, quenched with a saturated ammonium chloride solution, and after dissolving with a mixed solution of 150mL of ethyl acetate-dichloromethane 1:1, the insoluble matter was removed by filtration with celite, and after dissolving with 30mL of diethyl ether-ethyl acetate 1:1 solution washing of diatomaceous earth, organic phases were combined, washed with 5% saturated aqueous sodium bicarbonate solution and the aqueous phase was extracted with dichloromethane, and the combined organic phases were dried over anhydrous magnesium sulfate. The drying agent was removed by filtration and the solvent removed by rotary evaporation. Purification by silica gel column chromatography (petroleum ether: ethyl acetate = 4:1) gave D4-4 CN-CUMYL-butitaca as a white solid (190mg, 8.2%). ¹ H NMR(600MHz，Methanol-d4)δ8.01(s，1H)，7.51–7.46(m，2H)，7.34–7.27(m，2H)，7.22–7.16(m，1H)，4.54(t，J＝6.9Hz，2H)，2.47(t，J＝7.1Hz，2H)，2.17–2.05(m，2H)，1.81(s，6H)，1.69–1.59(m，2H). ¹³ C NMR(151MHz，cd3od)δ163.94，148.56，142.45，138.89，129.39，127.48，125.95，123.77，120.92，57.15，30.11，29.76，23.90，17.02.MS(EI)m/z:Calcd.for C ₂₁ H ₂₅ D4FN ₂ O ₃ [M]+364.2；Found:364.2。

The specific spectra are shown in FIGS. 1-4.

Example 2

The content of 1- (4-nitrile) -N- (2-phenylpropyl-2-yl) -1H-indazole-3-amide (4 CN-CUMYL-BUTINACA) in the hair is detected by taking D4-1- (4-nitrile) -N- (2-phenylpropyl-2-yl) -1H-indazole-3-amide (D4-4 CN-CUMYL-BUTINACA) as an internal standard.

(1) Detection conditions of liquid chromatography-mass spectrometry:

a) The instrument model is as follows: agilent 1290-6470QQQ;

b) A chromatographic column: poroshell120 PFP 3.0x100mm 1.9um;

c) Column temperature: 30 ℃;

d) Mobile phase: acetonitrile (0.01% formic acid), B: water (0.01% formic acid, 5% ammonium formate), gradient: 1min 10% by weight, 2min 50% by weight, A,4min 90% by weight, A,6min 95% by weight;

e) Flow rate: 0.5mL/min;

f) Sample introduction amount: 5 mu L of the solution;

g) An ion source: electrospray ion source, positive mode (ESI +);

h) The spraying voltage is 3500V;

i) Ion source temperature: 340 ℃;

j) Collision gas: and (4) nitrogen.

The ion pairs and corresponding conditions are shown in table 1:

TABLE 1

(2) Pretreatment and detection of samples

Cleaning hair sample with ultrapure water, detergent water and acetone, air drying, cutting into pieces, weighing 20.0mg each part, adding 1mL methanol (containing 10ng/mL D4-4 CN-CUMYL-BUTINACA), grinding, ultrasonic treating in a freezing ultrasonic instrument for 30min, centrifuging at 4000r for 5min, collecting supernatant 800 μ L, volatilizing under 60 deg.C water bath air flow, redissolving with 80 μ L methanol, filtering with 0.22 μ L filter membrane, and LC-MS/MS analyzing 5 μ L to obtain the spectrum shown in FIG. 4-5. Wherein the quantitative ion pair is 361.2/226.2, and the peak area ratios of the 4CN-CUMYL-BUTINACA and the internal standard D4-4CN-CUMYL-BUTINACA in the two detections are 272578/769265 and 242584/668782 respectively.

(3) Drawing of standard curve

Cleaning blank negative hair sample with ultrapure water, detergent water and acetone, air drying, cutting into pieces, weighing each part at 20.0mg, adding 1mL methanol (containing 10ng/mL D4-4 CN-CUMYL-BUTINACA), adding 20 μ L4 CN-CUMYL-BUTINACA standard reference substance at concentrations of 100, 200, 500, 2000 and 5000ng/mL, preparing hair addition samples at concentrations of 0.1, 0.2, 0.5, 2.0 and 5.0ng/mg, preparing each concentration in parallel at 3 parts, swirling for 3min, standing and soaking at room temperature for 30min, pretreating according to sample, and cutting into piecesAfter the process treatment, LC-MS/MS detection is carried out, and standard curves are made for the peak area ratio and the concentration of 4CN-CUMYL-BUTINACA and D4-4CN-CUMYL-BUTINACA, so as to obtain a spectrum shown in figure 6. The formula of the standard curve is Y = 0.714260X-0.009453, wherein X is the concentration of the sample to be detected, Y is the peak area obtained by liquid chromatography-mass spectrometry detection, b ₀ = 0.009453 is the intercept of the standard curve, b ₁ =0.714260 is the slope of the standard curve.

According to the quantitative relation between the peak area ratio and the concentration in the standard curve and the calculation formula:

and calculating the content of 4CN-CUMYL-BUTINACA in the sample to be detected to be 0.52ng/mg.

When the isotopic psychoactive substance labeled compound provided by the invention is used for detecting a psychoactive substance 4CN-CUMYL-BUTINACA in a detection material, a proper amount of the isotopic psychoactive substance labeled compound D4-4CN-CUMYL-BUTINACA is added into the detection material, appropriate pretreatment is carried out according to detection requirements, then liquid chromatography-mass spectrometry (LC-MS/MS) detection is carried out, the isotopic psychoactive substance labeled compound is used as an internal standard, and qualitative and quantitative detection of the substance to be detected is realized by comparing the peak area ratios of a target substance and the substance to be detected in a Multiple Reaction Monitoring (MRM) mode, so that the detected psychoactive substance is 4CN-CUMYL-BUTINAC, the specificity is high, and the sensitivity is high.

The foregoing shows and describes the general principles, principal features, and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.

Claims

1. An isotopically active substance labeled compound, wherein said labeled compound has the formula:

wherein R is ₁ 、R ₂ 、R ₃ 、R ₄ At least one of which is D, said psychotropic substance comprising 1- (4-nitrile) -N- (2-phenylpropyl-2-yl) -1H-indazole-3-amide (4 CN-CUMYL-BUTINACA).

2. The isotopically active substance labeled compound of claim 1, wherein the isotopically active substance labeled compound has the formula:

3. a method for preparing an isotopically active substance-labeled compound according to claim 1 or 2, comprising the steps of:

4. Use of the isotopically labeled compound of claim 1 or 2 for detecting the content of the toxic 1- (4-nitrile) -N- (2-phenylpropyl-2-yl) -1H-indazole-3-amide in biological samples or sewage.

5. A method for detecting the content of a toxic substance 1- (4-nitrile) -N- (2-phenylpropyl-2-yl) -1H-indazole-3-carboxamide in a biological detection material or sewage, which comprises the following steps: detecting the drug 1- (4-nitrile) -N- (2-phenylpropyl-2-yl) -1H-indazole-3-amide using the isotopically labeled drug compound of claim 1 or 2 as an internal standard.

6. The method of claim 5, comprising the steps of:

(2) Drawing a standard curve: adding 4CN-CUMYL-BUTINACA and D4-4CN-CUMYL-BUTINACA standard solutions with different proportions into a negative test material corresponding to a sample to be detected, performing LC-MS/MS detection after the same pretreatment step as the sample to be detected, and making a standard curve for the peak area ratio and the concentration of the 4CN-CUMYL-BUTINACA and the D4-4 CN-CUMYL-BUTINACA; wherein the internal standard is an isotopically drug-labeled compound of claim 1 or 2;

(3) Pretreatment and determination of a sample to be detected: and adding the isotope drug labeled compound into the sample to be detected as an internal standard, performing LC-MS/MS detection after the same pretreatment step as that used in drawing a standard curve to obtain quantitative parameters of the sample to be detected and the internal standard, and calculating by using a formula of the standard curve of an internal standard method to obtain the content of the sample to be detected.

7. The method according to claim 6, wherein in step (1), the mobile phase for the liquid chromatography-mass spectrometry detection is: acetonitrile (0.01% formic acid), B: water (0.01% formic acid, 5% ammonium formate), gradient: 1min 10% by weight, 2min 50% by weight, A,4min 90% by weight, A,6min 95% by weight; a chromatographic column: poroshell120 PFP 3.0x100mm 1.9um; column temperature: 30 ℃; flow rate: 0.5mL/min; sample introduction amount: 5 mu L of the solution; an ion source: electrospray ion source, positive mode (ESI +); the spraying voltage is 3500V; ion source temperature: 340 ℃; collision gas: nitrogen gas.

8. The method according to claim 6, wherein in the step (2), the mass concentration of the internal standard substance in the mixed standard solution is 0.1-99.9%; in the step (3), the mass concentration of the internal standard substance in the sample to be detected is 0.1-99.9%.

9. The method of claim 6, wherein in step (3), the internal standard method formula is calculated as:

wherein X is the concentration of a sample to be detected, Y is the peak area obtained by liquid chromatography-mass spectrometry detection, b ₀ Is the intercept of a standard curve, b ₁ Is the slope of the standard curve.

10. The method of claim 5, wherein the bioassay material or wastewater contains the 1- (4-nitrile) -N- (2-phenylpropyl-2-yl) -1H-indazole-3-amide in an amount of 10 ^-7 ％～10％。