CN107525859B - Method for screening rapid detection conditions of illegally added compound derivatives in health care products - Google Patents
Method for screening rapid detection conditions of illegally added compound derivatives in health care products Download PDFInfo
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- CN107525859B CN107525859B CN201710618621.XA CN201710618621A CN107525859B CN 107525859 B CN107525859 B CN 107525859B CN 201710618621 A CN201710618621 A CN 201710618621A CN 107525859 B CN107525859 B CN 107525859B
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- derivative
- derivatives
- dpa
- amlodipine
- sildenafil
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Abstract
本发明涉及医药分析技术领域，具体是一种筛选保健品中非法添加化合物衍生物快速检测条件的方法，涉及采用分子对接的方法，从整体上考虑配体(小分子)与受体(生物大分子)相互作用的好坏，从而发现具有潜在药理活性、易被作为非法添加的化合物；进一步结合密度泛函理论计算得到理论拉曼峰位，深入挖掘数据整理后得到的理论共有峰，为现场快速判断保健品中非法添加物提供方法。本发明的方法不需要人工合成对照品，应用范围广泛，实验成本低，操作简便、快速，适合现场快速检测，为保健食品中非法添加的衍生物的鉴定提供了更加坚固的保证。The invention relates to the technical field of medical analysis, in particular to a method for screening the rapid detection conditions of illegally added compound derivatives in health products, and relates to a method for using molecular docking. molecules) interactions, so as to discover compounds with potential pharmacological activity that are easily added illegally; the theoretical Raman peak positions were obtained by further combining with density functional theory calculations, and the theoretical common peaks obtained after in-depth data sorting were used for the scene. Quickly determine the provision of illegal additives in health products. The method of the invention does not require artificial synthesis of reference substances, has wide application range, low experimental cost, simple and fast operation, is suitable for rapid on-site detection, and provides a firmer guarantee for the identification of illegally added derivatives in health food.
Description
Technical Field
The invention relates to the technical field of medical analysis, in particular to a method for screening rapid detection conditions of illegally added compound derivatives in health care products, which relates to a method for calculating virtual docking by adopting a computer, wherein the quality of interaction between a ligand (micromolecule) and a receptor (biological macromolecule) is considered as a whole, and an optimal binding mode between the micromolecule and the receptor is found, so that a compound with potential pharmacological activity is found; and further combining a density functional theory to calculate the theoretical Raman peak position of the health product, and rapidly judging illegal additives in the health product for field detection personnel through the calculated common peak.
Background
The traditional Chinese medicine has stable efficacy and slow effect, and is produced according to the national medicine standard, and the difference of the effects of the medicines of various manufacturers is not too large. In order to enable the curative effect of the product of the patient to be better than that of other manufacturers, the western medicine components are doped in the product of the patient to enhance the medicine effect, so that the health care product which is mistakenly taken by the patient has the advantages of obvious effect and less side effect than western medicine. At present, many detection departments have adopted a liquid chromatography-mass spectrometry to establish chemical and pharmaceutical components which are illegally added in8 types of health care products such as blood sugar reduction, blood pressure reduction, blood fat reduction, kidney tonifying, yang strengthening, weight losing, cough and asthma relieving, swelling and pain relieving, mind tranquilizing and the like. With the increasing number of illegally added chemicals being tested in succession, many illegal manufacturers even have to adulterate chemical derivatives in healthcare products. In the beginning of 2005, it was first discovered and reported that an unknown compound, redfenafil, having sildenafil (viagra) -like action and the same parent nucleus structure was illegally added to health foods. The drug effect and toxicity of the derivatives with modified structures can be changed, and the derivatives are arbitrarily doped into health care products without drug effect and toxicity experiments of CFDA, so that great harm is brought to the drug safety. The complex traditional Chinese medicine and western medicine are combined for use, interaction may occur, and if the combination is not proper, some chemical reactions including drug effect enhancement, drug effect reduction, drug adverse reaction and even drug poisoning reaction are likely to occur. If the patient unknowingly takes the traditional Chinese medicine containing the chemical medicine components, the serious harm can be brought to the consumers.
However, the chemical derivatives are difficult to synthesize (the synthesized standard substance is not necessarily an illegally added substance), difficult to detect (leakage is easy to occur due to lack of the standard substance and corresponding detection means), and difficult to identify (for unknown compounds, the structure identification needs to be carried out by combining a plurality of large-scale instruments) which are problems to be overcome at present. In order to keep people healthy, it is necessary to establish a new detection strategy to prevent the bad actions of illegal addition in health food and even in beverages.
If the derivatives which are possibly added are synthesized to be prepared into standard products for detection personnel to use, the work is time-consuming and labor-consuming, and the derivatives which are more likely to be synthesized are not really chemical drugs which are added by illegal traders, but are drilled into the air. If a method is provided, a large amount of synthesis of series of derivatives is not needed, the biological activity of the derivatives can be known, and a characteristic identification spectrogram can be obtained, so that the method has great significance for laboratory or field detection.
Computer-aided drug design (CADD) is a commonly used drug development method, and the interaction between a drug and a target protein can be deeply understood by researching the crystal structure of a complex of the drug and the target protein, so that the most direct basis is provided for the design and structure optimization of high-activity drugs. The density general function Theory (DFT) is a quantum mechanical method for researching the electronic structure of a multi-electron system. In the continuous development process of the DFT method, the B3LYP method is provided to better describe the functional relationship between the electron correlation energy and the electron density, so that the DFT can be widely applied. Of course, both the CADD and DFT techniques are theoretical prediction methods, and certain experimental data is still required to verify the prediction accuracy.
Surface-enhanced Raman spectroscopy (SERS) is a very good experimental verification means, and the intensity of SERS is higher than 10 of Raman spectrum4～106The detection sensitivity is extremely high, and the method can be widely applied to analysis of trace substances; the SERS has rich spectral characteristic information, the band position of the SERS is not greatly different from that of the conventional Raman spectrum, and characteristic identification can be provided. Combining SERS with thinThe combination of Thin Layer Chromatography (TLC) can improve the characteristics and accuracy of SERS identification of certain specific compounds in complex mixtures through simple separation of TLC, and is widely applied to the fields of illegal addition of foods and medicines, active ingredient identification and the like.
Therefore, when the health product is detected, under the condition that the series derivative reference substances cannot be obtained, whether a certain chemical drug instead of a specific chemical drug is illegally added into the health product can be judged according to the DFT predicted value, a reliable theoretical basis is provided for field detection, the detection time is greatly saved, and even the expensive and difficultly obtained reference substances are not required to be relied on. In order to avoid the illegal addition of any derivative in health food and the occurrence of false positive phenomenon, the method carries out laboratory synthesis on a compound with a high scoring function after molecular docking calculation, establishes a TLC-SERS method to verify the reliability of DFT theory, and simultaneously carries out further confirmation on a suspected positive sample taken to a laboratory after field detection by adopting an UPLC-TOF/MS (Ultra-Performance liquid chromatography-Quadrupole Time-of-flight Time Mass Spectrometry) technology.
Finally, a 'field-laboratory' quick detection verification strategy for illegally adding unknown derivatives in the health care product is established.
Disclosure of Invention
The invention aims to provide a method for screening rapid detection conditions of illegally added compound derivatives in health products.
In order to achieve the purpose, the invention adopts the technical scheme that:
the computer aided design technology is adopted, the designed target compound and the action site thereof are subjected to butt joint research, the density generalized function theoretical calculation is carried out on the compound with high score of the computer butt joint scoring function, the theoretical Raman spectrum peak position is obtained, and the common theoretical Raman peak position of the series of derivatives is induced.
The invention provides a method for screening rapid detection conditions of illegally added compound derivatives in health products, which comprises the following steps:
the method comprises the following steps: designing a derivative target molecule by taking an illegally added compound parent nucleus as a key intermediate based on a structure-activity relationship method;
step two: evaluation of ligand molecule interaction with receptor
The drug design docking platform Discovery Studio3.0 is adopted to carry out the step one, namely, the docking of the ligand molecules and the active cavities of the action sites of the ligand molecules, and the effect of the combination of the ligand and the receptor is considered on the whole.
Selecting a Libdock molecular docking technology, wherein the docking method firstly calculates a hot zone diagram comprising molecular polarity and nonpolar parts aiming at a receptor active site; secondly, ligand molecules of different concepts are rigidly superimposed to the hot zone map respectively to form relatively suitable interactions; finally, energy optimization is performed, and the docking conformation with higher score is retained. Preferably, the high-grade derivative is selected according to the scoring function after docking;
step three: theoretical Raman spectrum of derivative predicted by density functional method
After molecular configuration is optimized by adopting GAUSS VIEW 5.0, a DFT method of B3LYP level is selected, and 6-31G of polarization functions of hydrogen atoms plus p polarization functions and heavy atoms plus d polarization functions in the system are obtained*(d, p) group, after configuration optimization is carried out on the derivative optimized in the step two, in a Linux system, the GAUSSIAN09 software is used for calculation;
step four: theoretical consensus peak of derivatives
According to the DFT calculation result, adopting GAUSSVIEW 5.0 software to carry out common peak induction on the derivatives; determining the common absorption peak of the derivative and judging the standard containing the derivative in the sample to be tested.
Preferably, the method further comprises the following steps:
step five: synthesis and structural verification of derivatives
Synthesizing the compound derivative optimized in the step two, and verifying the structure through nuclear magnetism and mass spectrum;
step six: method for verifying derivative binding kinetics assay
Sequentially sampling compounds with different concentrations by using a Biacore T200 molecular interaction analysis system, analyzing experimental data by using Biacore T200evaluation software, and fitting and calculating an experimental result by using a dynamic combination model;
step seven: establishment of conditions for TLC-SERS detection of derivatives
Weighing the derivative prepared in the fifth step, carrying out thin-layer separation, dropwise adding a surface reinforcing agent, wherein the surface reinforcing agent is gold sol, organic silver sol, silver sol or gold-silver composite gel, and scanning by adopting a portable Raman spectrometer;
step eight: processing non-blank spectrogram
Processing the non-blank spectrogram by standard spectrogram processing software, and selecting 400-1700cm-1The wave band is used as a characteristic wave band, the characteristic wave band is subjected to spectrogram smoothing and baseline correction by adopting OPUS5.0 software, and then vector normalization processing is carried out on the corrected spectral data to obtain a preprocessed surface enhanced Raman spectrogram;
step nine: discriminating the preprocessed surface enhanced Raman spectrogram
Integrating and summarizing the common peak obtained by TLC-SERS detection and the common peak obtained by the theoretical calculation of the step four DFT, determining the common absorption peak of the derivative and judging the standard of illegally adding compound derivatives in the sample to be detected.
More preferably, the method also comprises the following steps:
step ten: UPLC-QTOF/MS authentication
The derivatives are qualitatively analyzed by adopting a rapid separation ultra-high performance liquid chromatography-time-of-flight mass spectrometer to obtain the common fragment ions of the derivatives, and suspected positive samples detected on site can be confirmed for a laboratory under the condition of no standard substance.
Preferably, the health care product has the functions of tonifying yang, reducing blood pressure, losing weight, reducing blood sugar, relieving cough and asthma and the like.
More preferably, the illegal addition compound derivative is sildenafil analogue, amlodipine analogue, sibutramine analogue, metformin analogue or theophylline analogue.
In a preferred embodiment of the present invention, there is provided a method of screening a rapid test condition for sildenafil derivatives, comprising the steps of:
the method comprises the following steps: taking 3- (6, 7-dihydro-1-methyl-7-oxo-3-propyl-1H-pyrazolo [4,3-d ] pyrimidine-5-yl) -4-ethoxy-benzenesulfonyl serving as an intermediate of sildenafil as a key intermediate, designing target molecules of sildenafil derivatives (more than 50 are designed in the invention) based on a structure-activity relationship, and carrying out butt joint on the target molecules in the next step;
step two: virtual docking of ligands to phosphodiesterase type 5 (PDE-5) complex crystal structures
Adopting a drug design docking platform Discovery Studio3.0 to carry out the step of docking a target molecule with a crystal structure active cavity of a PDE-5 compound, and selecting a Libdock molecule docking technology, (the docking method firstly calculates a hot zone diagram comprising molecule polar parts and non-polar parts aiming at a receptor active site; in the embodiment of the invention, the total number of compounds with high scoring function is 11, and the compounds are respectively ethyl piperazine S-102, propyl piperazine S-109, butyl piperazine S-104, decyl piperazine S-110, piperidine substituent S-301, aromatic ring substituent S-400, methyl aromatic ring substituent S-401, ethyl aromatic ring substituent S-402, propyl aromatic ring substituent S-403, butyl aromatic ring substituent S-404 and 4-methylimidazole substituent S-501. (see Table 1).
Step three: theoretical Raman spectrum for predicting sildenafil derivative by density functional method
After molecular configuration is optimized by adopting GAUSS VIEW 5.0, a DFT method of B3LYP level is selected, and 6-31G of polarization functions of hydrogen atoms plus p polarization functions and heavy atoms plus d polarization functions in the system are obtained*(d, p) group, after configuration optimization is carried out on the sildenafil derivative optimized in the step two, in a Linux system, the sildenafil derivative is calculated by GAUSSIAN09 software;
step four: sildenafil derivative theoretical consensus peak
Performing theoretical common peak induction on the calculated theoretical Raman spectrogram of the sildenafil derivative by adopting GAUSSVIEW 5.0 software according to the DFT calculation result;
in the examples of the present invention, the total number of DFT theoretical Raman value peaks for summarizing the absorption peaks of sildenafil derivatives was 15, each 417,485,515,593,805,890,916,941,1010,1041,1150,1253,1263,1523,1550cm, based on the theoretical peak absorption intensity-1。
Preferably, the method for screening the sildenafil derivative rapid detection conditions further comprises the following steps: (i.e., verification of the above prediction method)
Step five: synthesis and structure verification of sildenafil derivative
Synthesizing the sildenafil derivative optimized in the second step, and verifying the structure through mass spectrum and nuclear magnetism;
in an embodiment of the present invention, the preparation method of the sildenafil derivative comprises: dissolving 5mmol of 3- (6, 7-dihydro-1-methyl-7-oxo-3-propyl-1H-pyrazolo [4,3-d ] pyrimidine-5-yl) -4-ethoxy-benzenesulfonyl in 30ml of chloroform, dissolving the mixture by stirring in an ice bath, and adding 10mmol of triethylamine. N-ethylpiperazine (5mmol), N-butylpiperazine (5mmol), N-isopropylpiperazine (5mmol), N-decylpiperazine (5mmol), 4-methylpiperidine (5mmol), aniline (5mmol), 4-methylaniline (5mmol), 4-ethylaniline (5mmol), 4-propylaniline (5mmol), 4-butylaniline (5mmol) and 4-methylimidazole (5mmol) were added thereto, respectively, and the mixture was stirred for reaction. And (5) carrying out post-treatment to obtain a pure product, and verifying the structure by nuclear magnetism and mass spectrum.
Step six: sildenafil derivative binding kinetics assay validation
A Biacore T200 molecular interaction analysis system was used. And (3) sequentially injecting compounds with different concentrations, analyzing experimental data by using Biacore T200evaluation software, fitting and calculating an experimental result by using a kinetic binding model, obtaining the binding activity of the derivative, and verifying the result of molecular docking calculation.
Step seven: establishing TLC-SERS detection conditions of sildenafil derivatives
Weighing the sildenafil derivative prepared in the fifth step, and carrying out thin-layer separation, wherein the volume ratio of dichloromethane to acetone to ammonia water is 20:1.5:0.2 in an unfolding system;
dropwise adding a surface reinforcing agent, wherein the surface reinforcing agent is gold sol, organic silver sol, silver sol or gold-silver composite gel, and scanning by adopting a portable Raman spectrometer; the method selects organic silver sol preferably, and adopts a portable Raman spectrometer to scan;
step eight: processing non-blank spectrogram
Processing the non-blank spectrogram by standard spectrogram processing software, and selecting 400-1700cm-1The wave band is used as a characteristic wave band, the characteristic wave band is subjected to spectrogram smoothing and baseline correction by adopting OPUS5.0 software, and then vector normalization processing is carried out on the corrected spectral data to obtain a preprocessed surface enhanced Raman spectrogram;
step nine: discriminating the preprocessed surface enhanced Raman spectrogram
Integrating and summarizing the common peak obtained by the seven TLC-SERS detection and the common peak obtained by the four DFT theoretical calculation, determining the common absorption peak of the sildenafil derivative and judging the standard of the sildenafil derivative contained in the sample to be detected;
in the embodiment of the invention, 9 common peaks obtained by TLC-SERS detection are 417, 813, 905, 927, 1002, 1159, 1233, 1529, 1560 and cm-1. Integrating and generalizing with common peak obtained by DFT theoretical calculation, namely 417 +/-10 cm-1，813±10cm-1，905±10cm-1，927±10cm-1，1002±10cm-1，1159±10cm-1，1233±10cm-1，1529±10cm-1，1560±10cm-1When the preprocessed surface-enhanced Raman spectrogram at least contains five absorption peaks of sildenafil derivatives, a field inspector can preliminarily judge that sildenafil analogues are doped in a sample to be detected and serve as a 'positive sample' to be verified in a laboratory (step ten). When the preprocessed surface enhanced Raman spectrum contains less than five absorption peaks, the field inspector can preliminarily judge that the sildenafil analog is not doped.
More preferably, the method for screening the sildenafil derivative rapid detection conditions further comprises the following laboratory verification steps:
step ten: UPLC-QTOF/MS laboratory validation
Further confirmation is carried out on the result of the field detection, and the occurrence of a false positive condition is avoided. And establishing a UPLC-TOF/MS detection method. The structure of the novel unknown sildenafil derivative is confirmed. By utilizing a QTOF/MS secondary mass spectrometry detection function and analyzing the cracking rule of sildenafil derivative fragments, the common fragment ions of the nafil derivatives are summarized, and judgment can be carried out according to the common fragment ions of the nafil derivatives under the condition of no standard substance during laboratory verification in the future.
And (3) carrying out qualitative analysis on the sildenafil derivative by adopting a rapid separation ultra-high performance liquid chromatography-time-of-flight mass spectrometer to obtain a total ion flow diagram and common fragment ions of the sildenafil derivative. The accurate molecular weight of the measured substance is determined by the quasi-molecular ion peak obtained by QTOF/MS, and a known chemical composition Database of the sildenafil derivatives is established by the agilent 'Formula Database Generator'. An analysis method for rapidly and accurately analyzing chemical components in the sildenafil derivatives illegally added in the traditional Chinese medicine is established.
In another preferred embodiment of the present invention, there is provided a method for screening rapid detection conditions for amlodipine derivatives, comprising the steps of:
the technical scheme is as follows:
taking amlodipine as a key intermediate, introducing compounds such as alkane substituent, aromatic ring substituent and the like on the amino group of amlodipine, and designing a novel structural type of amlodipine derivative. Calculating theoretical Raman spectra of derivatives thereof by using a density pan theory and summarizing theoretical Raman common peaks, and carrying out feasibility inference on the basis of predicted peaks. Meanwhile, the predicted peak and the actually measured peak are comprehensively compared, and the feasibility that the predicted peak calculated by DFT can replace the actually measured peak is proved when the derivatives of the substances cannot be obtained by field detection or cannot be synthesized in reality. Meanwhile, a laboratory UPLC-QTOF/MS verification method is established. And analyzing the cracking rule of the amlodipine substances by utilizing a QTOF/MS secondary mass spectrum detection function, and summarizing to obtain an MS common peak. The method comprises the following specific steps:
the method comprises the following steps: designer amlodipine derivatives
Analysis of the relationship between amlodipine structure (as shown in FIG. 25) and activity shows that 1, 4-dihydropyridine ring should be retained, carboxylic ester at 3, 5-position should be retained, and two-ring conformation of the compound should be retained. Thus, the most economical, convenient, and most likely to be derivatized is a structural modification at the amino group of amlodipine. Therefore, amlodipine is taken as a key intermediate, and compounds such as alkane substituent, aromatic ring substituent and the like are introduced on the amino group of amlodipine, so that a series of amlodipine derivatives with novel structural types are designed.
Step two, carrying out virtual docking on the amlodipine derivative and the calcium ion channel β 2 retarder
The drug design docking platform Discovery Studio3.0 is adopted to carry out the step one, the target molecule is docked with the calcium ion channel β 2 blocker active cavity, the Libdock molecule docking technology is selected, the amlodipine derivative with high grade is preferably selected according to the grading function after docking (table 3), and the docking result is shown in figure 26.
Step three: theoretical calculation of density function
And (3) adopting GAUSS VIEW 5.0 software to carry out molecular structure optimization on the 16 amlodipine derivatives with excellent virtual screening scores. The DFT method for selecting B3LYP level is characterized in that 16 amlodipine derivatives are subjected to configuration optimization on 6-31G (d, p) groups of a hydrogen atom plus p polarization function and a heavy atom plus d polarization function in a system and then are calculated by GAUSSIAN09 software in a Linux system.
Step four: theoretical consensus peak of amlodipine derivatives
According to the DFT calculation result, adopting GAUSSVIEW 5.0 software to carry out common peak induction on the amlodipine derivative. The DFT of 16 derivatives was found to have 28 common peaks, which are Δ ν 359, 421, 525, 571, 747, 792, 809, 836, 854, 872, 889, 955, 1001, 1011, 1032, 1049, 1102, 1117, 1156, 1187, 1199, 1267, 1282, 1334, 1422, 1451, 1479, 1486cm-1。
Preferably, the method for screening rapid detection conditions for amlodipine derivatives described above further comprises the following steps: (i.e., verification of the above prediction method)
Step five: synthesis and structure verification of amlodipine derivative
Synthesizing the amlodipine derivative optimized in the second step, and verifying the structure through nuclear magnetism and mass spectrum;
according to the second step, preferably 16 derivatives, the synthesis reaction is carried out. The amino position substituent groups are respectively butyl DPA-01, octyl DPA-02, propyl DPA-14, pentyl DPA-15, decyl DPA-16, 2-ethyltoluene DPA-03, 1-tert-butyl-4-ethylbenzene DPA-04, 1-ethyl-4-isopropylbenzene DPA-05, 1-ethyl-3-methoxy benzene DPA-06, 1-ethyl-2-fluorobenzene DPA-07, 1-ethyl-2, 3-fluorobenzene DPA-08, 1-ethyl-3, 5-fluorobenzene DPA-09, 1-ethyl-2-trifluorobenzene DPA-10, 1-ethyl-3-trifluorobenzene DPA-11, 1-ethyl-trifluoromethoxybenzene DPA-12, 1-chloro-3-ethylbenzene DPA-13.
Step six: amlodipine derivative animal activity assay
The derivatives are grouped, aliphatic chain substituents (DPA-01, 02, 14, 15, 16) and aromatic ring substituents (DPA-03-13) are selected from 2 groups, 2 target compounds (DPA-02, 15; DPA-06, 09) are selected from 2 groups respectively to carry out a whole animal antihypertensive test, and amlodipine is taken as a positive control. The results show that the four target compounds have better antihypertensive activity, and the antihypertensive activity is equivalent to that of amlodipine.
Step seven: establishing TLC-SERS detection conditions
Dividing the synthesized 16 derivatives into 2 groups according to the difference of polarities of the synthesized derivatives, wherein the first group comprises DPA-01, DPA-02, DPA-05, DPA-06, DPA-08 and DPA-14-16; the second group is DPA-03, DPA-04, DPA-7 and DPA-09-13, and the expansion system is as follows: acetone-n-hexane-dichloromethane ═ 5:4:1, ethyl acetate-methanol ═ 1: 1.
Dropwise adding a surface reinforcing agent, wherein the surface reinforcing agent is gold sol, silver sol (organic solvent or water) or gold-silver combined glue, and scanning by adopting a portable Raman spectrometer; the method selects organic silver sol preferably, and adopts a portable Raman spectrometer to scan;
step eight: processing non-blank spectrogram
Processing the non-blank spectrogram by adopting standard spectrogram processing software,selecting 400--1The wave band is taken as a characteristic wave band, and 300-1700cm is selected-1The wave band is used as a characteristic wave band, the characteristic wave band is subjected to spectrogram smoothing and baseline correction by adopting OPUS5.0 software, and then vector normalization processing is carried out on the corrected spectral data to obtain a preprocessed surface enhanced Raman spectrogram;
step nine: discriminating the preprocessed surface enhanced Raman spectrogram
Integrating and inducing the common peak obtained by the seven TLC-SERS detection and the common peak obtained by the four DFT theoretical calculation, determining the common absorption peak of the amlodipine derivative and judging the standard of the amlodipine derivative in the sample to be detected;
through SERS spectra, 11 SERS common peaks of 16 derivatives are found, namely delta upsilon 484, 555, 783, 798, 812, 903, 1038, 1100, 1207, 1268 and 1496cm-1. Integration and summarization of the consensus peaks obtained from DFT theory, Δ ν 792, 809, 889, 1032, 1049, 1102, 1199, 1267, 1486cm-1. When the preprocessed surface-enhanced Raman spectrogram at least contains absorption peaks of eight amlodipine derivatives, field detection personnel can preliminarily judge that amlodipine additives are doped in the traditional Chinese medicine to be detected for strengthening yang and serve as a 'positive sample' to be verified in a laboratory (step ten). When the preprocessed surface-enhanced Raman spectrogram contains less than six absorption peaks of the amlodipine additives, field detection personnel can judge that the amlodipine additives are not doped.
As can be seen from the structures of the 16 derivatives, groups such as alkane substituent, aromatic ring substituent and the like are introduced on the amino group of amlodipine, wherein DPA-01, 02, 14, 15 and 16 are different alkane substituents, and the others are different aromatic ring substituents. Because the derivatives have similar structures, the characteristic peaks of each compound are relatively fewer. The characteristic peak of DPA-01 is 1160cm-1. The characteristic peak of DPA-02 is 559cm-1. DPA-07 has a characteristic peak at 419, 1176 cm-1. The characteristic peak of DPA-09 is 505, 1320, 1626cm-1. The characteristic peak of DPA-13 is 1697cm-1. The characteristic peak of DPA-14 is 529cm-1. The characteristic peaks of DPA-15 are 307, 346 and 430cm-1。DPA-16The characteristic peak of (A) is 771cm-1. The derivative doped in the antihypertensive health care product can be judged through the characteristic peak.
More preferably, the method for screening rapid detection conditions for amlodipine derivatives as described above further comprises the following laboratory verification steps:
step ten: laboratory verification method for establishing UPLC-QTOF/MS
Further confirmation is carried out on the result of the field detection, and the occurrence of a false positive condition is avoided. And establishing a UPLC-TOF/MS detection method. Analyzing the cracking rule of amlodipine derivative fragments, and confirming the structure of the novel unknown amlodipine derivative.
And (3) carrying out qualitative analysis on the amlodipine derivative by adopting a rapid separation ultra-high performance liquid chromatography-time of flight mass spectrometer to obtain a total ion flow diagram. The accurate molecular weight of the measured substance is determined by the quasi-molecular ion peak obtained by QTOF/MS, and an amlodipine derivative known chemical composition Database is established by the agilent 'Formula Database Generator' software. An analysis method for rapidly and accurately analyzing chemical components in the traditional Chinese medicine in which the amlodipine derivative is illegally added is established.
Compared with the prior art, the technical scheme of the invention has the advantages and positive effects that:
1. no need of synthetic reference substance, and wide application range
Compared with the prior art, the technical scheme of the invention can realize detection without synthesizing a series of nafil derivatives and a derivative reference substance.
2. Low experimental cost
The computer-aided drug design can carry out simulated docking on the designed compound and target enzymes in a database to evaluate the combination quality of the compound and a target spot and correspondingly evaluate the activity of the compound; the DFT theoretical calculation result can visually reflect the information of molecular vibration, and along with the development of the DFT theory, a large amount of theoretical calculation is compared with the experimental result, so that the credibility of the calculation result is displayed. The invention combines two theoretical calculation methods for application, and under the condition that a series of derivative reference products are not needed (or not obtained), the derivative with better calculation result score is firstly subjected to DFT calculation to predict the theoretical Raman spectrum peak. In this way, in the laboratory or field test in the future, the tester can judge whether the sample is doped or not according to the common peak provided by the study, no matter what derivative or analogue is illegally added by illegal manufacturers.
3. Simple and quick operation, and is suitable for quick field detection
Substances after TLC separation can only judge whether doping phenomenon exists according to the spot and the specific shift value of the substances. If the screening result is further confirmed in the field when the sample is suspected to be positive, a portable instrument (such as a raman spectrometer) capable of providing a characteristic identification spectrum is needed for qualitative analysis, namely, surface enhanced raman spectroscopy. And during field detection, judging whether the health-care product is doped with illegal added compounds or derivatives thereof by combining the predicted common peaks of the non-species through the Raman peak position after TLC-SERS detection.
4. Provides firmer guarantee for the identification of illegally added derivatives in health food
Establishing a field-laboratory quick test verification strategy for illegally adding unknown derivatives in the health care product through five steps of investigation, prediction, detection, induction and confirmation. The method can be used for understanding the biological activity of the series of derivatives without synthesizing a large amount of the series of derivatives, obtaining a characteristic identification spectrogram of the series of derivatives, and has great significance for laboratory or field detection.
Drawings
FIG. 1 is a flow chart of a technical solution of embodiment 1 of the present invention;
FIG. 2 shows the docking results of Discovery Studio3.0 software on PDE-5 and nafil derivatives, which is the step of example 1 of the present invention;
FIG. 3 is the optimized molecular configuration of step two GAUSS VIEW 5.0 software for virtual screening of the best 11 sildenafil derivatives in example 1 of the present invention;
fig. 4 shows the configuration of sildenafil derivative optimized in step two of example 1, which is calculated by the GAUSSIAN09 software (Linux system) and compared with the actually measured raman spectrum;
FIG. 5 shows the general formula of the synthetic route and the structural formula of sildenafil derivative in step III of example 1;
FIG. 6 is a nuclear magnetic resonance spectrum of sildenafil derivative No. S-102 600HMZ in step III of example 1 of the present invention;
FIG. 7 is a nuclear magnetic resonance spectrum of sildenafil derivative No. S-104 600HMZ in step III of example 1 of the present invention;
FIG. 8 is a nuclear magnetic resonance spectrum of sildenafil derivative No. S-109 600HMZ in step III of example 1 of the present invention;
FIG. 9 is a nuclear magnetic resonance spectrum of sildenafil derivative No. S-110 600HMZ in step III of example 1 of the present invention;
FIG. 10 is a nuclear magnetic resonance spectrum of sildenafil derivative No. S-400 in step III of 600HMZ in example 1 of the present invention;
FIG. 11 is a nuclear magnetic resonance spectrum of sildenafil derivative No. S-401 600HMZ in step III of example 1 of the present invention;
FIG. 12 is a nuclear magnetic resonance spectrum of sildenafil derivative No. S-402 (600 HMZ) in step III according to example 1 of the present invention;
FIG. 13 is a nuclear magnetic resonance spectrum of sildenafil derivative No. S-403 600HMZ in step III according to example 1 of the present invention;
FIG. 14 is a nuclear magnetic resonance spectrum of sildenafil derivative No. S-404 (600 HMZ) in step III according to example 1 of the present invention;
FIG. 15 is a nuclear magnetic resonance spectrum of sildenafil derivative No. S-301 (600 HMZ) in step III according to example 1 of the present invention;
FIG. 16 is a nuclear magnetic resonance spectrum of sildenafil derivative No. S-501 600HMZ in step III of example 1 of the present invention;
FIG. 17 shows the result of TLC separation of sildenafil analog and derivative standard substance in step five of example 1 of the present invention: i-1. S-109; i-2, vardenafil; i-3. tadalafil; i-4. S-403; i-5. S-404; i-6. S-401; i-7. S-501; II-1. S-102; II-2. S-104; II-3. S-110; II-4. S-301; II-5. S-400; II-6. S-1402; II-7, sildenafil;
fig. 18 is a detection result after optimizing the detection conditions of the step five SERS in embodiment 1 of the present invention;
FIG. 19 shows SERS detection results of a pentasildenafil analog and derivative standard in example 1;
FIG. 20 shows the result of simulated sample TLC separation in step seven of example 1 of the present invention;
FIG. 21 is a simulated sample SERS acquisition spectrum in step seven of example 1 of the present invention;
FIG. 22 is a UPLC-QTOF/MS liquid phase total ion flow diagram in step eight of example 1 of the present invention.
FIG. 23 shows the result of TLC separation of sildenafil analog standard in step C of example 2 of the present invention: 1. sildenafil; 2. citric acid hydroxy-pimomosildenafil; 3. vardenafil; 4. pseudo-vardenafil; 5. homo sildenafil; 6. 1, Hondinafei; 7. tadalafil; 8. mixing;
fig. 24 shows a detection result after the SERS detection condition is optimized in step C in embodiment 2 of the present invention.
FIG. 25 is a flowchart of the embodiment 3 of the present invention;
FIG. 26 shows the docking of an amlodipine derivative with a calcium channel blocker β 2 receptor active cavity in step one of example 3 of the present invention;
FIG. 27 is the optimized molecular configuration of the GAUSS VIEW 5.0 software for virtually screening the best scoring 16 amlodipine derivatives in step two of example 3;
fig. 28a and 28b are graphs showing the configuration optimization of amlodipine derivatives in step two of example 3, calculated by GAUSSIAN09 software (Linux system), and compared with the actually measured raman spectrum;
FIG. 29 is a general synthesis formula and a structural formula of three amlodipine derivatives in the procedure of example 3;
FIG. 30 is a nuclear magnetic resonance spectrum of amlodipine DPA-01 derivative No. 600HMZ in step two of example 3;
FIG. 31 is a nuclear magnetic hydrogen spectrum of amlodipine DPA-02 derivative number 600HMZ in step two of example 3 of the present invention;
FIG. 32 is a nuclear magnetic resonance spectrum of amlodipine DPA-03 derivative number 600HMZ in step two according to example 3 of the present invention;
FIG. 33 is a nuclear magnetic resonance spectrum of amlodipine DPA-04 derivative No. 600HMZ in step two according to example 3 of the present invention;
FIG. 34 is a nuclear magnetic hydrogen spectrum of amlodipine DPA-05 derivative No. 600HMZ in step two of example 3;
FIG. 35 is a nuclear magnetic resonance spectrum of amlodipine DPA-06 derivative number 600HMZ in step two of example 3 according to the present invention;
FIG. 36 is a nuclear magnetic resonance spectrum of amlodipine DPA-07 derivative No. 600HMZ in step two according to example 3 of the present invention;
FIG. 37 is a nuclear magnetic resonance spectrum of amlodipine DPA-08 derivative 600HMZ in step two according to example 3 of the present invention;
FIG. 38 is a nuclear magnetic resonance spectrum of amlodipine DPA-09 derivative number 600HMZ in step two according to example 3 of the present invention;
FIG. 39 is a nuclear magnetic resonance spectrum of amlodipine DPA-10 derivative 600HMZ in step two according to example 3 of the present invention;
FIG. 40 is a nuclear magnetic resonance spectrum of amlodipine DPA-11 derivative No. 600HMZ in step two according to example 3 of the present invention;
FIG. 41 is a nuclear magnetic resonance spectrum of amlodipine DPA-12 derivative No. 600HMZ in step two of example 3;
FIG. 42 is a nuclear magnetic resonance spectrum of amlodipine DPA-13 derivative 600HMZ in step two according to example 3 of the present invention;
FIG. 43 is a nuclear magnetic resonance spectrum of amlodipine DPA-14 derivative No. 600HMZ in step two according to example 3 of the present invention;
FIG. 44 is a nuclear magnetic resonance spectrum of amlodipine DPA-15 derivative No. 600HMZ in step two of example 3 according to the present invention;
FIG. 45 is a nuclear magnetic resonance spectrum of amlodipine DPA-16 derivative No. 600HMZ in step two according to example 3 of the present invention;
FIG. 46 is a graph showing a comparison of the Systolic Blood Pressure (SBP) and Diastolic Blood Pressure (DBP) of the derivatives DPA-02, DPA-06, DPA-09 and DPA-15 with amlodipine at 6 hours after administration in step four of example 3;
FIG. 47 shows TLC separation of 16 amlodipine derivatives in step five in example 3 of the present invention; (ii) a
Fig. 48 is a SERS graph showing 16 amlodipine derivatives in step six in example 3;
FIG. 49 is a comparison of Raman spectra of sample No. 26 to be tested and sample No. DPA-12 in step seven of example 3 according to the present invention;
FIG. 50 is a diagram showing the total ion flow of amlodipine in step eight in example 3 of the present invention;
FIG. 51 shows the splitting pattern of nonaamlodipine in step 3 according to the present invention;
FIG. 52 is a sample extraction chromatogram of step No. 26 of step ten in example 3 of the present invention;
FIG. 53 is a second mass spectrum of sample No. 26 from step ten in example 3 of the present invention.
Detailed Description
The following will further explain the embodiments of the present invention with reference to the drawings.
Example 1
In this embodiment, a specific embodiment of the technical solution of fig. 1 is further explained. The software used includes: drug design platform Discovery Studio 3.0; GAUSSIAN09 package.
In this embodiment, the apparatus used includes: portable Raman spectrometer (BWS415, B)&W Tek inc., usa), excitation wavelength 785nm, resolution 5cm-1The spectrum coverage range is 175-3200cm-1(ii) a WFH-203B three-purpose ultraviolet analyzer (Shanghai Jingke industries, Ltd.); ultrasonic instrument (KUDOS-SK5200HP, China); centrifuges (HERAEUS, FRESCO17 Centrifuge; Thermo Scientific, USA); analytical balance (METTLER AE240, china); standard spectrum processing software OPUS 5.0.
In this embodiment, the materials used include: thin layer chromatography separation plate F254(10X 20cm) (Merck, Darmstadt, Germany); silver nitrate, trisodium citrate (AR, chemical agents ltd, national drug group); other used reagents are analytically pure and purchased from chemical reagents of national drug group, Inc.; chromatographically pure formic acid was purchased from Tedia (USA)) (ii) a Mass Spectrometry pure methanol was purchased from Fisher Scientific (USA); ultrapure water was produced by Milli-Q Academic ultrapure water system (Millipore, USA). The column was an ACQUITY UPLCTM BEH C18column (2.1 mm. times.100 mm,1.7 μm, Waters, Milford, Mass.).
The method comprises the following steps:
designing about 50 substituents such as piperazines, aromatic rings, aliphatic chains and the like at the position of methylpiperazine in a sildenafil structure, butting 50 designed target molecules with a crystal structure active cavity of a phosphodiesterase type 5 (PDE-5) sildenafil compound by adopting drug design software Discovery studio3.0 based on a computer aided design method, selecting Libdock molecular docking technology, and selecting 11 compounds with high Libdock scoring function, namely ethylpiperazine S-102, propylpiperazine S-109, butylpiperazine S-104, sunflower-based piperazine S-104, piperidine substituent S-301, aromatic ring substituent S-400, methyl aromatic ring substituent S-401, ethyl aromatic ring substituent S-402, propyl aromatic ring substituent S-403, butyl aromatic ring substituent S-404 and 4-methylimidazole substituent S-501 (Table 1), the docking results for the 11 derivatives are shown in figure 2.
TABLE 1 LibDock docking scoring results
Step two:
the molecular configuration was optimized using GAUSS VIEW 5.0 (fig. 3). In the calculation process, a DFT method of B3LYP level is selected, and the DFT method is composed of a correlation function of Lee-yang-Parr and a hybrid exchange function of Becke. The 6-31G (d, p) group of the polarization function of hydrogen atom plus p polarization function and heavy atom plus d in the system is calculated by the software GAUSSIAN09 in the Linux systematicness after the configuration optimization of 11 sildenafil derivatives. Comparing the calculated theoretical raman spectrum with the actually measured raman spectrum.
The results of DFT calculation, common peak of Raman measured values (FIG. 4) and TLC-SERS measured values of 11 sildenafil derivatives were integrated and analyzed by alignment. It was found that there were 41 common peaks in the DFT theoretical Raman value, each of which wasIn the examples of the present invention, the total number of DFT theoretical Raman value peaks for summarizing the absorption peaks of sildenafil derivatives was 15, each 417,485,515,593,805,890,916,941,1010,1041,1150,1253,1263,1523,1550cm, based on the theoretical peak absorption intensity-1。
9 common peaks obtained by TLC-SERS detection are 417, 813, 905, 927, 1002, 1159, 1233, 1529, 1560 and cm-1. Integrating and generalizing with common peak obtained by DFT theoretical calculation, namely 417 +/-10 cm-1，813±10cm-1，905±10cm-1，927±10cm-1，1002±10cm-1，1159±10cm-1，1233±10cm-1，1529±10cm-1，1560±10cm-1From the results, the measured values are substantially similar to the theoretical values (allowable error is + -10 cm)-1)。
And when the preprocessed surface-enhanced Raman spectrogram at least contains five absorption peaks, judging that the traditional Chinese medicine to be detected for strengthening Yang is doped with the sildenafil derivative, and when the preprocessed surface-enhanced Raman spectrogram contains less than five absorption peaks, judging that the traditional Chinese medicine to be detected for strengthening Yang is not doped with the sildenafil derivative.
Step three:
the synthesis and separation studies of the compounds were performed on the 11 preferable sildenafil derivatives to verify the predictive ability of Libdock and DFT calculations. The general formula of the synthetic route is shown in figure 5.
(1) Preparation of S-102
Dissolving 5mmol of 3- (6, 7-dihydro-1-methyl-7-oxo-3-propyl-1H-pyrazolo [4,3-d ] pyrimidine-5-yl) -4-ethoxy-benzenesulfonyl in 30ml of chloroform, carrying out ice bath, stirring for dissolving, adding 10mmol of triethylamine and 5mmol of N-ethylpiperazine in 25ml of chloroform, clarifying, pouring into a reaction bottle, stirring for reaction, and completely reacting (monitoring the reaction progress by TLC). And pouring the reactants into a separating funnel, washing with a saturated sodium bicarbonate solution, washing with a sodium chloride solution, washing with water, separating out an organic layer, drying with anhydrous magnesium sulfate, adding a proper amount of n-hexane into the organic layer, slowly separating out crystals, filtering, and drying to obtain 0.9g of a colorless solid. The yield was 36.8%.
1H NMR(600MHz,CDCL3)，δ10.83(s,1H), δ 8.79(d, J ═ 2.40Hz,1H), δ 7.80(dd, J ═ 2.40,8.70Hz,1H), δ 7.13(d, J ═ 8.76Hz,1H), δ 4.36(q, J ═ 6.98Hz,2H), δ 4.26(s,3H), δ 3.09(s,4H), δ 2.91(t, J ═ 15.12Hz,3H), δ 2.52(s,4H), δ 2.4(q, J ═ 7.11Hz,2H), δ 1.85(q, J ═ 7.56Hz,2H), δ 1.87-1.82(m,2H), δ 1.63(t, J ═ 6.99Hz,3H), δ 1.03-0.99 (t, 6H) (see fig.). HRMS [ M + H ]]489.61。
(2) Preparation of S-104
2.1g (5mmol) of 3- (6, 7-dihydro-1-methyl-7-oxo-3-propyl-1H-pyrazolo [4,3-d ] pyrimidin-5-yl) -4-ethoxy-benzenesulfonyl was dissolved in 30ml of chloroform at room temperature with stirring, 1.01g (10mmol) of triethylamine and 0.71g (5mmol) of N-butylpiperazine were added thereto with stirring, and the mixture was refluxed for 2 hours to complete the reaction (the progress of the reaction was monitored by TLC). And (3) post-treatment: transferring the reaction solution into a separating funnel, washing 20ml by using a saturated sodium bicarbonate solution, washing 20ml by using a saturated sodium chloride solution, washing by using water, drying an organic layer by using anhydrous magnesium sulfate, decoloring by using activated carbon, and evaporating chloroform to obtain a crude product. Adding dichloromethane 20ml into the crude product, dissolving, adding n-hexane 10ml, clarifying, crystallizing, filtering, and drying to obtain colorless solid 1.15 g. The yield was 44.5%.
1H NMR(600MHz,CDCL3) δ 10.82(s,1H), δ 8.83(d, J ═ 2.4Hz,1H), δ 7.84(dd, J ═ 2.43,8.73Hz,1H), δ 7.15(d, J ═ 8.12Hz,1H), δ 4.38(q, J ═ 6.96Hz,2H), δ 4.28(s,3H), δ 3.12(brs,4H), δ 2.93(t, J ═ 15.06Hz,2H), δ 2.54(brs,4H), δ 2.34(brs,2H), δ 1.89-1.84(m,2H), δ 1.66(t, J ═ 6.96Hz,3H), δ 1.41(brs,2H), δ 1.30-1.27(m,2H), δ 1.03(t, J ═ 6.96Hz,3H), δ 1.41(brs,2H), δ 1.30-1.27(m,2H), δ 7.03 (t, J ═ 6.88H), 7.7H) (fig. 7H), δ 7H). HRMS [ M + H ]]517.51。
(3) Preparation of S-109
Dissolving 5mmol of 3- (6, 7-dihydro-1-methyl-7-oxo-3-propyl-1H-pyrazolo [4,3-d ] pyrimidine-5-yl) -4-ethoxy-benzenesulfonyl in 30ml of chloroform, stirring for dissolving at room temperature, adding 10mmol of triethylamine and 5mmol of N-isopropylpiperazine, stirring for reacting, and refluxing for 2H to complete the reaction (TLC monitors the reaction progress). And (3) post-treatment: transferring the reaction solution into a separating funnel, washing 20ml by using a saturated sodium bicarbonate solution, washing 20ml by using a saturated sodium chloride solution, washing by using water, drying an organic layer by using anhydrous magnesium sulfate, decoloring by using activated carbon, and evaporating chloroform to obtain a crude product. Adding dichloromethane 20ml into the crude product, dissolving, adding n-hexane 10ml, clarifying, crystallizing, filtering, and drying to obtain colorless solid 0.7 g. The yield was 27.9%.
1H NMR (600MHz, CDCl3), δ 10.81(s,1H), δ 8.83(d, J ═ 2.46Hz,1H), δ 7.83(dd, J ═ 2.46,8.71Hz,1H), δ 7.15(d, J ═ 8.76Hz,1H), δ 4.38(q, J ═ 7.00Hz,2H), δ 4.28(s,3H), δ 3.10(brs,4H), δ 2.93(t, J ═ 15.12Hz,2H), δ 2.69-2.67(m,1H), δ 2.62(brs,4H), δ 1.89-1.85(m,2H), δ 1.65(t, J ═ 6.99Hz,3H), δ 1.03(t, J ═ 7.38, 3H), δ 1.00(d, 6.6 Hz,6H) (fig. 6H). HRMS [ M + H ]]503.68。
(4) Preparation of S-110
Dissolving 5mmol of 3- (6, 7-dihydro-1-methyl-7-oxo-3-propyl-1H-pyrazolo [4,3-d ] pyrimidine-5-yl) -4-ethoxy-benzenesulfonyl in 30ml of chloroform, stirring for dissolving, adding 10mmol of triethylamine and 5mmol of N-decylpiperazine, stirring for reacting, and refluxing for 2H to complete the reaction (TLC monitors the reaction progress). And (3) post-treatment: transferring the reaction solution into a separating funnel, washing 20ml by using a saturated sodium bicarbonate solution, washing 20ml by using a saturated sodium chloride solution, washing by using water, drying an organic layer by using anhydrous magnesium sulfate, decoloring by using activated carbon, and evaporating chloroform to obtain a crude product. Adding dichloromethane 20ml into the crude product, dissolving, adding n-hexane 10ml, clarifying, crystallizing, filtering, and drying to obtain colorless solid 0.9 g. The yield was 30.0%.
1H NMR(600MHz,CDCL3) δ 10.81(s,1H), δ 8.83(d, J ═ 2.22Hz,1H), δ 7.83(dd, J ═ 2.36,8.76Hz,1H), δ 7.14(d, J ═ 8.76Hz,1H), δ 4.37(q, J ═ 7.00Hz,2H), δ 4.28(s,3H), δ 3.10(brs,4H), δ 2.93(t, J ═ 15.12Hz,2H), δ 2.53(brs,4H), δ 2.31(t, J ═ 14.82Hz,2H), δ 1.89-1.83(m,2H), δ 1.65(t, J ═ 6.96Hz,3H), δ 1.40(brs,2H), δ 1.26(q, J ═ 7.02, 2H), δ 1.86 (t, J ═ 3H), δ 1.3H) (fig. 7.02, 3H), δ 1H, 3H, δ 1H, δ 3H, and δ 3H (q, 3H). HRMS [ M + H ]]601.35。
(5) Preparation of S-400
Dissolving 5mmol of 3- (6, 7-dihydro-1-methyl-7-oxo-3-propyl-1H-pyrazolo [4,3-d ] pyrimidine-5-yl) -4-ethoxy-benzenesulfonyl in 30ml of chloroform, stirring for dissolving at room temperature, adding 10mmol of triethylamine and 5mmol of aniline, stirring for reacting, and refluxing for 2H to complete the reaction (TLC monitors the reaction progress). And (3) post-treatment: transferring the reaction solution into a separating funnel, washing 20ml by using a saturated sodium bicarbonate solution, washing 20ml by using a saturated sodium chloride solution, washing by using water, drying an organic layer by using anhydrous magnesium sulfate, decoloring by using activated carbon, and evaporating chloroform to obtain a crude product. Adding dichloromethane 20ml into the crude product, dissolving, adding n-hexane 10ml, clarifying, crystallizing, filtering, and drying to obtain colorless solid 0.7 g. The yield was 29.9%.
1H NMR(600MHz,CDCL3) δ 10.79(s,1H), δ 8.86(d, J ═ 2.40Hz,1H), δ 7.79(dd, J ═ 6.30,11.22Hz,1H), δ 7.26-7.24(m,2H), δ 7.15-7.10(m,3H), δ 7.01(t, J ═ 7.74Hz,2H), δ 4.30(q, J ═ 6.98Hz,2H), δ 4.26(s,3H), δ 2.78(t, J ═ 15.18Hz,2H), δ 1.87-1.81(m,2H), δ 1.59(t, J ═ 6.96Hz,3H), δ 1.03(t, J ═ 7.38Hz,3H) (see fig. 10). HRMS [ M + H ]]468.17。
(6) Preparation of S-401
Dissolving 5mmol of 3- (6, 7-dihydro-1-methyl-7-oxo-3-propyl-1H-pyrazolo [4,3-d ] pyrimidine-5-yl) -4-ethoxy-benzenesulfonyl in 30ml of chloroform, stirring for dissolving at room temperature, adding 10mmol of triethylamine and 5mmol of 4-methylaniline, stirring for reacting, and refluxing for 2H to complete the reaction (TLC monitors the reaction progress). And (3) post-treatment: transferring the reaction solution into a separating funnel, washing 20ml by using a saturated sodium bicarbonate solution, washing 20ml by using a saturated sodium chloride solution, washing by using water, drying an organic layer by using anhydrous magnesium sulfate, decoloring by using activated carbon, and evaporating chloroform to obtain a crude product. Adding dichloromethane 20ml into the crude product, dissolving, adding n-hexane 10ml, clarifying, crystallizing, filtering, and drying to obtain colorless solid 0.8 g. The yield was 33.2%.
1H NMR (600MHz, DMSO), δ 12.12(s,1H), δ 10.14(s,1H), δ 7.77(d, J ═ 2.46Hz,1H), δ 7.80(dd, J ═ 2.52,8.64Hz,1H), δ 7.27(d, J ═ 8.88Hz,1H), δ 7.05-7.01(m,4H), δ 4.15(s,3H), δ 4.14(q, J ═ 7.02Hz,2H), δ 2.79(t, J ═ 7.38Hz,2H), δ 2.19(s,3H), δ 1.79-1.73(m,2H), δ 1.31(t, J ═ 6.96Hz,3H), δ 0.96(t, J ═ 7.38Hz, 3H). (see fig. 11). HRMS [ M + H ]]482.19。
(7) Preparation of S-402
Dissolving 5mmol of 3- (6, 7-dihydro-1-methyl-7-oxo-3-propyl-1H-pyrazolo [4,3-d ] pyrimidine-5-yl) -4-ethoxy-benzenesulfonyl in 30ml of chloroform, stirring for dissolving at room temperature, adding 10mmol of triethylamine and 5mmol of 4-ethylaniline, stirring for reacting, and refluxing for 2H to complete the reaction (TLC monitors the reaction progress). And (3) post-treatment: transferring the reaction solution into a separating funnel, washing 20ml by using a saturated sodium bicarbonate solution, washing 20ml by using a saturated sodium chloride solution, washing by using water, drying an organic layer by using anhydrous magnesium sulfate, decoloring by using activated carbon, and evaporating chloroform to obtain a crude product. Adding dichloromethane 20ml into the crude product, dissolving, adding n-hexane 10ml, clarifying, crystallizing, filtering, and drying to obtain colorless solid 0.8 g. The yield was 33.3%.
1H NMR (600MHz, DMSO), δ 12.13(s,1H), δ 10.17(s,1H), δ 7.97(d, J ═ 2.46Hz,1H), δ 7.83(dd, J ═ 2.28,8.82Hz,1H), δ 7.27(, J ═ 8.94Hz,1H), δ 7.08-7.04(m,4H), δ 4.16(s,3H), δ 4.15(q, J ═ 7.02Hz,2H), δ 2.79(t, J ═ Hz,7.56H,2H), δ 2.48(q, J ═ Hz,7.56H,2H), δ 1.79-1.72(m,2H), δ 1.31(t, J ═ 6.96Hz,3H), δ 1.09(t, J ═ 7.56H,2H), δ 1.95 (t, 3H), δ 1.38H) (fig. 12H, 3H). HRMS [ M + H ]]496.20。
(8) Preparation of S-403
Dissolving 5mmol of 3- (6, 7-dihydro-1-methyl-7-oxo-3-propyl-1H-pyrazolo [4,3-d ] pyrimidine-5-yl) -4-ethoxy-benzenesulfonyl in 30ml of chloroform, stirring for dissolving at room temperature, adding 10mmol of triethylamine and 5mmol of 4-propylaniline, stirring for reacting, and refluxing for 2H to complete the reaction (TLC monitors the reaction progress). And (3) post-treatment: transferring the reaction solution into a separating funnel, washing 20ml by using a saturated sodium bicarbonate solution, washing 20ml by using a saturated sodium chloride solution, washing by using water, drying an organic layer by using anhydrous magnesium sulfate, decoloring by using activated carbon, and evaporating chloroform to obtain a crude product. Adding dichloromethane 20ml into the crude product, dissolving, adding n-hexane 10ml, clarifying, crystallizing, filtering, and drying to obtain colorless solid 1.1 g. The yield was 43.1%.
1H NMR (600MHz, DMSO), δ 12.12(s,1H), δ 10.1478(s,1H), δ 7.93(d, J ═ 2.46Hz,1H), δ 7.82(dd, J ═ 2.46,8.88Hz,1H), δ 7.27(d, J ═ 8.94Hz,1H), δ 7.06-7.02(m,4H), δ 4.16(s,3H), δ 4.15(q, J ═ 7.02Hz,2H), δ 2.78(t, J ═ 7.38Hz,2H), δ 2.43(t, J ═ 7.44Hz,2H), δ 1.77-1.73(m,2H), δ 1.50-1.46(m,2H), δ 1.31(t, J ═ 6.96, 3H), δ 0.95(t, 3H), δ 1.81 (t, 3H), δ 1.13H) (fig. 13H, 13H). HRMS [ M + H ]]510.22
(9) Preparation of S-404
Dissolving 5mmol of 3- (6, 7-dihydro-1-methyl-7-oxo-3-propyl-1H-pyrazolo [4,3-d ] pyrimidine-5-yl) -4-ethoxy-benzenesulfonyl in 30ml of chloroform, stirring for dissolving at room temperature, adding 10mmol of triethylamine and 5mmol of 4-butylaniline, stirring for reacting, and refluxing for 2H to complete the reaction (TLC monitors the reaction progress). And (3) post-treatment: transferring the reaction solution into a separating funnel, washing 20ml by using a saturated sodium bicarbonate solution, washing 20ml by using a saturated sodium chloride solution, washing by using water, drying an organic layer by using anhydrous magnesium sulfate, decoloring by using activated carbon, and evaporating chloroform to obtain a crude product. Adding dichloromethane 20ml into the crude product, dissolving, adding n-hexane 10ml, clarifying, crystallizing, filtering, and drying to obtain colorless solid 0.7 g. The yield was 26.7%.
1H NMR (600MHz, DMSO), δ 12.12(s,1H), δ 10.15(s,1H), δ 7.95(d, J ═ 2.46Hz,1H), δ 7.83(dd, J ═ 2.52,8.82Hz,1H), δ 7.27(d, J ═ 8.94Hz,1H), δ 7.05-7.02(m,4H), δ 4.16(s,3H), δ 4.15(q, J ═ 6.96Hz,2H), δ 2.78(t, J ═ 15.00Hz,2H), δ 2.44(t, J ═ 15.42Hz,2H), δ 1.77-1.73(m,2H), δ 1.46-1.41(m,2H), δ 1.31(t, J ═ 6.96, 3H), δ 1.24 (m,2H), δ 1.35H) (fig. 7H, 3H), δ 7.35H, 3H, δ,3H, fig. HRMS [ M + H ]]524.23。
(10) Preparation of S-301
Dissolving 5mmol of 3- (6, 7-dihydro-1-methyl-7-oxo-3-propyl-1H-pyrazolo [4,3-d ] pyrimidine-5-yl) -4-ethoxy-benzenesulfonyl in 30ml of chloroform, stirring for dissolving at room temperature, adding 10mmol of triethylamine and 5mmol of 4-methylpiperidine, stirring for reacting, and refluxing for 2H to complete the reaction (TLC monitors the reaction progress). And (3) post-treatment: transferring the reaction solution into a separating funnel, washing 20ml by using a saturated sodium bicarbonate solution, washing 20ml by using a saturated sodium chloride solution, washing by using water, drying an organic layer by using anhydrous magnesium sulfate, decoloring by using activated carbon, and evaporating chloroform to obtain a crude product. Adding dichloromethane 20ml into the crude product, dissolving, adding n-hexane 10ml, clarifying, crystallizing, filtering, and drying to obtain colorless solid 1.2 g. The yield was 50.6%.
1H NMR(600MHz,CDCL3)，δ10.85(s,1H)，δ8.79(d,J＝2.4Hz,1H)，δ7.82(dd,J＝2.46,8.70Hz,1H)，δ7.13(d,J＝8.82Hz,1H)，δ4.36(q,J＝6.98Hz,2H)，δ4.25(s,3H)，δ3.78(d,J＝11.4Hz,2H)，δ2.92(t,J＝11.4Hz,2H)，δ2.33(t,J＝11.4Hz,2H), δ 1.87-1.83(m,2H), δ 1.67(d, J ═ 9.78Hz,2H), δ 1.63(t, J ═ 6.96Hz,3H), δ 1.33-1.29(m,3H), δ 1.01(t, J ═ 7.35Hz,3H), δ 0.91(d, J ═ 5.40Hz,3H) (see fig. 15). HRMS [ M + H ]]474.22。
(11) Preparation of S-501
Dissolving 5mmol of 3- (6, 7-dihydro-1-methyl-7-oxo-3-propyl-1H-pyrazolo [4,3-d ] pyrimidine-5-yl) -4-ethoxy-benzenesulfonyl in 30ml of chloroform, stirring for dissolving at room temperature, adding 10mmol of triethylamine and 5mmol of 4-methylimidazole, stirring for reacting, and refluxing for 2H to complete the reaction (TLC monitors the reaction progress). And (3) post-treatment: transferring the reaction solution into a separating funnel, washing 20ml by using a saturated sodium bicarbonate solution, washing 20ml by using a saturated sodium chloride solution, washing by using water, drying an organic layer by using anhydrous magnesium sulfate, decoloring by using activated carbon, and evaporating chloroform to obtain a crude product. Adding dichloromethane 20ml into the crude product, dissolving, adding n-hexane 10ml, clarifying, crystallizing, filtering, and drying to obtain colorless solid 0.75 g. The yield was 33.0%.
1H NMR (600MHz, DMSO), δ 12.26(s,1H), δ 8.26(s,1H), δ 8.16(s,1H), δ 8.14-8.12(m,1H), δ 7.44(s,1H), δ 7.41-7.39(m,1H), δ 4.20(q, J ═ 6.98Hz,2H), δ 4.15(s,3H), δ 2.76(t, J ═ 7.5Hz,2H), δ 2.06(s,3H), δ 1.73-1.72(m,2H), δ 1.29(t, J ═ 6.96Hz,3H), δ 0.94(t, J ═ 7.35Hz,3H) (see fig. 16). HRMS [ M + H ]]457.17。
Step four: sildenafil derivative binding kinetics assay validation
A Biacore T200 molecular interaction analysis system was used. The compounds with different concentrations are sequentially injected, experimental data are analyzed by Biacore T200evaluation software, a kinetic binding model is adopted to fit and calculate experimental results, the binding activity of the derivatives is obtained, and the results of molecular docking calculation are verified (Table 2).
TABLE 2 sildenafil derivative binding kinetics assay results
"-" indicates that the compound affinity cannot be accurately calculated at the experimental concentration
Step five: establishing TLC-SERS detection conditions
Performing TLC separation on the control (as shown in FIG. 17), and sucking 1 μ L of the control mixed solution respectively, and spotting on the same thin layer chromatography separation plate F254(10X 20cm) divided into 2 groups: group (1) is vardenafil, tadalafil, S-109, S-403, S-404, S-401, S-501; and the group (2) is prepared by taking 1 mu l of sildenafil citrate, S-102, S-104, S-110, S-301, S-400 and S-402 respectively, adopting optimized TLC (thin layer chromatography) development conditions, spotting at a position 1-2 cm away from the bottom of the thin layer plate, and adopting dichloromethane, acetone and ammonia water to develop for 25min at a ratio of 20:1.5:0.2 (v/v). And taking out the thin layer plate after the thin layer plate is unfolded to be 7cm away from the top of the thin layer plate, airing, and inspecting thin layer spots under an ultraviolet lamp at 365 nm.
TLC separation results: group (1) S-109, vardenafil, tadalafil, S-403, S-404, S-401, S-501RfValues of 0.13,0.02,0.24,0.43,0.45,0.40,0.22, respectively; group (2) S-102, S-104, S-110, S-301, S-400, S-402, sildenafil citrate RfThe values are 0.24,0.51,0.69,0.75,0.35,0.41,0.10, respectively.
Control product R in positioning chromatographic stripfAnd (3) dropping 4 mu L of organic silver sol at the position with the same value, collecting signals at the spot position where the silver sol is dropped by using a portable Raman spectrometer, and determining that the SERS spectrum is finally carried out by adopting the integration time of 20s and the laser power of 80mW (figure 19) after the experimental conditions are optimized (figure 18). The number of times of collection was 3.
Step six: data analysis method
And (3) performing data preprocessing on the obtained spectrum by adopting Matlab 13.0 software, and selecting a spectrum section (400-1700 cm & lt-1 & gt), smoothing (Sgolay method), baseline correction (airPLS method) and Normalization (Min-Max Normalization method). The origine version 8.0 was used for mapping.
Step seven: simulated positive sample for TLC-SERS detection
The simulated positive samples were subjected to TLC separation (see FIG. 20), and 1. mu.L of the control mixed solution was pipetted onto the same thin layer chromatography separation plate F254(10 multiplied by 20cm), and adopting optimized TLC (thin layer chromatography) development conditions to perform the thin layer plate at a position 1-2 cm away from the bottom of the thin layer plateSpotting was performed for 25min using dichloromethane, acetone, ammonia (20: 1.5: 0.2) (v/v). And taking out the thin layer plate after the thin layer plate is unfolded to be 7cm away from the top of the thin layer plate, airing, and inspecting thin layer spots under an ultraviolet lamp at 254 nm.
Sample No. 19, R with S-109fSERS spectra of the same positions are 419, 484, 553, 630, 658, 712, 904, 926, 1098, 1259, 1293, 1312, 1560 and 1583cm-1Is at a peak, with R of S-301fThe same-position SERS spectrum is 483, 556, 725, 819, 1237, 1339 and 1528cm-1Is at a peak, with R of S-400fThe same position SERS spectrum has peaks at 525, 570, 733, 753, 910, 948, 1040, 1096, 1236, 1266, 1530, 1562cm-1, and R of S-501fSame position SERS spectra are obtained at 418, 484, 535, 819, 926, 990, 1009, 1185, 1236, 1332 and 1529cm-1The peak is substantially consistent with the common peak obtained in the summary of the present study, which indicates that sample No. 19 contains sildenafil derivatives (see fig. 21).
Step eight: establishment of UPLC-QTOF/MS detection method for sildenafil derivative structure (as shown in FIG. 22)
Liquid phase conditions: the column was an ACQUITY UPLCTM BEH C18column (2.1 mm. times.100 mm,1.7m, Waters, Milford, MA). Mobile phase a was 0.1% aqueous formic acid and mobile phase B was 0.1% acetonitrile formic acid, using gradient elution, the procedure was: 0-2 min, 5-5% of B; 2-28 min, 5-95% B; 28-30 min, 95% B. The analysis time was 30 min. The column temperature was 40 ℃, the flow rate was 0.35ml/min, and the sample volume was 2. mu.l.
Mass spectrum conditions: the ion source is an ESI source, the positive ion mode is adopted, and the mass spectrum scanning range is 100-1100 m/z; detecting parameters: the ionization voltage is 4000V, the flow rate of the drying gas is 11l/min, and the temperature of the drying gas is 350 ℃; atomizer pressure was 45psig, cracker voltage was 120V, skimmer 60V, eight-step rod rf voltage 750V. Internal standard ions of m/z 121.0509 and m/z 922.0098 were selected for real-time mass number correction. Before each sample measurement, the mass axis was calibrated using a tuning mix (Turning mix). Experimental data was collected and analyzed using MassHunter software version B.03.00(Agilent technologies, USA) software. Further MS/MS analysis was performed, adjusting the collision energy between 10-30V depending on the ion condition.
Step nine: analysis of the mass spectrum cleavage law of the derivative structure
By setting the fragment voltage, the secondary mass spectrometry analysis was performed on the excimer ions of sildenafil (see table 3), and the cleavage pattern of the derivative was similar to that of sildenafil. The 11 sildenafil derivatives share fragment ions of m/z377, 329, 311, 299, 283, 269, 166. S-102, 109, 104, 110 is mainly substituted by piperazine species with a common fragment ion m/z of 461, 99, 84, 72, 58; s-400-404 is mainly substituted by aromatic hydrocarbon substances, and the m/z of the common fragment ion is 360, 347.
TABLE 3 sildenafil derivatives major fragment ions
In conclusion, according to the invention, sildenafil derivatives are used as research objects, CADD is used as a basis for screening derivative structures, DFT theoretical calculation is used as a characteristic identification spectrogram peak, TLC-SERS experiments are adopted for verification, and virtual screening and prediction research of health food doped PDE-5 inhibitors is carried out. The comprehensive, rapid and accurate analysis strategy of 'prediction-detection-confirmation' is established, and the method has great theoretical guiding significance and application value.
Example 2
Step A, pretreatment of a sample to be detected: weighing 1mg of a traditional Chinese medicine to be detected, dissolving in 10ml of methanol, fixing the volume for 3 minutes, placing in an ultrasonic instrument after 5 minutes of vortex, extracting for 30 minutes, centrifuging for 10 minutes (5000rpm, 4 ℃), preserving in a refrigerator at 4 ℃ and detecting;
step B, pipetting 1. mu.L of the solution, and spotting the solution on a thin-layer chromatography separation plate F254(10 × 20cm) (Merck, Darmstadt, Germany), 1 μ l of each standard sample sildenafil citrate, hydroxymanometalafil citrate, vardenafil, pseudo-vardenafil, manometalafil, reddenafil and tadalafil was sampled at a distance of 1-2 cm from the bottom of the thin layer plate under optimized TLC development conditions, and developed for 25min with dichloromethane, acetone, ammonia water, 20:1.5:0.2 (v/v). Thin layer plate top 7 to be unfoldedcm, taking out, air drying, and inspecting thin layer spots under an ultraviolet lamp at 254 nm.
And step C, taking out after the development is completed, airing, and inspecting thin-layer spots under an ultraviolet lamp at 254nm (figure 23). In the R of the analoguefAt the value: 0.108, 0.036, 0.072, 0.373, 0.169, 0.024 and 0.253 parts of surface reinforcing agent is dripped, the surface reinforcing agent is 3 mu L of water gel, and a portable Raman spectrometer is adopted to collect signals of spot positions at which the surface reinforcing agent is dripped. (integration time 5s, laser power 90 mW. acquisition times was 3 (fig. 24).
Step D, processing the non-blank spectrogram
Processing the non-blank spectrogram by standard spectrogram processing software, and selecting 400-1700cm-1The wave band is taken as a characteristic wave band, and 400-1700cm is selected-1The wave band is used as a characteristic wave band, the characteristic wave band is subjected to spectrogram smoothing and baseline correction by adopting OPUS5.0 software, and then vector normalization processing is carried out on the corrected spectral data to obtain a preprocessed surface enhanced Raman spectrogram;
step E, distinguishing the preprocessed surface enhanced Raman spectrogram
The common peak of sildenafil and analogues is 813 +/-10 cm-1，905±10cm-1，927±10cm-1，1002±10cm-1，1159±10cm-1，1233±10cm-1，1529±10cm-1，1560±10cm-1When the preprocessed surface-enhanced Raman spectrogram at least contains five absorption peaks of sildenafil derivatives, judging that sildenafil additives are doped in the traditional Chinese medicine to be detected for strengthening yang, and using the samples as 'positive samples' to be verified in a laboratory; and when the pretreated surface enhanced Raman spectrum contains less than five absorption peaks of the sildenafil additives, judging that the sildenafil additives are not doped.
Example 3
In this embodiment, a specific embodiment of the embodiment of fig. 25 will be further explained. The software used includes: drug design platform Discovery Studio 3.0; the GAUSSIAN09 package; standard spectrum processing software OPUS 5.0.
In this embodiment, adoptThe materials used included: thin layer plate GF254(coating thickness: 0.2 mm. + -. 0.03mm, silica gel particle size (8. + -. 2) m ≧ 80%), Nicotiyou silica gel development Co., Ltd.); silver nitrate, trisodium citrate (AR, chemical agents ltd, national drug group); other used reagents are analytically pure and purchased from chemical reagents of national drug group, Inc.; chromatographically pure formic acid was purchased from Tedia (usa); mass Spectrometry pure methanol was purchased from Fisher Scientific (USA); ultrapure water was produced by Milli-QACAdemic ultrapure water system (Millipore, USA). The column was an ACQUITY UPLCTM BEH C18column (2.1 mm. times.100 mm,1.7 μm, Waters, Milford, Mass.). Polyvinylpyrrolidone (PVP) is subpackaged at GFA import, and every 100g of the PVP is dissolved by heparinized physiological saline with 200IU/ml to prepare heparinized PVP solution; 1 g/piece of heparin sodium injection (sigma import split charging); ketamine hydrochloride injection 0.1g/2ml (Shanghai Western medicine industry Co., Ltd.).
In this embodiment, the apparatus used includes: portable Raman spectrometer (BWS415, B)&W Tek inc., usa), excitation wavelength 785nm, resolution 5cm-1The spectrum coverage range is 175-3200cm-1(ii) a WFH-203B three-purpose ultraviolet analyzer (Shanghai Jingke industries, Ltd.); ultrasonic instrument (KUDOS-SK5200HP, China); centrifuges (HERAEUS, FRESCO17 Centrifuge; Thermo Scientific, USA); analytical balance (METTLER AE240, china).
In this example, the laboratory animal SHR-SP was introduced from Japan by the pharmacological research laboratory of the college of medicine of the second university of military medicine. 32 week old SHR-SP, females, were used, 7 per group. During the experiment, the room temperature of the animal room is kept at about 22 ℃, the relative humidity is about 70%, and the illumination is automatically carried out from 8 am to 8 am.
Step one, taking amlodipine as a key intermediate, and mixing the designed derivative with a calcium channel blocker β2Receptor activity cavity docking, a Libdock molecular docking technology is selected, 16 amlodipine derivatives with high scoring functions are preferably selected according to the scoring functions after docking (table 4), and docking results are shown in a figure 26.
TABLE 4 LibDock docking scoring results
Step two:
the molecular configuration was constructed using the GAUSSIAN09 package, GAUSS VIEW 3.0 (fig. 27). In the calculation process, a DFT method of B3LYP level is selected, and the DFT method is composed of a correlation function of Lee-yang-Parr and a hybrid exchange function of Becke. The 6-31G (d, p) group of the polarization function of hydrogen atom plus p polarization function and heavy atom plus d polarization function in the system is adopted to carry out configuration optimization on 16 amlodipine derivatives, and then the configuration optimization is carried out on the Linux systematicness by using GAUSSIAN09 software. The calculated theoretical raman spectrum was compared with the actually measured raman spectrum (fig. 28).
According to the DFT calculation result, the GAUSSVIEW 3.0 software is adopted to summarize the common peak of the amlodipine derivatives. The DFT of 16 derivatives was found to have 28 common peaks, which are Δ ν 359, 421, 525, 571, 747, 792, 809, 836, 854, 872, 889, 955, 1001, 1011, 1032, 1049, 1102, 1117, 1156, 1187, 1199, 1267, 1282, 1334, 422, 1451, 1479 and 1486cm-1。
Step three: taking amlodipine as a key intermediate, introducing compounds such as alkane substituent, aromatic ring substituent and the like on the amino group of amlodipine, preferably selecting 16 amlodipine derivatives according to the Libdock scoring function, and carrying out synthesis and separation research. Adding 17.65mmol of amlodipine besylate and 100ml of dichloromethane into a 250ml round bottom bottle, dissolving and clarifying, adding 35.3mmol of triethylamine, stirring for 10 minutes at room temperature, carrying out ice bath, adding 21.2mmol of a halide intermediate, stirring, carrying out ice bath, and carrying out stirring reaction for 1 hour. The reaction progress was monitored by TLC (developing solvent: dichloromethane: methanol: 4.7: 0.3; petroleum ether: ethyl acetate: 3.5: 1.5); the reaction was stirred at room temperature for 2 hours until the reaction was complete. And (3) post-treatment: if the product is separated out, directly filtering, collecting the target substance, weighing, and recrystallizing with acetone or ethyl acetate to obtain the pure product. If no product precipitated, the dichloromethane layer was washed with water, and then with a saturated sodium bicarbonate solution, followed by further washing with water and drying with anhydrous magnesium sulfate. The organic layer was evaporated to dryness, 100ml of diethyl ether was added, 100ml of petroleum ether was added, and the mixture was stirred at room temperature to precipitate a crude solid. The crude product is separated by silica gel column chromatography or medium pressure column to obtain pure product. The general formula of the amlodipine derivative is shown in figure 29.
(1) DPA-01 Structure validation (FIG. 30)
1H NMR(600MHz,DMSO)，δ8.96(s,1H)，δ8.81(s,1H)，δ7.32(dd,J＝7.8,1.7Hz,1H)，δ7.25(dd,J＝1.20,7.98Hz,1H)，δ7.21(td,J＝7.6,1.3Hz,1H)，δ7.22-7.19(m,1H)，δ5.28(s,1H)，δ4.68(d,J＝13.7Hz,1H)，δ4.55(d,J＝13.7Hz,1H)，δ4.02-3.90(m,2H)，δ3.80-3.68(m,2H)，δ3.48(s,3H)，δ3.30(s,3H)，δ3.14(brs,2H)，δ2.97-2.82(m,2H)，δ2.34(s,3H)，δ1.63-1.61(m,2H)，δ1.33-1.30(m,2H)，δ1.09(t,J＝7.1Hz,3H)，δ0.88(t,J＝7.4Hz,3H)。HRMS[M+H]465.16。
(2) DPA-02 Structure validation (FIG. 31)
1H NMR(600MHz,DMSO)，δ8.91(s,1H)，δ7.34(dd,J＝7.8,1.7Hz,1H)，δ7.26(dd,J＝8.0,1.3Hz,1H)，δ7.20(td,J＝7.6,1.3Hz,1H)，δ7.16-7.08(m,1H)，δ5.28(s,1H)，δ4.63(d,J＝15.0Hz,1H)，δ4.52(d,J＝15.0Hz,1H)，δ3.97-3.93(m,2H)，δ3.50-3.48(m,2H)，δ3.49(s,3H)，δ2.63-2.66(m,2H)，δ2.51-2.50(m,2H)，δ2.27(s,3H)，δ1.89(s,1H)，δ1.40-1.38(m,2H)，δ1.27-1.23(m,10H)，δ1.09(t,J＝7.1Hz,3H)，δ0.88(t,J＝7.4Hz,3H)。HRMS[M+H]521.28。
(3) DPA-03 (fig. 32)
1H NMR(600MHz,DMSO)，δ9.23(s,1H)，δ8.76(s,1H)，δ7.51(s,J＝7.1Hz,1H)，δ7.32(dd,J＝7.8,1.5Hz,1H)，δ7.30(d,J＝6.8,1H)，δ7.25-7.24(m,3H)，δ7.22-7.19(m,1H)，δ7.11(t,J＝6.8Hz,1H)，δ5.29(s,1H)，δ4.69(d,J＝13.9Hz,1H)，δ4.57(d,J＝13.9Hz,1H)，δ4.16(s,2H)，δ3.99-3.94(m,2H)，δ3.80-3.77(m,2H)，δ3.49(s,3H)，δ3.22(s,2H)，δ2.37(s,3H)，δ2.32(s,3H)，δ1.08(t,J＝7.01Hz 3H)。
HRMS[M+H]513.33。
(4) DPA-04 (fig. 33)
1H NMR(600MHz,DMSO)，δ9.22(s,1H)，δ8.77(s,1H)，δ7.47(d,J＝7.1Hz,2H)，δ7.42(d,J＝8.3Hz,2H)，δ7.32(dd,J＝7.8,1.6Hz,1H)，δ7.26(d,J＝7.9Hz,1H)，δ7.21(td,J＝7.6,1.2Hz,1H)，δ7.11(td,J＝7.6,1.5Hz,1H)，δ5.29(s,1H)，δ4.65(d,J＝13.9Hz,1H)，δ4.53(d,J＝13.9Hz,1H)，δ4.10(s,2H)，δ4.04-3.87(m,2H)，δ3.72(s,2H)，δ3.48(s,3H)，δ3.09(s,2H)，δ2.32-2.31(m,3H)，δ1.28(s,9H)，δ1.08(t,J＝7.22Hz 3H)。HRMS[M+H]555.39。
(5) DPA-05 (FIG. 34)
1H NMR(600MHz,DMSO)，δ9.31(s,1H)，δ8.77(s,1H)，δ7.47(s,2H)，δ7.32(dd,J＝7.8,1.7Hz,1H)，δ7.29(d,J＝7.9Hz,2H)，δ7.26(d,J＝8.0Hz,1H)，δ7.24-7.18(m,1H)，δ7.11(m,1H)，δ5.29(s,1H)，δ4.67(d,J＝13.8Hz,1H)，δ4.54(d,J＝13.8Hz,1H)，δ4.12(s,2H)，δ4.01-3.89(m,2H)，δ3.74(s,2H)，δ3.49(s,3H)，δ3.12(s,2H)，δ2.90(s,1H)，δ2.33(s,3H)，δ1.19(d,J＝6.9Hz,6H)，δ1.08(t,J＝7.1Hz,3H)。HRMS[M+H]541.28。
(6) DPA-06 (fig. 35)
1H NMR(600MHz,DMSO)，δ9.64-9.03(m,1H)，δ9.01-8.46(m,1H)，δ7.49-7.29(m,2H)，δ7.25(d,J＝7.6Hz,1H)，δ7.20(t,J＝7.5Hz,2H)，δ7.13-7.08(m,2H)，δ6.97(d,J＝8.0Hz,1H)，δ5.28(s,1H)，δ4.68(d,J＝13.9Hz,1H)，δ4.54(d,J＝13.8Hz,1H)，δ4.14(s,2H)，δ3.99-3.90(m,2H)，δ3.76(s,3H)，δ3.73(s,2H)，δ3.48(s,3H)，δ3.18-3.10(m,2H)，δ2.32(s,3H)，δ1.08(t,J＝7.1Hz,3H)。HRMS[M+H]529.18。
(7) DPA-07 (FIG. 36)
1H NMR(600MHz,DMSO)，δ8.76(s,1H)，δ7.48-7.42(m,1H)，δ7.32(dd,J＝7.8,1.7Hz,1H)，δ7.28-7.26(m,1H)，δ7.27-7.22(m,1H)，δ7.19(td,J＝7.5,1.3Hz,1H)，δ7.15(dt,J＝6.8,3.4Hz,1H)，δ7.13-7.12(m,1H)，δ7.10-7.08(m,1H)，δ5.27(s,1H)，δ4.62(d,J＝14.9Hz,1H)，δ4.52(d,J＝14.9Hz,1H)，δ4.00-3.88(m,2H)，δ3.76(s,2H)，δ3.53(t,J＝5.3Hz,2H)，δ3.48(s,3H)，δ2.69(t,J＝5.2Hz,2H)，δ2.19(s,3H)，δ1.08(t,J＝7.1Hz,3H)。HRMS[M+H]517.12。
(8) DPA-08 (FIG. 37)
1H NMR(600MHz,DMSO)，δ8.77(s,1H)，δ7.32(dd,J＝7.8,1.7Hz,1H)，δ7.29-7.26(m,2H)，δ7.32(dd,J＝7.8,1.7Hz,1H)，δ7.20-7.17(m,1H)，δ7.17-7.12(m,1H)，δ7.11-7.08(m,1H)，δ5.27(s,1H)，δ4.67(d,J＝13.8Hz,1H)，δ4.52(d,J＝13.8Hz,1H)，δ3.95-3.82(m,2H)，δ3.80(s,2H)，δ3.53-3.51(m,2H)，δ3.47(s,3H)，δ2.63(t,J＝5.32Hz,2H)，δ2.20(s,3H)，δ1.08(t,J＝7.1Hz,3H)。HRMS[M+H]535.05。
(9) DPA-09 (fig. 38)
1H NMR(600MHz,DMSO)，δ8.77(s,1H)，δ7.32(dd,J＝7.8,1.7Hz,1H)，δ7.25(dd,J＝8.0,1.3Hz,1H)，δ7.19(td,J＝7.6,1.3Hz,1H)，δ7.13-7.08(m,1H)，δ7.07-7.01(m,3H)，δ5.28(s,1H)，δ4.63(d,J＝14.8Hz,1H)，δ4.53(d,J＝14.8Hz,1H)，δ4.00-3.89(m,2H)，δ3.74(s,2H)，δ3.55-3.49(m,2H)，δ3.48(s,3H)，δ2.65(t,J＝5.3Hz,2H)，δ2.64(s,1H)，δ2.22(s,3H)，δ1.08(t,J＝7.1Hz,3H)。HRMS[M+H]535.07。
(10) DPA-10 (fig. 39)
1H NMR(600MHz,DMSO)，δ9.57(s,1H)，δ8.76(s,1H)，δ7.99(d,J＝7.5Hz,1H)，δ7.81(d,J＝8.04,1H)，δ7.77(t,J＝7.3Hz,1H)，δ7.63(t,J＝7.3,1H)，δ7.34(dd,J＝7.8,1.6Hz,1H)，δ7.27(dd,J＝8.0,1.2Hz,1H)，δ7.21(td,J＝7.6 1.2Hz,1H)，δ7.12(td,J＝7.6,1.7Hz,1H)，δ5.30(s,1H)，δ4.71(d,J＝13.9Hz,1H)，δ4.58(d,J＝13.9Hz,1H)，δ4.31(s,2H)，δ4.02-3.91(m,2H)，δ3.78(s,2H)，δ3.50(s,3H)，δ3.23(s,2H)，δ2.32(s,3H)，δ1.10(t,J＝7.1Hz,3H)。HRMS[M+H]567.18
(11) DPA-11 (fig. 40)
1H NMR(600MHz,DMSO)，δ9.3(s,1H)，δ8.77(s,1H)，δ7.47(s,1H)，δ7.97(s,1H)，δ7.78(s,1H)，δ7.71(d,J＝7.5Hz,1H)，δ7.36(dd,J＝7.8,1.6Hz,1H)，δ7.30(d,J＝8.0Hz,1H)，δ7.24(t,J＝7.0Hz,1H)，δ7.15(t,J＝7.6Hz,1H)，δ5.33(s,1H)，δ4.73(d,J＝14.1Hz,1H)，δ4.59(d,J＝13.8Hz,1H)，δ4.30(s,2H)，δ4.04-3.95(m,2H)，δ3.78(s,2H)，δ3.49(s,3H)，δ3.14(s,2H)，δ2.32(s,3H)，δ1.09(t,J＝7.1Hz,3H)。HRMS[M+H]567.17。
(12) DPA-12 (fig. 41)
1H NMR(600MHz,DMSO)，δ9.52(s,1H)，δ8.85(s,1H)，δ7.72(s,2H)，δ7.42(d,J＝8.2Hz,2H)，δ7.31(dd,J＝7.8,1.6Hz,1H)，δ7.22-7.20(m,2H)，δ7.13-7.06(m,1H)，δ5.28(s,1H)，δ4.67(d,J＝13.8Hz,1H)，δ4.54(d,J＝13.8Hz,1H)，δ4.21(s,2H)，δ4.00-3.87(m,2H)，δ3.75(s,2H)，δ3.48(s,3H)，δ3.15(s,2H)，δ2.33(s,3H)，δ1.08(t,J＝7.1Hz,3H)。HRMS[M+H]583.14。
(13) DPA-13 (FIG. 42)
1H NMR(600MHz,DMSO)，δ9.17(s,1H)，δ8.81(s,1H)，δ7.67(s,1H)，δ7.50(s,1H)，δ7.46-7.39(m,2H)，δ7.32(dd,J＝7.8,1.6Hz,1H)，δ7.27-7.17(m,2H)，δ7.13-7.07(m,1H)，δ5.28(s,1H)，δ4.67(d,J＝13.9Hz,1H)，δ4.54(d,J＝13.9Hz,1H)，δ4.13(s,2H)，δ4.00-3.88(m,2H)，δ3.72-3.70(m,2H)，δ3.48(s,3H)，δ3.07(s,2H)，δ2.32(s,3H)，δ1.08(t,J＝7.1Hz,3H)。HRMS[M+H]533.10。
(14) DPA-14 (FIG. 43)
1H NMR(600MHz,DMSO)，δ8.93(s,1H)，δ8.79(s,1H)，δ7.32(dd,J＝7.8,1.7Hz,1H)，δ7.25(dd,J＝8.0,1.3Hz,1H)，δ7.24-7.19(m,1H)，δ7.12-7.09(m,1H)，δ5.29(s,1H)，δ4.69(d,J＝13.7Hz,1H)，δ4.55(d,J＝13.7Hz,1H)，δ4.02-3.90(m,2H)，δ3.72(dd,J＝9.7,4.0Hz,2H)，δ3.48(s,3H)，δ3.14(s,2H)，δ2.92-2.82(m,2H)，δ2.33(s,3H)，δ1.66(dd,J＝15.3,7.5Hz,2H)，δ1.09(t,J＝7.1Hz,3H)，δ0.90(t,J＝7.5Hz,3H)。HRMS[M+H]451.15。
(15) DPA-15 (fig. 44)
1H NMR(600MHz,DMSO)，δ8.92(s,1H)，δ8.83(s,1H)，δ7.32(dd,J＝7.8,1.7Hz,1H)，δ7.25(d,J＝7.9Hz,1H)，δ7.24-7.18(m,1H)，δ7.12-7.09(m,1H)，δ5.29(s,1H)，δ4.68(d,J＝13.7Hz,1H)，δ4.55(d,J＝13.7Hz,1H)，δ4.02-3.90(m,2H)，δ3.76-3.69(m,2H)，δ3.48(s,3H)，δ3.14(s,2H)，δ2.96-2.82(m,2H)，δ2.34(s,3H)，δ1.65(s,2H)，δ1.32-1.20(m,4H)，δ1.09(t,J＝7.1Hz,3H)，δ0.86(t,J＝7.0Hz,3H)。HRMS[M+H]479.18。
(16) DPA-16 (FIG. 45)
1H NMR(600MHz,DMSO)δ8.94(s,1H)，δ8.80(s,1H)，δ7.32(dd,J＝7.8,1.7Hz,1H)，δ7.25(d,J＝8.0Hz,1H)，δ7.23-7.19(m,1H)，δ7.11-7.09(m,1H)，δ5.29(s,1H)，δ4.68(d,J＝13.7Hz,1H)，δ4.55(d,J＝13.7Hz,1H)，δ4.02-3.90(m,2H)，δ3.72(tq,J＝11.0,5.5Hz,2H)，δ3.48(s,3H)，δ3.14(s,2H)，δ2.96-2.82(m,2H)，δ2.33(s,3H)，δ1.63(dd,J＝15.1,7.5Hz,2H)，δ1.25(d,J＝13.7Hz,12H)，δ1.09(t,J＝7.1Hz,3H)，δ0.84(t,J＝6.9Hz,3H)。HRMS[M+H]535.24。
Step four: activity assay of amlodipine derivatives
The derivatives are grouped, aliphatic chain substituents (DPA-01, 02, 14, 15, 16) and aromatic ring substituents (DPA-03-13) are selected from 2 groups, 2 target compounds (DPA-02, 15; DPA-06, 09) are selected from 2 groups respectively to carry out a whole animal antihypertensive test, and amlodipine is taken as a positive control.
The medicine is taken through a gastric fistula with the dose of 2mg/kg and the administration volume of 4ml/kg, and the medicine is weighed, dissolved by DMSO (10ml), and mixed with 0.5 percent CMC-Na. The temperature and humidity of the laboratory are constant (20-25 ℃), constant humidity (60%), bright and sound-proof. The rat which recovers 24 hours after operation is placed in an organic glass cylinder (the diameter of the cylinder is 30cm, the height is 24cm, a round hole with the diameter of 15cm is arranged in the middle of a cylinder cover, a long and narrow gap with the width of 0.8cm is arranged between the hole and the edge of the cover for the arterial catheter to pass through, the arterial catheter is pulled out of a metal needle, then heparinized PVP liquid is discharged, a small amount of 200IU/ml heparinized saline is used for pushing blood in the catheter back, then the blood is connected with a pressure transducer through a rotating device and a perfusion three-way pipe, meanwhile, the perfusion three-way pipe is connected with a constant speed pump, an isotonic glucose solution (20IU/ml) is injected by heparin injection at the speed of 0.5ml/h, air bubbles are discharged from the whole pipeline system so as not to influence blood pressure waveforms, each pulse blood pressure signals are converted into electric signals through the pressure transducer and amplified through a physiological recorder, each pulse systolic pressure (SBP) and diastolic pressure (diastolic pressure) are recorded in real time through a computer, DBP), cardiac interval (HP), etc. Data statistical analysis: the measurements are expressed as mean ± standard deviation (mean ± SD). The paired t test method is adopted for comparison before and after experimental administration. The results showed that each of the four target compounds had better antihypertensive activity (fig. 46), which was comparable to amlodipine (table 5).
TABLE 5 Effect of amlodipine and derivatives on systolic and diastolic blood pressure in SHR rats 6 hours after administration
n＝7，*p<0.05,**p<0.01
Step five: TLC-SERS detection method
Preparation of control solutions: accurately weighing appropriate amount of the derivatives and standard, adding 1mL of methanol for dissolving, respectively making into reference solution with concentration of 1mg/mL, storing in refrigerator at 4 deg.C, and inspecting.
Preparation of a sample to be tested: weighing 26 Chinese medicinal materials for lowering blood pressure, dissolving in 10ml methanol, diluting to constant volume for 3 min, vortexing for 5min, placing in ultrasonic instrument, extracting for 30min, centrifuging for 5min (5000rpm,10 deg.C), storing in refrigerator at 4 deg.C, and testing.
TLC separation: dividing the synthesized 16 derivatives into 2 groups according to the difference of polarities of the synthesized derivatives, wherein the first group comprises DPA-01, DPA-02, DPA-05, DPA-06, DPA-08 and DPA-14-16; the second group is DPA-03, DPA-04, DPA-7 and DPA-09-13, and the expansion system is as follows: acetone-n-hexane-dichloromethane ═ 5:4:1, ethyl acetate-methanol ═ 1:1 (fig. 47). Rf values of the derivatives are (DPA-01-16): 0.24,0.52,0.29,0.21,0.69,0.64,0.50,0.41,0.60,0.77,0.54,0.36,0.41,0.27,0.39,0.56.
Step six: common peak and characteristic peak of derivative
After SERS detection, the 16 derivatives were found to have 11 SERS common peaks, i.e., Δ ν 484, 555, 783, 798, 812, 903, 1038, 1100, 1207, 1268, 1496cm, by SERS spectroscopy (fig. 48)-1. Integration and summarization of the consensus peaks obtained from DFT theory, Δ ν 792, 809, 889, 1032, 1049, 1102, 1199, 1267, 1486cm-1. When the preprocessed surface-enhanced Raman spectrogram at least contains absorption peaks of eight amlodipine derivatives, field detection personnel can preliminarily judge that amlodipine additives are doped in the traditional Chinese medicine to be detected for strengthening yang and serve as a 'positive sample' to be verified in a laboratory (step ten). When the pretreated surface enhanced Raman spectrum containsAnd the absorption peaks of less than six amlodipine additives exist, and field detection personnel judge that the amlodipine additives are not doped.
As can be seen from the structures of the 16 derivatives, groups such as alkane substituent, aromatic ring substituent and the like are introduced on the amino group of amlodipine, wherein DPA-01, 02, 14, 15 and 16 are different alkane substituents, and the others are different aromatic ring substituents. Because the derivatives have similar structures, the characteristic peaks of each compound are relatively fewer. The characteristic peak of DPA-01 is 1160cm-1. The characteristic peak of DPA-02 is 559cm-1. DPA-07 has a characteristic peak at 419, 1176 cm-1. The characteristic peak of DPA-09 is 505, 1320, 1626cm-1. The characteristic peak of DPA-13 is 1697cm-1. The characteristic peak of DPA-14 is 529cm-1. The characteristic peaks of DPA-15 are 307, 346 and 430cm-1. The characteristic peak of DPA-16 is 771cm-1. The derivative doped in the antihypertensive health care product can be judged through the characteristic peak.
Step seven: true sample detection
And detecting whether amlodipine or derivatives thereof are added in 26 real antihypertensive traditional Chinese medicines (No. 1-26) by using the established TLC-SERS method and the summarized common peak. The sample nos. 1 to 26 were subjected to TLC separation, and 1 μ L of each control mixed solution was pipetted onto the same silica gel plate (HSGF254, 10 × 10cm), and developed under conditions optimized by TLC using acetone-n-hexane-dichloromethane at a ratio of 5:4:1 and ethyl acetate-methanol at a ratio of 1: 1. Taking out after development, air drying, and positioning under ultraviolet lamp (254 nm).
Based on the above common peaks as reference, the results showed that sample number 26 detected 494, 557, 788, 807, 896, 1038, 1093, 1188, 1269, 1498cm in raman wavenumber-1Common peak Δ v with amlodipine is 484, 555, 783, 798, 812, 903, 1038, 1100, 1207, 1268, 1496cm-1Correspondingly, the illegal addition of amlodipine substances in the sample is indicated. Further analysis revealed that sample No. 26 had a Raman spectrum peak very similar to that of DPA-12, and as shown in FIG. 49, it was initially assumed that sample No. 26 was doped with the derivative DPA-12.
Step eight: establishment of illegal addition amlodipine derivative UPLC-QTOF/MS analysis method in antihypertensive health care products (figure 50)
Liquid phase conditions: the column was an ACQUITY UPLCTM BEH C18column (2.1 mm. times.100 mm,1.7 μm, Waters, Milford, Mass.). Mobile phase a was 0.1% aqueous formic acid and mobile phase B was 0.1% acetonitrile formic acid, using gradient elution, the procedure was: 0-2 min, 5-5% of B; 2-9 min, 5-25% of B; 9-20 min, 25-55% B; 60-95% B for 25-28 min; 28-30 min, 95% -95% B. The analysis time was 30 min. The column temperature was 40 ℃, the flow rate was 0.35ml/min, and the sample volume was 2. mu.l.
Mass spectrum conditions: the ion source is an ESI source, the positive ion mode is adopted, and the mass spectrum scanning range is 100-1100 m/z; detecting parameters: the ionization voltage is 4000V, the flow rate of the drying gas is 11l/min, and the temperature of the drying gas is 350 ℃; atomizer pressure was 45psig, cracker voltage was 120V, skimmer 60V, eight-step rod rf voltage 750V. Internal standard ions of m/z 121.0509 and m/z 922.0098 were selected for real-time mass number correction. Before each sample measurement, the mass axis was calibrated using a tuning mix (Turning mix). Experimental data was collected and analyzed using MassHunter software version B.03.00(Agilent technologies, USA) software.
Step nine: law of cleavage of derivatives
Molecular formulas were calculated in the 5ppm range using MassHunter Quantitativeanalysis data analysis software based on the determination of the exact excimer ion, e.g., [ M + H ] +. Secondary mass spectrometry was performed on the excimer ions of amlodipine by setting the fragment voltage (secondary mass spectrum and cleavage pathway are shown in fig. 51). Amlodipine major fragment ions m/z392, 360, 334, 316, 288, 260, 208. The main cleavage pathways are: firstly, an oxygen atom on a pyridine ring has a strong electron-withdrawing effect, so that a COCH3 bond is broken, and fragment ions with m/z of 392 are generated; second, cleavage of the C ═ O bond followed by elimination yielded a fragment ion with m/z of 360, elimination of two CH3 on the pyridine ring yielded a fragment ion with m/z of 334, followed by rearrangement of the pyridine ring to yield a cleavage product with m/z of 294. Another cleavage route is the loss of fragment ions on the pyridine ring, resulting in a product with m/z of 238, followed by rearrangement of the pyridine ring, resulting in a product with m/z of 206.
By setting fragment voltage, secondary mass spectrometry analysis is carried out on the excimer ions of the 16 amlodipine derivatives, and the cracking path is similar to that of amlodipine. By secondary fragment ion analysis (as in table 6), the 16 amlodipine derivatives were found to have a total fragment ion of m/z392, 360, 334, 316, 288, 260, 208. Wherein, DPA-08 and DPA-09, DPA-10 and DPA-11 are isomers, but m/z 141, 376 and 475 are characteristic fragment ions of DPA-09; m/z 507, which is the characteristic fragment ion of DPA-11.
Table 6 amlodipine derivative major fragment ions
Step ten: UPLC-QTOF/MS analysis of real samples
And carrying out further mass spectrum verification on the No. 26 sample suspected to be positive in TLC-SERS quick detection result according to the established UPLC-QTOF/MS separation condition.
The retention time of sample No. 26 and DPA-12 were both 16.2min as shown by comparison with the standard liquid chromatogram, indicating that the retention times were the same (see FIG. 52). The m/z of the main fragment ions of the sample No. 26 is 551.1554, 334.0836, 218.0786 and 175.0360 by comparison with a standard secondary mass spectrum; the major fragment ions m/z of DPA-12 were 551.1558, 334.0839, 218.0788, 175.0362 (see FIG. 53). The fragment ions of the two are basically similar, which indicates that the sample No. 26 is doped with the amlodipine derivative with the structure of DPA-12.
While the preferred embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that the invention is not limited thereto, and that various changes and modifications may be made without departing from the spirit of the invention, and the scope of the appended claims is to be accorded the full range of equivalents.
Claims (3)
1. A method for screening rapid detection conditions of illegally added compound derivatives in health products is characterized by comprising the following steps:
the method comprises the following steps: designing a derivative target molecule by taking an illegally added compound parent nucleus as a key intermediate based on a structure-activity relationship method;
step two: evaluation of ligand molecule interaction with receptor
Adopting a drug design docking platform Discovery Studio3.0 to perform the step one, namely docking the ligand molecules with the active cavities of the action sites, selecting a Libdock molecule docking technology, and preferably selecting a high-grade derivative according to a scoring function after docking;
step three: theoretical Raman spectrum of derivative predicted by density functional method
After molecular configuration is optimized by adopting GAUSS VIEW 5.0, a DFT method of B3LYP level is selected, and 6-31G of polarization functions of hydrogen atoms plus p polarization functions and heavy atoms plus d polarization functions in the system are obtained*(d, p) group, after configuration optimization is carried out on the derivative optimized in the step two, in a Linux system, the GAUSSIAN09 software is used for calculating;
step four: theoretical consensus peak of derivatives
According to the DFT calculation result, adopting GAUSSVIEW 5.0 software to carry out common peak induction on the derivatives; determining a common absorption peak of the derivative and judging a standard that the sample to be detected contains the derivative of the illegal addition compound;
step five: synthesis and structural verification of derivatives
Synthesizing the compound derivative optimized in the step two, and verifying the structure through nuclear magnetism and mass spectrum;
step six: method for verifying derivative binding kinetics assay
Sequentially sampling compounds with different concentrations by using a Biacore T200 molecular interaction analysis system, analyzing experimental data by using Biacore T200evaluation software, and fitting and calculating an experimental result by using a dynamic combination model;
step seven: establishment of conditions for TLC-SERS detection of derivatives
Weighing the derivative prepared in the fifth step, carrying out thin-layer separation, dropwise adding a surface reinforcing agent, wherein the surface reinforcing agent is gold sol, organic silver sol, silver sol or gold-silver composite gel, and scanning by adopting a portable Raman spectrometer;
step eight: processing non-blank spectrogram
Processing the non-blank spectrogram by standard spectrogram processing software, and selecting 400-1700cm-1The wave band is used as a characteristic wave band, the characteristic wave band is subjected to spectrogram smoothing and baseline correction by adopting OPUS5.0 software, and then vector normalization processing is carried out on the corrected spectral data to obtain a preprocessed surface enhanced Raman spectrogram;
step nine: discriminating the preprocessed surface enhanced Raman spectrogram
Integrating and summarizing the common peak obtained by TLC-SERS detection and the common peak obtained by the theoretical calculation of the step four DFT, determining the common absorption peak of the derivative and judging the standard of illegally adding compound derivatives in the sample to be detected;
step ten: UPLC-QTOF/MS authentication
And (3) qualitatively analyzing the derivative by adopting a rapid separation ultra-high performance liquid chromatography-time-of-flight mass spectrometer to obtain common fragment ions of the derivative, and confirming a suspected 'positive sample' detected on site.
2. The method for screening the rapid detection conditions of the illegally added compound derivative in the health product according to claim 1, wherein the health product has the functions of strengthening yang, lowering blood pressure, losing weight, lowering blood sugar, and relieving cough and asthma.
3. The method for screening rapid test conditions for illegally added compound derivatives in health products according to claim 2, wherein the illegally added compound derivatives are sildenafil analogs, amlodipine analogs, sibutramine analogs, metformin analogs, theophylline analogs.
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| KR101425772B1 (en) | 2010-11-23 | 2014-08-06 | 엘지전자 주식회사 | Method for encoding and decoding images, and device using same |
| CN110057809A (en) * | 2019-05-29 | 2019-07-26 | 桂林电子科技大学 | One kind non-contact, rapid detection system and detection method for the fine Raman spectrum of substance |
| CN114113418B (en) * | 2021-12-30 | 2024-11-26 | 山西省公安厅刑事科学技术研究所 | Simultaneous determination of eight illegally added chemical drugs in food by ultra-high performance liquid chromatography-tandem mass spectrometry |
| CN117825436A (en) * | 2023-12-30 | 2024-04-05 | 上海新泊地化工技术服务有限公司 | Analysis method for unknown western medicine components in general traditional Chinese medicine |
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