CN106928079B - Trefanocide haptens and its preparation method and application - Google Patents
Trefanocide haptens and its preparation method and application Download PDFInfo
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- CN106928079B CN106928079B CN201710177395.6A CN201710177395A CN106928079B CN 106928079 B CN106928079 B CN 106928079B CN 201710177395 A CN201710177395 A CN 201710177395A CN 106928079 B CN106928079 B CN 106928079B
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C229/00—Compounds containing amino and carboxyl groups bound to the same carbon skeleton
- C07C229/02—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton
- C07C229/04—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated
- C07C229/06—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton
- C07C229/18—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton the nitrogen atom of the amino group being further bound to carbon atoms of six-membered aromatic rings
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
Abstract
The invention discloses trefanocide haptens and its preparation method and application, have the following structure formula:Wherein, R is-(CH2)n, n=1-4 or-CH=CH- or
Description
Technical field
The present invention relates to immunological technique fields, and in particular to trefanocide haptens and its preparation method and application.
Background technique
Trefanocide (trifluralin) is dinitroaniline (dinitroaniline) before the important selective bud of one kind
Herbicide is widely used in the crops such as soybean, barley, vegetables and fruit, prevents and kill off annual gramineous weed and part broadleaf weeds.
Though trefanocide belongs to low-toxin farm chemicals, long-term exposure experiment shows to cause to damage to liver and kidney, the meeting under high dose exposure
Cause weight and the height of fetus relatively low and increase risk of miscarriage.Because of being widely used for trefanocide pesticide, make it in soil and water
Residual in body is generally existing, can especially be enriched in vivo in aquatic products (fish, shrimp, crab), to eat to aquatic products
Safety belt carrys out huge hidden danger.
In order to avoid the potential hazard of Basalin, many countries are used for being limited.European Union 2015
New limit standard is formulated trefanocide, the maximum residue limit of trefanocide in water fruits and vegetables, cereal and animal-derived food
It (MRLs) is 0.01mg/kg.Japan is 0.001mg/kg to trefanocide maximum residue limit in import aquatic products.
The method that the detection of trefanocide is analyzed usually using instrument in agricultural product at present, such as ultraviolet spectra, high-efficient liquid phase color
Spectrum and LC-MS, gas-chromatography, Capillary Electrophoresis etc..It is accurate, sensitive that above analysis method has the characteristics that, but due to instrument
Device is expensive, operating cost is high-leveled and difficult to popularize in an all-round way.It was verified that carrying out primary dcreening operation to a large amount of samples with rapid detection method, then
Carrying out instrument confirmation to wherein positive sample is the effective means for improving detection efficiency, reducing cost, is tallied with the national condition.Existing
Have in Fast Detection Technique, the immunoassay technology based on Ag-Ab specific recognition due to it is sensitive, quick, accurate,
It is low in cost and can realize the advantages such as on-site test, it is considered to be 21 century is most competitive and the ultramicron of challenge detects skill
Art.
Document foreign countries about trefanocide immune detection only have several reports.Bruce Riggle was attempted in 1991 first
The enzyme-linked immune analytic method for trefanocide, 0.1~1.0ppm of the range of linearity (ng/mL) are constructed, while can detecte portion
Divide the metabolin of trefanocide.Sz é k again attempt to and establish to trefanocide immune detection for á cs et al 2000
ELISA method, the linear range of detection increase (0.1~100ng/mL), the cross reaction with other structures analog
Rate≤5%, and have detected the trefanocide content in surface water and fruit juice.Sz é k á cs seminar established again in 2003 and is based on
Spectral detection (the optical waveguide lightmode spectroscopic that the light wave of immunosensor mediates
Detection, OWLS) method, although the method substantially increases detection sensitivity (2 × 10-7~3 × 10-5ng/mL),
It is such method because instrument condition problem is difficult in actually detected middle application.Currently, the country has no related trefanocide immune detection
Relevant report.Country's detection is more the absence of the product of trefanocide pesticide residue immunity detection reagent in the market.
Major part drug, toxin, environmental contaminants molecular mass are smaller now, belong to haptens, most of haptens
It needs that immunogenicity could be generated after being coupled with protein.The conventional method for preparing hapten antibody at present is that selection has toxicity
The original shape drug of meaning is learned as determinand, is synthesized by design and retains determinand molecular characterization or similar structures feature simultaneously
Haptens with active group prepares artificial immunogen by haptens and macro-molecular protein carrier conjugation, while through dynamic
The immune preparation of object is directed to the specific antibody of haptens.And the offer of haptens is step the most key in immunochemistry analysis
Suddenly, reasonable hapten design and preparation are beneficial to the raising of the quality of antibody.
Summary of the invention
It can be universally used in the partly anti-of trefanocide detection it is an object of the invention to overcome the deficiencies of the prior art and provide a series of
Original can make after the coupled carrier protein of haptens body generate potency high, can identify the special of trefanocide pesticide apokoinou construction
Property antibody.
It is a further object to provide the preparation method and application of the haptens.
The technical problems to be solved by the invention are achieved by the following technical programs:
A kind of trefanocide haptens, which is characterized in that have the following structure formula:
Wherein, R is-(CH2)n, n=1-4 or-CH=CH- or
Trefanocide haptens of the present invention specifically has structural formula shown in following 1c, 2c, 3c, 4c, 5c and 6c:
The following are the descriptions of the preparation process of hapten molecule of the invention.
1. reaction equation involved in hapten synthesis of the present invention is expressed as follows:
1c:R=- (CH2)-1
2c:R=- (CH2)-2N=1
3c:R=- (CH2)-3N=2
4c:R=- (CH2)-4N=3
5c:R=- (CH=CH)-
6c:
2. steps are as follows for six kinds of trefanocide hapten synthesis according to the present invention:
(1) when trefanocide haptens structure is 1c, 2c, 3c or 4c, synthesis step includes: in the presence of a catalyst, to incite somebody to action
Substrate is alkylated with alkylating reagent to react, and obtains secondary amine intermediate after purification, and secondary amine intermediate is hydrolyzed reaction and obtains
The second intermediate is obtained, the second intermediate with chloro- 3, the 5- dinitro-p-trifluorotoluene hybrid reaction of 4-, obtains target half again after purified
Antigen molecule.
Further, ethyl acrylate is dissolved in chloroform, n-propylamine is added, wherein ethyl acrylate and n-propylamine rub
You are stirred to react than being 1: 1.5,40 DEG C of oil baths, obtain the secondary amine intermediate containing ester group；By the secondary amine intermediate dissolution containing ester group
In tetrahydrofuran, sodium hydrate aqueous solution is added, is stirred at room temperature and is hydrolyzed, obtains the second intermediate；To among above-mentioned second
Chloro- 3, the 5- dinitro-p-trifluorotoluene of 4- is added in body, is stirred at room temperature, carries out soda acid after reaction and falls to extract, merge organic phase,
It is dry with anhydrous sodium sulfate, solvent evaporated is rotated, is obtained hapten molecule (1c).
Further, 2-Pyrrolidone is dissolved in DMF, sequentially adds 60% sodium hydride solution and n-Propyl Bromide, wherein
The molar ratio of 2-Pyrrolidone, sodium hydride and n-Propyl Bromide are as follows: 1: 1.2: 1.3, under nitrogen protection, 70 DEG C of oil baths, stirring is instead
It answers；After reaction, reaction mixture is extracted, organic phase is merged, anhydrous sodium sulfate is dry, rotates solvent evaporated, obtains ester-containing
The secondary amine intermediate of base；4N sodium hydrate aqueous solution is added into the secondary amine intermediate containing ester group, is stirred overnight in 100 DEG C of oil baths
It is hydrolyzed, obtains the second intermediate；Chloro- 3, the 5- dinitro-p-trifluorotoluene of 4- is dissolved in tetrahydrofuran, is added above-mentioned second
The molar ratio of intermediate, chloro- 3, the 5- dinitro-p-trifluorotoluene of 4- and the second intermediate is 1: 1.5, and reaction is stirred at room temperature；It has reacted
It is diluted with water after complete, dilute hydrochloric acid is added to be acidified and be extracted with ethyl acetate, organic phase saturated common salt water washing, anhydrous sodium sulfate is done
It is dry, solvent evaporated is rotated, is obtained hapten molecule (2c).
Further, valerolactam is dissolved in DMF, sequentially adds 60% sodium hydride solution and n-Propyl Bromide, wherein penta
The molar ratio of lactams, sodium hydride and n-Propyl Bromide are as follows: 1: 1.2: 1.2, under nitrogen protection, 70 DEG C of oil baths are stirred to react；Instead
It after answering, is diluted with water, and adds acid for adjusting pH to occur sediment to 2-3, be extracted with ethyl acetate, then use saturated salt solution
Washing, anhydrous sodium sulfate is dry, rotates solvent evaporated, obtains the secondary amine intermediate containing ester group；To the secondary amine intermediate containing ester group
37% hydrochloric acid of middle addition and water, 100 DEG C of oil baths, condensing reflux are hydrolyzed, and obtain the second intermediate；By the chloro- 3,5- dinitro of 4-
Base benzotrifluoride is dissolved in tetrahydrofuran, and above-mentioned second intermediate, triethylamine and water, chloro- 3, the 5- dinitro fluoroform of 4- is added
The molar ratio of benzene and the second intermediate is 1:1.2, and reaction is stirred at room temperature；It is diluted with water after fully reacting, adds dilute hydrochloric acid acidification simultaneously
It is extracted with ethyl acetate, organic phase saturated common salt water washing, anhydrous sodium sulfate is dry, rotates solvent evaporated, obtains haptens
Molecule (3c).
Further, caprolactam is dissolved in DMF, sequentially adds 60% sodium hydride solution and n-Propyl Bromide, wherein penta
The molar ratio of lactams, sodium hydride and n-Propyl Bromide are as follows: 1:1.2:1.2, under nitrogen protection, 70 DEG C of oil baths are stirred to react；Instead
It is diluted with water after answering completely, dilute hydrochloric acid is added to be acidified and be extracted with ethyl acetate, organic phase saturated common salt water washing, anhydrous slufuric acid
Sodium is dry, rotates solvent evaporated, obtains the secondary amine intermediate containing ester group；37% hydrochloric acid is added into the secondary amine intermediate containing ester group
And water, 100 DEG C of oil baths, condensing reflux are hydrolyzed, and obtain the second intermediate；By the chloro- 3,5- dinitro-p-trifluorotoluene dissolution of 4-
In tetrahydrofuran, above-mentioned second intermediate, triethylamine and water, chloro- 3, the 5- dinitro-p-trifluorotoluene of 4- and the second intermediate is added
Molar ratio be 1:3, reaction is stirred at room temperature；It is diluted with water after fully reacting, adds dilute hydrochloric acid to be acidified and be extracted with ethyl acetate, have
Machine mutually uses saturated common salt water washing, and anhydrous sodium sulfate is dry, rotates solvent evaporated, obtains hapten molecule (4c).
The present inventor attempts discovery by many-sided research, when trefanocide haptens structure is 1c, by appropriate
Its preparation process condition of variable and parameter generate the secondary amine intermediate containing ester group, carboxyl are protected not react with other groups,
This step substantially increases the purity and yield of trefanocide haptens.When trefanocide haptens structure is 2c, 3c or 4c, inventor
It is selected in numerous substrates and alkylating reagent, by its preparation process condition of appropriate variable and parameter, is greatly improved
The purity and yield of trefanocide haptens.
(2) when trefanocide haptens structure is 5c or 6c, synthesis step includes: that substrate and n-propylamine pass through acylation
Reaction, generates the secondary amine intermediate containing ester group, and the secondary amine intermediate containing ester group is anti-with chloro- 3, the 5- dinitro-p-trifluorotoluene of 4- again
It answers, obtains the second intermediate after purified, reaction is hydrolyzed in the second intermediate again.
Further, 4- crotonic ethyl bromide is dissolved in ethyl acetate, reactor is placed in ice-methanol and is cooled to -15
DEG C, n-propylamine is slowly added to syringe, and the molar ratio of 4- crotonic ethyl bromide and n-propylamine is 1:3, stirring at normal temperature reaction.Instead
After answering, ethyl acetate extracts reaction mixture, then is washed with saturated sodium chloride solution, and organic phase is dry with anhydrous sodium sulfate
It is dry, solvent evaporated is rotated, the secondary amine intermediate containing ester group is obtained；Secondary amine intermediate containing ester group is dissolved in tetrahydrofuran, is added
Enter chloro- 3, the 5- dinitro-p-trifluorotoluene of 4- and triethylamine, be stirred at room temperature reaction, chloro- 3, the 5- dinitro-p-trifluorotoluene of 4- with it is ester-containing
The molar ratio of the secondary amine intermediate of base is 1:1.5；It is diluted with water after fully reacting, dilute hydrochloric acid is added to be acidified and is extracted with ethyl acetate
Take, organic phase saturated common salt water washing, anhydrous sodium sulfate is dry, rotate solvent evaporated, residue through column chromatographic isolation and purification,
It rotates solvent evaporated and obtains hapten molecule (5c).
Further, 4- bromomethyl-benzoic acid methyl ester is dissolved in ethyl acetate, reactor is placed in ice-methanol and is cooled down
To -15 DEG C, n-propylamine is slowly added to syringe, the molar ratio of 4- bromomethyl-benzoic acid methyl ester and n-propylamine is 1:2.5.Reaction
After, reaction mixture is extracted with ethyl acetate, then washed with saturated sodium chloride solution, organic phase merges, anhydrous sodium sulfate
It is dry, solvent evaporated is rotated, the secondary amine intermediate containing ester group is obtained；Secondary amine intermediate containing ester group is dissolved in tetrahydrofuran,
Chloro- 3, the 5- dinitro-p-trifluorotoluene of 4- and triethylamine is added, is stirred at room temperature reaction, chloro- 3, the 5- dinitro-p-trifluorotoluene of 4- with contain
The molar ratio of the secondary amine intermediate of ester group is 1:1.5；It is diluted with water after fully reacting, dilute hydrochloric acid is added to be acidified and is extracted with ethyl acetate
Take, organic phase saturated common salt water washing, anhydrous sodium sulfate is dry, rotate solvent evaporated, residue through column chromatographic isolation and purification,
It rotates solvent evaporated and obtains hapten molecule (6c).
It is by above-mentioned trefanocide haptens and carrier the present invention also provides a kind of trefanocide artificial immunogen and coating antigen
Albumen coupling obtains.
The present invention also provides a kind of trefanocide antibody, the trefanocide antibody is prepared using above-mentioned trefanocide artificial immunogen
It obtains, the trefanocide antibody is polyclonal antibody, monoclonal antibody or genetic engineering antibody.
The invention has the following beneficial effects:
(1) six kinds of haptens of the invention are derivative connection arm, active carboxyl, with carrier in amino sites
Body can be made to generate potency height after albumen coupling, can identify the specific antibody of trefanocide pesticide apokoinou construction.
(2) hapten compound of the invention, not only simple synthetic method, purity are higher, but also it is suitable to can apply to synthesis
In the antigen system of animal immune, the blank of domestic trefanocide immunological detection method technical field is compensated for, is exempted from for trefanocide
The further development of epidemic disease detection method is laid a good foundation.
(3) present invention improves the preparation method of trefanocide haptens, and synthesis step is simple, required experiment condition
Mildly, preparation method of the present invention is simple and feasible, cost is relatively low, and the purity and yield of haptens are higher.
Specific embodiment
The present invention will now be described in detail with reference to examples, and the examples are only preferred embodiments of the present invention,
It is not limitation of the invention.
Embodiment 1: the synthetic method of trefanocide hapten compound 1c is as follows:
(1) ethyl acrylate (3600mg, 36mmol) is dissolved in the chloroform of 30mmol, be added n-propylamine (3191.94mg,
54mmol) 40 DEG C of stirring 12h of oil bath obtain the secondary amine intermediate 1a containing ester group.
(2) 1a (239mg, 1.5mmol) is dissolved in 6ml tetrahydrofuran, is added NaOH aqueous solution (120mg, 3mmol),
It is stirred at room temperature, is hydrolyzed, 1b is obtained after hydrolysis.
(3) the chloro- benzotrifluoride of 3,5- dinitro -4- (270mg, 1mmol), stirring at normal temperature are directly added into 1b.With than
Example is n-hexane: ethyl acetate=2:1 chromatographic solution carries out contact plate tracking, falls to extract to carry out soda acid after the reaction was completed, acid extraction
Organic phase merges, dry with anhydrous sodium sulfate.40 DEG C are spin-dried for, and are n-hexane with chromatographic solution: ethyl acetate: acetic acid=2:1:
Silica gel plate separates on 0.015, obtains object 1c, weighing, yield 75.6%.ESI-MS analysis(negative):m/
z 366.2[M+H]—；1H NMR(600MHz,CDCl3)δ8.03(s,2H)；3.29 (t, J=6.3Hz, 2H)；2.89–2.84(m,
2H)；2.57–2.51(m,2H)；1.60–1.50(m,2H)；0.81 (t, J=7.4Hz, 3H).
Embodiment 2: the synthetic method of trefanocide hapten compound 2c is as follows:
(1) 2-Pyrrolidone (1000mg, 11mmol) is dissolved in 10mlDMF, and 13.2mmol NaH (60%), positive bromine third is added
Alkane (1758.9mg, 14.3mmol), under nitrogen protection, 70 DEG C of oil baths are stirred overnight.70ml ethyl acetate is added in reaction solution,
50ml saturated ammonium chloride solution is extracted, and organic phase merges, and anhydrous sodium sulfate is dry.40 DEG C are spin-dried for, and obtain containing the secondary of ester group
Amine intermediate 2a.
(2) 13ml 4N NaOH aqueous solution is added in 2a (846mg, 6mmol) and is stirred overnight carry out water in 100 DEG C of oil baths
Solution obtains 2b.
(3) the chloro- benzotrifluoride of 3,5- dinitro -4- (270mg, 1mmol) is dissolved in 6ml tetrahydrofuran, 2b is added
(190.5mg, 1.5mmol), is stirred at room temperature.It is diluted with 30ml water, adds 1M hydrochloric acid tune pH to 2-3, sediment occurs.Use second
Acetoacetic ester is extracted twice, and each ethyl acetate dosage is 30ml, and precipitating dissolves, and 10ml saturated salt solution is washed, organic phase
Merge, it is dry with anhydrous sodium sulfate.40 DEG C are spin-dried for, and are n-hexane with chromatographic solution: the upper silica gel plate of ethyl acetate=1:1 is divided
From, object 2c is obtained, is weighed, yield 82.4%.ESI-MS analysis(negative):m/z380.1[M+H]—；1H
NMR(600MHz,CDCl3)δ8.00(s,2H)；3.07–2.98(m,2H)；2.94–2.86(m,2H)；2.34 (t, J=7.2Hz,
2H)；1.89–1.79(m,2H)；1.55 (dd, J=14.9,7.5Hz, 2H)；0.81 (t, J=7.4Hz, 3H).
Embodiment 3: the synthetic method of trefanocide hapten compound 3c is as follows:
(1) valerolactam (2180mg, 20mmol) is dissolved in 10mlDMF, and 24mmol NaH (60%), n-Propyl Bromide is added
(2950mg, 24mmol), 70 DEG C of oil bath stirrings, reacts 4-5 hours under nitrogen protection.It is diluted with 50ml water, adds 1M salt
Acid adjusts pH to 2-3, and sediment occurs.It being extracted with ethyl acetate twice, each ethyl acetate dosage is 50ml, and precipitating dissolves,
15ml saturated salt solution is washed, and organic phase merges, dry with anhydrous sodium sulfate.40 DEG C are spin-dried for, and obtain the secondary amine containing ester group
3a, weighing, yield 55%.
(2) be added 37% hydrochloric acid of 2.5ml and 2.5ml water in 3a (846mg, 6mmol), 100 DEG C of oil baths be condensed back into
Row hydrolysis 4-5 hours, obtains hydrolysate 3b.
(3) the chloro- benzotrifluoride of 3,5- dinitro -4- (270mg, 1mmol) is dissolved in 6ml tetrahydrofuran, 3b is added
(235mg, 1.2mmol), triethylamine (404mg, 4mmol) and 1ml water, stirring at normal temperature are overnight.It is diluted, is added with 30ml water
1M hydrochloric acid tune pH to 2-3, sediment occur.It is extracted with ethyl acetate twice, each ethyl acetate dosage is 30ml, is precipitated molten
Solution, is washed with 10ml saturated salt solution, and organic phase merges, dry with anhydrous sodium sulfate.40 DEG C are spin-dried for, and are positive with chromatographic solution
Hexane: ethyl acetate: the upper silica gel plate of acetic acid=4:1:0.02 is separated, and obtains object 3c, and weigh 72mg, and yield is
65.3%.ESI-MS analysis(negative):m/z 394.3[M+H]—；1H NMR(600MHz,CDCl3)δ8.06(s,
2H)；3.04 (t, J=6.9Hz, 2H)；3.01–2.94(m,2H)；2.36 (t, J=6.8Hz, 2H)；1.74–1.50(m,6H)；
0.88 (t, J=7.4Hz, 3H).
Embodiment 4: the synthetic method of trefanocide hapten compound 4c is as follows:
(1) caprolactam (2260mg, 20mmol) is dissolved in 10mlDMF, and 24mmol NaH (60%), n-Propyl Bromide is added
(2950mg, 24mmol), 70 DEG C of oil bath stirrings, reacts 2h under nitrogen protection.It is diluted with 50ml water, adds 1M hydrochloric acid tune pH
To 2-3, sediment occurs.It is extracted with ethyl acetate twice, each ethyl acetate dosage is 50ml, and precipitating dissolution is full with 15ml
It is washed with saline solution, organic phase merges, dry with anhydrous sodium sulfate.40 DEG C are spin-dried for, and obtain the secondary amine intermediate containing ester group
4a, weighing, yield 55%.
(2) 37% hydrochloric acid of 2.5ml and 2.5ml water is added in 4a (930mg, 6mmol), and 100 DEG C of oil baths, which are condensed back, to be carried out
Hydrolyse over night obtains hydrolysate 4b.
The chloro- benzotrifluoride of (3) 3,5- dinitro -4- (450mg, 1.67mmol) is dissolved in 15ml tetrahydrofuran, and 4b is added
(1047.5mg, 5mmol), triethylamine (1686.7mg, 16.7mmol) and 2ml water, stirring at normal temperature are overnight.It is carried out with 90ml water
Dilution, adds 1M hydrochloric acid tune pH to 2-3, and sediment occurs.It is extracted with ethyl acetate twice, each ethyl acetate dosage is 90m, is sunk
It forms sediment and dissolves, washed with 30ml saturated salt solution, organic phase merges, dry with anhydrous sodium sulfate.40 DEG C are spin-dried for, use just oneself
Alkane: ethyl acetate=20:1,10:1,5:1,4:1 chromatography liquid proportional carries out column chromatography for separation, and 40 DEG C are spin-dried for, and obtains object
4c, weigh 73.2mg, yield 58%.ESI-MS analysis(negative)m/z 408.3[M+H]—；1H NMR
(600MHz,CDCl3)δ8.06(s,2H)；3.1 (t, J=7.2Hz, 2H)；2.98-2.93(m,2H)；2.34 (t, J=7.2Hz,
2H)；1.64-1.57(m,6H)；1.37-1.29(m,2H)；0.88 (t, J=7.8Hz, 3H).
Embodiment 5: the synthetic method of trefanocide hapten compound 5c is as follows:
(1) 4- crotonic ethyl bromide (965mg, 5mmol) is dissolved in 15ml ethyl acetate, and it is (pre- that reactor is placed in ice-methanol
First freeze) in be cooled to -15 DEG C, be slowly added to n-propylamine (885mg, 15mmol) with syringe, stirring at normal temperature 4-5 hours.With
The extraction of 80ml ethyl acetate, 30ml washing, the washing of 10ml saturated sodium chloride solution, organic phase are dry with anhydrous sodium sulfate.It is lower than
30 DEG C are spin-dried for, and obtain the secondary amine intermediate 5a containing ester group, are weighed as 874mg.
(2) 5a (256.5mg, 1.5mmol) is dissolved in 5ml tetrahydrofuran, and the chloro- benzotrifluoride of 3,5- dinitro -4- is added
(270mg, 1mmol), triethylamine (101mg, 1mmol), is stirred overnight at room temperature.It is n-hexane with chromatographic solution: ethyl acetate=10:
1 is tracked, and 70ml water is added after fully reacting and is diluted, and adds 1M hydrochloric acid tune pH to 2-3, and sediment occurs.With acetic acid second
Ester is extracted twice, and each ethyl acetate dosage is 70ml, and precipitating dissolution is washed with 20ml saturated salt solution, organic to be harmonious
And it is dry with anhydrous sodium sulfate.40 DEG C are spin-dried for, with n-hexane: ethyl acetate=40:1,20:1,10:1 chromatography liquid proportional into
Row column chromatography for separation, 40 DEG C are spin-dried for, and obtain intermediate 5b, and weigh 276.6mg, yield 68.29%.
(3) it is stirred in 50 DEG C of oil bath pans that 10ml tetrahydrofuran, 37% hydrochloric acid of 13ml is added in 5b (352mg, 0.87mmol)
Night is n-hexane with chromatographic solution: ethyl acetate: acetic acid=2:1:0.01 is tracked, and 120ml water is added after fully reacting and carries out
Dilution, adds 1M hydrochloric acid tune pH to 2-3, and sediment occurs.It is extracted with 120ml*2 ethyl acetate, precipitating dissolution is saturated with 40ml and is eaten
Salt water is washed, and organic phase merges, dry with anhydrous sodium sulfate.40 DEG C are spin-dried for, and are petroleum ether with chromatographic solution: ethyl acetate:
Acetic acid=10:1:0.05,10:2:0.05,10:4:0.05 sequence carries out column chromatography for separation, is spin-dried for, and weigh 122.9mg, yield
It is 76.9%.ESI-MS analysis(negative)m/z 378.2[M+H]—；1H NMR(600MHz,CDCl3)δ8.10(s,
2H)；7.12–6.72(m,1H)；6.03 (d, J=15.7Hz, 1H)；3.02–2.90(m,2H)；2.17(s,2H)；1.66–1.48
(m,2H)；0.86 (dd, J=15.9,8.5Hz, 3H).
Embodiment 6: the synthetic method of trefanocide hapten compound 6c is as follows:
(1) 4- bromomethyl-benzoic acid methyl ester (916.28mg, 4mmol) is dissolved in 12ml ethyl acetate, by reactor as ice-
It is cooled to -15 DEG C in methanol, is slowly added to n-propylamine (591.1mg, 10mmol) with syringe and reacts 4-5 hours.With 90ml second
Acetoacetic ester extraction, 30ml washing, the washing of 10ml saturated sodium chloride solution, organic phase merge, and anhydrous sodium sulfate is dry.Lower than 30 DEG C
It is spin-dried for, obtains the secondary amine intermediate 6a containing ester group, weigh 725mg, yield 87.5%.
(2) 6a (310.77mg, 1.5mmol) is dissolved in 6ml tetrahydrofuran, and the chloro- benzotrifluoride of 3,5- dinitro -4- is added
(270mg, 1mmol), triethylamine (101mg, 1mmol), is stirred overnight at room temperature.Reaction solution is added 80ml water and is diluted, and adds 1M
Hydrochloric acid tune pH to 2-3, sediment occur.It being extracted with ethyl acetate twice, each ethyl acetate dosage is 80ml, and precipitating dissolves,
It is washed with 30ml saturated salt solution, organic phase merges, dry with anhydrous sodium sulfate.40 DEG C are spin-dried for, and are positive oneself with chromatographic solution
Alkane: ethyl acetate=40:1,20:1,10:1 sequence carries out column chromatography for separation, is spin-dried for obtaining 6b, is weighed as 338.9mg, yield
It is 76.9%.
(3) 6b (150mg, 0.34mmol) is dissolved in 7.5ml tetrahydrofuran, 6.8ml 1mol/L sodium hydroxide solution, room temperature
Be stirred overnight, be n-hexane with chromatographic solution: ethyl acetate: acetic acid=2:1:0.01 is tracked, and reaction is completed, and carries out soda acid
, acid extraction organic phase merges, and 40 DEG C are spin-dried for, and obtains crude product 118.8mg, carries out thin-layer chromatography separation, obtains object 6c, claims
Weight 105.8mg, yield 86%.ESI-MS analysis(negative)m/z 428.4[M+H]—；1H NMR(600MHz
CDCl3)δ8.08(s,2H)；8.06 (d, J=8.4Hz, 2H)；4.28(s,2H)；2.93–2.88(m,2H)；0.87 (d, J=
7.1Hz,2H)；0.82 (d, J=7.2Hz, 3H).
Embodiments of the present invention above described embodiment only expresses, the description thereof is more specific and detailed, but can not
Therefore limitations on the scope of the patent of the present invention are interpreted as, as long as skill obtained in the form of equivalent substitutions or equivalent transformations
Art scheme should all be fallen within the scope and spirit of the invention.
Claims (1)
1. a kind of synthetic method of trefanocide haptens, which is characterized in that the trefanocide haptens has the following structure formula:
Haptens structure be 3c when, synthetic method is as follows: valerolactam is dissolved in DMF, sequentially add 60% sodium hydride solution and
N-Propyl Bromide, wherein the molar ratio of valerolactam, sodium hydride and n-Propyl Bromide are as follows: 1:1.2:1.2, under nitrogen protection, 70 DEG C
Oil bath is stirred to react；After reaction, it is diluted with water, and adds acid for adjusting pH to 2-3, sediment occur, extracted with ethyl acetate
It takes, then with saturated common salt water washing, anhydrous sodium sulfate is dry, rotates solvent evaporated, obtains the secondary amine intermediate containing ester group；Xiang Han
37% hydrochloric acid and water, 100 DEG C of oil baths are added in the secondary amine intermediate of ester group, condensing reflux is hydrolyzed, and obtains the second intermediate；
Chloro- 3, the 5- dinitro-p-trifluorotoluene of 4- is dissolved in tetrahydrofuran, is added above-mentioned second intermediate, triethylamine and water, 4- chloro- 3,
The molar ratio of 5- dinitro-p-trifluorotoluene and the second intermediate is 1:1.2, and reaction is stirred at room temperature；It is diluted with water after fully reacting,
Dilute hydrochloric acid is added to be acidified and be extracted with ethyl acetate, organic phase saturated common salt water washing, anhydrous sodium sulfate is dry, and rotation is evaporated molten
Agent obtains hapten molecule；
When haptens structure is 5c, synthetic method includes the following: that 4- crotonic ethyl bromide is dissolved in ethyl acetate, and reactor is set
- 15 DEG C are cooled in ice-methanol, is slowly added to n-propylamine, the molar ratio of 4- crotonic ethyl bromide and n-propylamine with syringe
For 1:3, stirring at normal temperature reaction, after reaction, ethyl acetate extracts reaction mixture, then is washed with saturated sodium chloride solution,
Organic phase is dry with anhydrous sodium sulfate, rotates solvent evaporated, obtains the secondary amine intermediate containing ester group；Among the secondary amine containing ester group
Body is dissolved in tetrahydrofuran, and chloro- 3, the 5- dinitro-p-trifluorotoluene of 4- and triethylamine is added, reaction, chloro- 3, the 5- bis- of 4- is stirred at room temperature
The molar ratio of nitro-trifluoromethyl toluene and the secondary amine intermediate containing ester group is 1:1.5；It is diluted with water after fully reacting, adds dilute hydrochloric acid sour
Change and be extracted with ethyl acetate, organic phase saturated common salt water washing, anhydrous sodium sulfate is dry, rotates solvent evaporated, residue
Through column chromatographic isolation and purification, rotates solvent evaporated and obtain hapten molecule；
When haptens structure is 6c, synthetic method includes the following: that 4- bromomethyl-benzoic acid methyl ester is dissolved in ethyl acetate, will be reacted
Device is placed in ice-methanol and is cooled to -15 DEG C, is slowly added to n-propylamine, 4- bromomethyl-benzoic acid methyl ester and n-propylamine with syringe
Molar ratio be 1:2.5 reaction mixture is extracted with ethyl acetate, then washed with saturated sodium chloride solution after reaction,
Organic phase merges, and anhydrous sodium sulfate is dry, rotates solvent evaporated, obtains the secondary amine intermediate containing ester group；By the secondary amine containing ester group
Intermediate is dissolved in tetrahydrofuran, and chloro- 3, the 5- dinitro-p-trifluorotoluene of 4- and triethylamine is added, and is stirred at room temperature reaction, 4- chloro- 3,
The molar ratio of 5- dinitro-p-trifluorotoluene and the secondary amine intermediate containing ester group is 1:1.5；It is diluted with water after fully reacting, adds dilute salt
Acid is acidified and is extracted with ethyl acetate, organic phase saturated common salt water washing, and anhydrous sodium sulfate is dry, rotates solvent evaporated, residual
Excess rotates solvent evaporated and obtains hapten molecule through column chromatographic isolation and purification.
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