CN109956895A - A kind of procymidone haptens and its synthetic method and application - Google Patents
A kind of procymidone haptens and its synthetic method and application Download PDFInfo
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- CN109956895A CN109956895A CN201910194037.5A CN201910194037A CN109956895A CN 109956895 A CN109956895 A CN 109956895A CN 201910194037 A CN201910194037 A CN 201910194037A CN 109956895 A CN109956895 A CN 109956895A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D209/00—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom
- C07D209/02—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom condensed with one carbocyclic ring
- C07D209/52—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom condensed with one carbocyclic ring condensed with a ring other than six-membered
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/02—Food
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
Abstract
The invention discloses a kind of procymidone haptens and its synthetic method and applications, synthesize (1) using methyl α-chloroacrylate, methyl methacrylate as starting material and synthesize 1,2- dimethyl -1,2- cyclopropane dicarboxylic acid's methyl esters；1,2- dimethyl -1,2- cyclopropane dicarboxylic acid methyl esters is using generation 1,2- dimethylcyclopropane -1,2- dicarboxylic anhydride after hydrolysis and intramolecular condensation；It (2) is intermediate of the Material synthesis with arm with the chloro- 5- nitrophenol of 3-, compound 3, which obtains intermediate B through reduction reaction；(3) 1,2- dimethylcyclopropane -1,2- dicarboxylic anhydride and intermediate B pass through the cyclization of condensation reaction twice and a hydrolysis obtains final goal object.In the present invention, the procymidone haptens of synthesis had both utmostly remained the feature structure of procymidone, so that the immunogenicity of procymidone haptens is remarkably reinforced, and has the carboxyl that can be coupled with carrier, basis is provided for the subsequent various immunoassay methods for establishing procymidone.
Description
Technical field
The present invention relates to technical field of food safety detection, more particularly to a kind of procymidone haptens and its synthesis side
Method and application.
Background technique
Procymidone (Procymidone) is also known as Sukeling, kills mould benefit, SP 751011 etc., belongs to hypotoxicity fungicide,
Protection and therapeutic effect are had both, the lasting period is longer, resistance to rainwater punching；It can be used for preventing and treating the grey mold of cucumber, eggplant, tomato, onion etc.
Disease, lettuce, capsicum stem rot, sclerotinia sclerotiorum etc. is also used for the anti-of the fruit storages phase such as citrus, banana, apple, pears, peach
It is rotten fresh-keeping.A small amount of pesticide residue not will lead to acute poisoning, but the vegetables of excessive pesticide residues are eaten for a long time, may be to human body
Health generates certain adverse effect, and country also exposes leek, the exceeded situation of procymidone in garlic stems often in the recent period.
The detection method of existing procymidone mainly has high performance liquid chromatography (HPLC), liquid phase-mass spectrometry (LC-MS)
Method, gas chromatography-mass spectrometry (GC-MS) etc..Instrument equipment operation is complicated, at high cost, right in above-mentioned detection method
Operator's technical requirements are high, and cannot show immediately as a result, not being suitable for food, commodity inspection, epidemic prevention, husbandry sector person to suspection
Object carries out quick on-line checking and monitoring.
Compared with above-mentioned detection method, immunology detection technology have it is economical, quickly, technical essential it is low, it is easy to operate and
On-site test etc. can be achieved a little.Immunoassay detection technique developed in the fields of environment and food inspection in recent years
A kind of novel quick and precisely detection method, has been increasingly becoming the main of the quick selective mechanisms of countries in the world noxious residual chemicals
One of method.
When establishing immunological detection method and application detection method detection procymidone residual quantity, key technology is energy
The antibody of high specificity, high sensitivity is enough got, and to realize this target, precondition must exactly synthesize, prepare conjunction
Suitable procymidone haptens.But the currently, domestic relevant report for being directed to procymidone haptens not yet.
Summary of the invention
In order to make up the defect of prior art, the present invention provides a kind of procymidone haptens and its synthetic method and application,
Design a kind of synthetic method of procymidone haptens for the design feature of procymidone, synthesize procymidone haptens, for into
One-step synthesis procymidone artificial antigen induces immune animal to generate antibody, to establish the various immunoassay methods of procymidone.
The technical problems to be solved by the invention are achieved by the following technical programs:
A kind of procymidone haptens, which is characterized in that shown in its structural formula such as formula (I):
Wherein, n is-CH2 group numbers, and n is the integer of 1-5.
The present invention also provides the synthetic methods of above-mentioned procymidone haptens, include the following steps:
S1, provides intermediate A, and the intermediate A has structural formula；
S2, provides intermediate B, and the intermediate B has structural formula；
S3 reacts the intermediate A and intermediate B, obtains compound 5, and the compound 5 has structural formula；
The compound 5 is carried out condensation reaction by S4, obtains compound 6, and the compound 6 has structural formula；
The compound 6 is hydrolyzed reaction, obtains the procymidone haptens by S5.
Further, step S3 such as includes the following steps: to weigh at the intermediate A and intermediate B of amount of substance, uses organic solvent
It is reacted after dissolution, after reaction solvent evaporated, crosses column purification and obtain the compound 5；Wherein, the organic solvent is
One of anhydrous pyridine, tetrahydrofuran and methylene chloride are a variety of；The reaction temperature is 50-100 DEG C.
Further, the step S4 includes the following steps: for the compound 5 to be dissolved in methylene chloride, is added de-
Water condensing agent, back flow reaction 0.5-5h, after completion of the reaction, solvent evaporated crosses column purification and obtains the compound 6；It is wherein described
Dehydrating condensation agent is N, N'- Dicyclohexylcarbodiimide, EDCHCl or N, N '-carbonyl dimidazoles；The compound 5 and de-
The ratio between amount of substance of water condensing agent is 1:1.
Further, the step S5 includes the following steps: for the compound 6 to be dissolved in methylene chloride, is added three
It is reacted after fluoroacetic acid, after completion of the reaction, solvent evaporated, after ethyl alcohol is added, solvent evaporated again, washed, dry, mistake column
The procymidone haptens is obtained after purification；Wherein, the volume ratio of the methylene chloride and trifluoroacetic acid is 2:1, reaction time
For 0.5-5h.
Further, in the step S1, the intermediate A the preparation method comprises the following steps:
(1) it is reacted by methyl methacrylate and methyl α-chloroacrylate, obtains compound 1, the compound 1 has
Structural formula；
(2) reaction is hydrolyzed in the compound 1, obtains compound 2, the compound 2 has structural formula；
(3) compound 2 is subjected to condensation reaction, obtains intermediate A.
Further, the compound 1 the preparation method comprises the following steps: reaction dissolvent is added in the reaction vessel, in ice-water bath item
Under part, sodium hydride is added, methyl α-chloroacrylate and methyl methacrylate are slowly added dropwise after a certain period of time, drips for stirring
It is reacted at room temperature overnight after finishing, after completion of the reaction, is isolated and purified, obtain the compound 1；Wherein the reaction dissolvent is first
One of benzene, methylene chloride and DMF or a variety of；The sodium hydride, methyl α-chloroacrylate and methyl methacrylate
The ratio between amount of substance is (2-5): 1:(1-1.3)；It is described to isolate and purify operation are as follows: ethyl alcohol is added, after stirring 10-15min, is added
PH value of solution is tuned into 5-6 with dilute hydrochloric acid, is extracted with ethyl acetate by water, collects ethyl acetate layer, washed, drying process
Vacuum distillation removes reaction dissolvent and ethyl acetate afterwards, and residue obtains the compound 1 through oil pump vacuum distillation purifying.
Further, the compound 2 the preparation method comprises the following steps: Weigh Compound 1, after being dissolved with ethyl alcohol, it is molten that alkalinity is added
Liquid is heated to reflux, and reaction is hydrolyzed；Vacuum distillation removes ethyl alcohol after reaction, and water is added, is extracted with ethyl acetate, point
Water layer is separated out, water layer adjusts pH to 2-3 with dilute hydrochloric acid, then is extracted with ethyl acetate, and obtains after washed, dry, revolving described
Compound 2；Wherein, it is 10% or more sodium hydroxide or potassium hydroxide solution that the alkaline solution, which uses mass concentration,
Dosage is 15 times of 10- of 1 mass of compound.
Further, in the step (3) the step of condensation reaction are as follows: acetic anhydride is added in the compound 2, reflux is anti-
It answers, after reaction, acetic anhydride is distilled off, residue is evaporated under reduced pressure through oil pump, crystallization obtains the intermediate A；Wherein, institute
The time for stating back flow reaction is 2-5h；The temperature that acetic anhydride is distilled off is 160-180 DEG C.
Further, in the step S2, the intermediate B the preparation method comprises the following steps:
(1) the chloro- 5- nitrophenol of 3- and compound 3 are reacted, obtains compound 4, the compound 3 has structural formula, wherein the X in compound 3 is halogen, and the compound 4 has structural formula:
(2) compound 4 is subjected to nitro-reduction reaction, obtains intermediate B.
Further, the compound 4 the preparation method comprises the following steps: the chloro- 5- nitrophenol of 3- and the compound 3 are dissolved in
In DMF, add Anhydrous potassium carbonate, reacted at 50-110 DEG C, after reaction, be added distilled water, with ethyl acetate into
Row extraction collects ethyl acetate layer and obtains the compound 4 using column chromatography for separation after washed, dry, revolving processing；
Wherein, the ratio between amount of substance of the chloro- 5- nitrophenol of the 3-, compound 3 and Anhydrous potassium carbonate is 1:(1-3): (1.5-
10).
The present invention also provides application of the above-mentioned procymidone haptens in the analysis of procymidone immune detection.
The invention has the following beneficial effects:
In the present invention, the derivative linking arm on the phenyl ring of procymidone, derivative linking arm has carboxyl, and the procymidone half of synthesis is anti-
Original had both utmostly remained the feature structure of procymidone, so that the immunogenicity of procymidone haptens is remarkably reinforced, and had
The carboxyl that can be coupled with carrier；The procymidone artificial antigen obtained after procymidone haptens and carrier conjugation goes immune dynamic
Object is more advantageous to stimulation animal immune response and generates specific stronger, the higher antibody of sensitivity, for subsequent procymidone of establishing
Various immunoassay methods provide basis.
The synthetic method of procymidone haptens in the present invention, the raw material used are easy to get, and operation is relatively simple, react item
Part is easily controllable.Procymidone synthesis of semiantigen of the invention, the purity and high income of procymidone haptens, the amino of synthesis
For the yield of acid herbicides haptens up to 50% or more, whole synthesis cost has more advantage.
Detailed description of the invention
Fig. 1 is the synthetic route of procymidone haptens in the embodiment of the present invention 1.
Specific embodiment
In a first aspect, the present invention provides a kind of procymidone haptens, which is characterized in that shown in its structural formula such as formula (I):
Wherein, n is-CH2 group numbers, and n is the integer of 1-5.
In the present invention, derivative linking arm, derivative linking arm have carboxyl, the procymidone of synthesis on the phenyl ring of procymidone
Haptens had both utmostly remained the feature structure of procymidone, so that the immunogenicity of procymidone haptens is remarkably reinforced, again
With the carboxyl that can be coupled with carrier；The procymidone artificial antigen obtained after procymidone haptens and carrier conjugation goes to exempt from
Epidemic disease animal is more advantageous to stimulation animal immune response and generates specific stronger, the higher antibody of sensitivity, rotten mould for subsequent foundation
The various immunoassay methods of benefit provide basis.
Procymidone haptens of the invention, not only simple synthetic method, purity are higher, but also can apply to synthesis and be suitable for moving
The immune antigen system of object, compensates for the blank of domestic procymidone immunological detection method technical field, for the immune inspection of procymidone
The further development of survey method is laid a good foundation.
Second aspect, the present invention also provides the synthetic methods of above-mentioned procymidone haptens, include the following steps:
S1, provides intermediate A, and the intermediate A has structural formula；
S2, provides intermediate B, and the intermediate B has structural formula, wherein n
For-CH2 group numbers, n is the integer of 1-5；
S3 reacts the intermediate A and intermediate B, obtains compound 5, and the compound 5 has structural formula, wherein n is-CH2 group numbers, and n is the integer of 1-5；
The compound 5 is carried out condensation reaction by S4, obtains compound 6, and the compound 6 has structural formula, wherein n is-CH2 group numbers, and n is the integer of 1-5；
The compound 6 is hydrolyzed reaction, obtains the procymidone haptens by S5.
The present invention rationally designs the synthetic method of procymidone haptens, the raw material used according to the design feature of procymidone
It is easy to get, operation is relatively simple, and reaction condition is easily controllable.Procymidone synthesis of semiantigen of the invention, procymidone half
The purity and high income of antigen, the yield of the amino acids herbicide haptens of synthesis up to 50% or more, whole synthesis at
This has more advantage.
Wherein, in the step S1, the intermediate A the preparation method comprises the following steps:
(1) it is reacted by methyl methacrylate and methyl α-chloroacrylate, obtains compound 1, the compound 1 has
Structural formula；
Preferably, in order to further increase the yield and reaction efficiency of compound 1, the compound 1 the preparation method comprises the following steps: anti-
It answers and reaction dissolvent is added in container, under the conditions of ice-water bath, sodium hydride is added, alpha-chloro third is slowly added dropwise in stirring after a certain period of time
E pioic acid methyl ester and methyl methacrylate react at room temperature overnight after being added dropwise, after completion of the reaction, are isolated and purified, obtained
The compound 1.Wherein the reaction dissolvent include but is not limited to be one of toluene, methylene chloride and DMF or a variety of；Institute
Stating the ratio between amount of substance of sodium hydride, methyl α-chloroacrylate and methyl methacrylate is (2-5): 1:(1-1.3), it is more excellent
Selection of land, the ratio between the sodium hydride, amount of substance of methyl α-chloroacrylate and methyl methacrylate are 2:1:1.2；It will be each
Raw material dosage controls within the above range, is on the one hand conducive to the yield for further increasing compound 1, is on the one hand conducive to avoid
Raw material excess waste.It is described to isolate and purify operation preferably are as follows: ethyl alcohol is added, after stirring 10-15min, water is added, it will with dilute hydrochloric acid
PH value of solution is tuned into 5-6, is extracted with ethyl acetate, collects ethyl acetate layer, is evaporated under reduced pressure and removes after washed, drying process
Reaction dissolvent and ethyl acetate, residue obtain the compound 1 through oil pump vacuum distillation purifying.
(2) reaction is hydrolyzed in the compound 1, obtains compound 2, the compound 2 has structural formula；
Preferably, the compound 2 the preparation method comprises the following steps: Weigh Compound 1, after being dissolved with ethyl alcohol, alkaline solution, heating is added
Reflux, is hydrolyzed reaction；Vacuum distillation removes ethyl alcohol after reaction, and water is added, is extracted with ethyl acetate, separation water outlet
Layer, water layer adjusts pH to 2-3 with dilute hydrochloric acid, then is extracted with ethyl acetate, and obtains the compound after washed, dry, revolving
2.The step is conducive to further increase the reaction efficiency and reaction stability of compound 2.Wherein, the alkaline solution uses matter
Amount concentration is 10% or more sodium hydroxide or potassium hydroxide solution, and dosage is 10-15 times of 1 mass of compound.
Within the above range by the control of each raw material dosage, be on the one hand conducive to the yield for further increasing compound 2, be on the one hand conducive to
Avoid raw material excess waste.
(3) compound 2 is subjected to condensation reaction, obtains intermediate A.
In the present invention, compound 2 carries out condensation reaction, the intramolecular dehydration cyclization of compound 2.The side of the condensation reaction
Method and condition can be the conventional method and condition of the such reaction in this field, following methods and condition specifically preferred according to the invention: will
Acetic anhydride is added in the compound 2, and acetic anhydride is distilled off after reaction in back flow reaction, and residue is depressurized through oil pump to be steamed
It evaporates, crystallize and obtain the intermediate A；Wherein, the time of the back flow reaction is 2-5h, it is highly preferred that the back flow reaction
Time is 3h；The temperature that acetic anhydride is distilled off is 160-180 DEG C, more preferably 170 DEG C.Under the step, reaction has higher
Efficiency and stability.
In the step S2, the intermediate B the preparation method comprises the following steps:
(1) the chloro- 5- nitrophenol of 3- and compound 3 are reacted, obtains compound 4, the compound 3 has structural formulaWherein, the X in compound 3 is halogen, and n is-CH2 group numbers, and n is the integer of 1-5,
The compound 4 has structural formula, wherein n is-CH2 group numbers, n 1-5
Integer；
Preferably, the compound 4 the preparation method comprises the following steps: the chloro- 5- nitrophenol of 3- and the compound 3 are dissolved in DMF, then
Anhydrous potassium carbonate is added, is reacted at 50-110 DEG C, after reaction, distilled water is added, is extracted with ethyl acetate, receive
Collect ethyl acetate layer and obtains the compound 4 using column chromatography for separation after washed, dry, revolving processing；Wherein, described
The ratio between amount of substance of the chloro- 5- nitrophenol of 3-, compound 3 and Anhydrous potassium carbonate is 1:(1-3): (1.5-10), more preferably
Ground, the ratio between the chloro- 5- nitrophenol of the 3-, amount of substance of compound 3 and Anhydrous potassium carbonate are 1:1.5:2.Each raw material is used
Amount control within the above range, is on the one hand conducive to the yield for further increasing compound 1, is on the one hand conducive to avoid raw material mistake
Degree waste.
(2) compound 4 is subjected to nitro-reduction reaction, obtains intermediate B.
The nitro-reduction reaction is the nitro and reduced iron powder, metallic tin, protochloride of compound 4 in organic solvent
The mixture of one or more of tin, sulfur-containing compound, metal hydride, CO react or amino, institute is made in catalytic hydrogenation
The method and condition for stating nitro-reduction reaction can be the conventional method and condition of the such reaction in this field, and the present invention is especially excellent
Select following methods and condition:
Reduction reaction is carried out in organic solvent under the conditions of by the compound 4 existing for the hydrogen and catalyst, and reaction terminates
Afterwards, it filtered, be evaporated organic solvent, column purification excessively obtains intermediate B；Wherein, it is preferred that the catalyst is palladium carbon, described
Organic solvent be methanol；The quality of the catalyst is the 10% of the quality of the compound 4.
The step S3 such as includes the following steps: to weigh at the intermediate A and intermediate B of amount of substance, is dissolved with organic solvent
After reacted, solvent evaporated after reaction crosses column purification and obtains the compound 5；Wherein, the organic solvent include but
It is not limited to one of anhydrous pyridine, tetrahydrofuran and methylene chloride or a variety of；The reaction temperature is preferably 50-100 DEG C,
More preferably 80 DEG C.Under the step, intermediate A and intermediate B can more fully react, to further improve compound
5 yield and reaction efficiency.
The step S4 includes the following steps: for the compound 5 to be dissolved in methylene chloride, and dehydrating condensation agent is added,
Back flow reaction 0.5-5h, after completion of the reaction, solvent evaporated crosses column purification and obtains the compound 6；The wherein dehydrating condensation agent
It is including but not limited to N, N'- Dicyclohexylcarbodiimide, EDCHCl or N, N '-carbonyl dimidazoles；5 He of compound
The ratio between amount of substance of dehydrating condensation agent is 1:1.Under the step, reaction has higher efficiency and stability.
The step S5 includes the following steps: for the compound 6 to be dissolved in methylene chloride, and it is laggard that trifluoroacetic acid is added
Row reaction, after completion of the reaction, solvent evaporated, after ethyl alcohol is added, solvent evaporated again is obtained after washed, dry, mistake column purification
The procymidone haptens；Wherein, the volume ratio of the methylene chloride and trifluoroacetic acid is 2:1, reaction time 0.5-5h.It should
Under step, reaction has higher efficiency and stability.
The third aspect, the present invention also provides application of the above-mentioned procymidone haptens in the analysis of procymidone immune detection.
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
A kind of synthetic method of procymidone haptens, includes the following steps:
S1, provides intermediate A, and the intermediate A is 1,2- dimethyl -1,2- cyclopropane acid anhydrides；
The intermediate A the preparation method comprises the following steps:
(1) 100mL toluene is added in 250mL there-necked flask, under the conditions of ice-water bath, 0.2mol sodium hydride is added, stirs 30min
After 0.1mol methyl α-chloroacrylate and 0.12mol methyl methacrylate is slowly added dropwise, reacted at room temperature after being added dropwise
10mL ethyl alcohol is added after completion of the reaction in night, after stirring 10-15min, 200mL water is added, pH value of solution is tuned into 5- with dilute hydrochloric acid
6, it is extracted with 200mL ethyl acetate, collects ethyl acetate layer, ethyl acetate layer is through saturated common salt water washing, anhydrous slufuric acid
Vacuum distillation removes toluene and ethyl acetate after sodium is dried, and residue is evaporated under reduced pressure through oil pump and purifies, and obtains 1,2- diformazan
Base -1,2- cyclopropane dicarboxylic acid's methyl esters, i.e. compound 1；
(2) 10g compound 1 is weighed, after the dissolution of 30mL ethyl alcohol, the sodium hydroxide solution that 100g mass concentration is 10% is added,
It is refluxed overnight at 80 DEG C, reaction is hydrolyzed；Vacuum distillation removes ethyl alcohol after reaction, and water is added, is extracted with ethyl acetate,
Water-yielding stratum is separated, water layer adjusts pH to 2-3 with dilute hydrochloric acid, then is extracted with ethyl acetate, through saturated common salt water washing, anhydrous slufuric acid
Compound 2 is obtained after sodium drying, revolving；
(3) dissolution of 20mL acetic anhydride is added in compound 2 described in 8g, 170 DEG C of back flow reaction 2h are distilled off after reaction
Acetic anhydride, residue is evaporated under reduced pressure through oil pump, crystallization obtains the intermediate A；
S2, provides intermediate B, the intermediate B the preparation method comprises the following steps:
(1) the chloro- 5- nitrophenol of 0.02mol 3- and the 0.03mol 4- bromo-butyric acid tert-butyl ester are dissolved in 15mL DMF, are added
0.04mol Anhydrous potassium carbonate is reacted at 80 DEG C, after reaction, 200mL distilled water is added, is extracted with ethyl acetate
It takes, collects ethyl acetate layer, after saturated common salt water washing, anhydrous sodium sulfate drying, revolving processing, using column chromatography for separation
Obtain compound 4；
(2) 2g compound 4 is dissolved with methanol, 0.2g palladium carbon is added, then be passed through hydrogen, stirring is reacted at room temperature, is reacted
After, it filtered, be evaporated methanol, column purification excessively obtains intermediate B；
S3 the intermediate A and intermediate B of amount of substance such as weighs, after being dissolved with anhydrous pyridine, is reacted at 80 DEG C, reaction knot
Solvent evaporated after beam crosses column purification and obtains the compound 5；
Compound 5 is dissolved in methylene chloride by S4, and dehydrating condensation agent is added, and back flow reaction 1h is evaporated molten after completion of the reaction
Agent crosses column purification and obtains compound 6；Wherein the dehydrating condensation agent is N, N'- Dicyclohexylcarbodiimide；The compound 5
It is 1:1 with the ratio between the amount of substance of dehydrating condensation agent；
Compound 6 is dissolved in methylene chloride by S5, and room temperature carries out reaction 1h after trifluoroacetic acid is added, and after completion of the reaction, is evaporated
Solvent, after ethyl alcohol is added, solvent evaporated again obtains the procymidone haptens after washed, dry, mistake column purification；Wherein,
The volume ratio of the methylene chloride and trifluoroacetic acid is 2:1.
Embodiment 2
A kind of synthetic method of procymidone haptens, includes the following steps:
S1, provides intermediate A, and the intermediate A is 1,2- dimethyl -1,2- cyclopropane acid anhydrides；
The intermediate A the preparation method comprises the following steps:
(1) 100mL methylene chloride is added in 250mL there-necked flask, under the conditions of ice-water bath, 0.5mol sodium hydride, stirring is added
0.1mol methyl α-chloroacrylate and 0.1mol methyl methacrylate are slowly added dropwise after 30min, it is anti-that rear room temperature is added dropwise
It should stay overnight, after completion of the reaction, 20mL ethyl alcohol be added, after stirring 10-15min, 200mL water be added, with dilute hydrochloric acid by pH value of solution tune
It at 5-6, is extracted with 200mL ethyl acetate, collects ethyl acetate layer, ethyl acetate layer is through saturated common salt water washing, anhydrous
Vacuum distillation removes methylene chloride and ethyl acetate after sodium sulphate is dried, and residue is evaporated under reduced pressure through oil pump and purifies, obtains
1,2- dimethyl -1,2- cyclopropane dicarboxylic acid's methyl esters, i.e. compound 1；
(2) 10g compound 1 is weighed, after the dissolution of 30mL ethyl alcohol, the potassium hydroxide solution that 120g mass concentration is 10% is added,
It is refluxed overnight at 80 DEG C, reaction is hydrolyzed；Vacuum distillation removes ethyl alcohol after reaction, and water is added, is extracted with ethyl acetate,
Water-yielding stratum is separated, water layer adjusts pH to 2-3 with dilute hydrochloric acid, then is extracted with ethyl acetate, through saturated common salt water washing, anhydrous slufuric acid
Compound 2 is obtained after sodium drying, revolving；
(3) dissolution of 20mL acetic anhydride is added in compound 2 described in 8g, 160 DEG C of back flow reaction 5h are distilled off after reaction
Acetic anhydride, residue is evaporated under reduced pressure through oil pump, crystallization obtains the intermediate A；
S2, provides intermediate B, the intermediate B the preparation method comprises the following steps:
(1) the chloro- 5- nitrophenol of 0.02mol 3- and 0.06mol 2- bromo-acetic acid tert-butyl are dissolved in 15mL DMF, are added
0.03mol Anhydrous potassium carbonate is reacted at 50 DEG C, after reaction, 200mL distilled water is added, is extracted with ethyl acetate
It takes, collects ethyl acetate layer, after saturated common salt water washing, anhydrous sodium sulfate drying, revolving processing, using column chromatography for separation
Obtain compound 4；
(2) 2g compound 4 is dissolved with methanol, 0.2g palladium carbon is added, then be passed through hydrogen, stirring is reacted at room temperature, is reacted
After, it filtered, be evaporated methanol, column purification excessively obtains intermediate B；
S3 the intermediate A and intermediate B of amount of substance such as weighs, after being dissolved with tetrahydrofuran, is reacted at 50 DEG C, reaction knot
Solvent evaporated after beam crosses column purification and obtains the compound 5；
The compound 5 is dissolved in methylene chloride by S4, addition dehydrating condensation agent, back flow reaction 0.5h, after completion of the reaction,
Solvent evaporated crosses column purification and obtains compound 6；Wherein the dehydrating condensation agent is EDCHCl；The compound 5 and dehydration contracting
The ratio between amount of substance of mixture is 1:1；
Compound 6 is dissolved in methylene chloride by S5, and room temperature carries out reaction 0.5h after trifluoroacetic acid is added, and after completion of the reaction, is steamed
Dry solvent, after ethyl alcohol is added, solvent evaporated again obtains the procymidone haptens after washed, dry, mistake column purification；Its
In, the volume ratio of the methylene chloride and trifluoroacetic acid is 2:1.
Embodiment 3
A kind of synthetic method of procymidone haptens, includes the following steps:
S1, provides intermediate A, and the intermediate A is 1,2- dimethyl -1,2- cyclopropane acid anhydrides；
The intermediate A the preparation method comprises the following steps:
(1) 100mLDMF is added in 250mL there-necked flask, under the conditions of ice-water bath, 0.4mol sodium hydride is added, stirs 30min
After 0.1mol methyl α-chloroacrylate and 0.13mol methyl methacrylate is slowly added dropwise, reacted at room temperature after being added dropwise
15mL ethyl alcohol is added after completion of the reaction in night, after stirring 10-15min, 200mL water is added, pH value of solution is tuned into 5- with dilute hydrochloric acid
6, it is extracted with 200mL ethyl acetate, collects ethyl acetate layer, ethyl acetate layer is through saturated common salt water washing, anhydrous slufuric acid
Vacuum distillation removes DMF and ethyl acetate after sodium is dried, and residue is evaporated under reduced pressure through oil pump and purifies, and obtains 1,2- diformazan
Base -1,2- cyclopropane dicarboxylic acid's methyl esters, i.e. compound 1；
(2) 10g compound 1 is weighed, after the dissolution of 30mL ethyl alcohol, the sodium hydroxide solution that 150g mass concentration is 12% is added,
It is refluxed overnight at 80 DEG C, reaction is hydrolyzed；Vacuum distillation removes ethyl alcohol after reaction, and water is added, is extracted with ethyl acetate,
Water-yielding stratum is separated, water layer adjusts pH to 2-3 with dilute hydrochloric acid, then is extracted with ethyl acetate, through saturated common salt water washing, anhydrous slufuric acid
Compound 2 is obtained after sodium drying, revolving；
(3) dissolution of 20mL acetic anhydride is added in compound 2 described in 8g, 180 DEG C of back flow reaction 3h are distilled off after reaction
Acetic anhydride, residue is evaporated under reduced pressure through oil pump, crystallization obtains the intermediate A；
S2, provides intermediate B, the intermediate B the preparation method comprises the following steps:
(1) the chloro- 5- nitrophenol of 0.02mol 3- and the 0.02mol 3- bromo-propionic acid tert-butyl ester are dissolved in 15mL DMF, are added
0.2mol Anhydrous potassium carbonate is reacted at 110 DEG C, after reaction, 200mL distilled water is added, is extracted with ethyl acetate
It takes, collects ethyl acetate layer, after saturated common salt water washing, anhydrous sodium sulfate drying, revolving processing, using column chromatography for separation
Obtain compound 4；
(2) 2g compound 4 is dissolved with methanol, 0.2g palladium carbon is added, then be passed through hydrogen, stirring is reacted at room temperature, is reacted
After, it filtered, be evaporated methanol, column purification excessively obtains intermediate B；
S3 the intermediate A and intermediate B of amount of substance such as weighs, after being dissolved with methylene chloride, is reacted at 100 DEG C, reacts
After solvent evaporated, cross column purification obtain the compound 5；
The compound 5 is dissolved in methylene chloride by S4, and dehydrating condensation agent is added, and back flow reaction 5h steams after completion of the reaction
Dry solvent crosses column purification and obtains compound 6；Wherein the dehydrating condensation agent is N, N '-carbonyl dimidazoles；5 He of compound
The ratio between amount of substance of dehydrating condensation agent is 1:1；
Compound 6 is dissolved in methylene chloride by S5, and room temperature carries out reaction 5h after trifluoroacetic acid is added, and after completion of the reaction, is evaporated
Solvent, after ethyl alcohol is added, solvent evaporated again obtains the procymidone haptens after washed, dry, mistake column purification；Wherein,
The volume ratio of the methylene chloride and trifluoroacetic acid is 2:1.
Embodiment 4
A kind of synthetic method of procymidone haptens, includes the following steps:
S1, provides intermediate A, and the intermediate A is 1,2- dimethyl -1,2- cyclopropane acid anhydrides；
The intermediate A the preparation method comprises the following steps:
(1) 100mL toluene is added in 250mL there-necked flask, under the conditions of ice-water bath, 0.3mol sodium hydride is added, stirs 30min
After 0.1mol methyl α-chloroacrylate and 0.1mol methyl methacrylate is slowly added dropwise, reacted at room temperature after being added dropwise
10mL ethyl alcohol is added after completion of the reaction in night, after stirring 10-15min, 200mL water is added, pH value of solution is tuned into 5- with dilute hydrochloric acid
6, it is extracted with 200mL ethyl acetate, collects ethyl acetate layer, ethyl acetate layer is through saturated common salt water washing, anhydrous slufuric acid
Vacuum distillation removes toluene and ethyl acetate after sodium is dried, and residue is evaporated under reduced pressure through oil pump and purifies, and obtains 1,2- diformazan
Base -1,2- cyclopropane dicarboxylic acid's methyl esters, i.e. compound 1；
(2) 10g compound 1 is weighed, after the dissolution of 30mL ethyl alcohol, the potassium hydroxide solution that 120g mass concentration is 15% is added,
It is refluxed overnight at 80 DEG C, reaction is hydrolyzed；Vacuum distillation removes ethyl alcohol after reaction, and water is added, is extracted with ethyl acetate,
Water-yielding stratum is separated, water layer adjusts pH to 2-3 with dilute hydrochloric acid, then is extracted with ethyl acetate, through saturated common salt water washing, anhydrous slufuric acid
Compound 2 is obtained after sodium drying, revolving；
(3) dissolution of 20mL acetic anhydride is added in compound 2 described in 8g, 170 DEG C of back flow reaction 3h are distilled off after reaction
Acetic anhydride, residue is evaporated under reduced pressure through oil pump, crystallization obtains the intermediate A；
S2, provides intermediate B, the intermediate B the preparation method comprises the following steps:
(1) the chloro- 5- nitrophenol of 0.02mol 3- and the 0.04mol 6- bromocaproic acid tert-butyl ester are dissolved in 15mL DMF, are added
0.1mol Anhydrous potassium carbonate is reacted at 70 DEG C, after reaction, 200mL distilled water is added, is extracted with ethyl acetate
It takes, collects ethyl acetate layer, after saturated common salt water washing, anhydrous sodium sulfate drying, revolving processing, using column chromatography for separation
Obtain compound 4；
(2) 2g compound 4 is dissolved with methanol, 0.2g palladium carbon is added, then be passed through hydrogen, stirring is reacted at room temperature, is reacted
After, it filtered, be evaporated methanol, column purification excessively obtains intermediate B；
S3 the intermediate A and intermediate B of amount of substance such as weighs, after being dissolved with methylene chloride, is reacted at 70 DEG C, reaction knot
Solvent evaporated after beam crosses column purification and obtains the compound 5；
The compound 5 is dissolved in methylene chloride by S4, and dehydrating condensation agent is added, and back flow reaction 3h steams after completion of the reaction
Dry solvent crosses column purification and obtains compound 6；Wherein the dehydrating condensation agent is N, N '-carbonyl dimidazoles；5 He of compound
The ratio between amount of substance of dehydrating condensation agent is 1:1；
Compound 6 is dissolved in methylene chloride by S5, and room temperature carries out reaction 3h after trifluoroacetic acid is added, and after completion of the reaction, is evaporated
Solvent, after ethyl alcohol is added, solvent evaporated again obtains the procymidone haptens after washed, dry, mistake column purification；Wherein,
The volume ratio of the methylene chloride and trifluoroacetic acid is 2:1.
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 (10)
1. a kind of procymidone haptens, which is characterized in that shown in its structural formula such as formula (I):
Wherein, n is-CH2 group numbers, and n is the integer of 1-5.
2. the synthetic method of procymidone haptens as described in claim 1, which is characterized in that it includes the following steps:
S1, provides intermediate A, and the intermediate A has structural formula；
S2, provides intermediate B, and the intermediate B has structural formula；
S3 reacts the intermediate A and intermediate B, obtains compound 5, and the compound 5 has structural formula；
The compound 5 is carried out condensation reaction by S4, obtains compound 6, and the compound 6 has structural formula；
The compound 6 is hydrolyzed reaction, obtains the procymidone haptens by S5.
3. the synthetic method of procymidone haptens as claimed in claim 2, which is characterized in that the step S4 includes following step
It is rapid: the compound 5 being dissolved in methylene chloride, dehydrating condensation agent is added, back flow reaction 0.5-5h steams after completion of the reaction
Dry solvent crosses column purification and obtains the compound 6；Wherein the dehydrating condensation agent be N, N'- Dicyclohexylcarbodiimide,
EDCHCl or N, N '-carbonyl dimidazoles；The ratio between amount of substance of the compound 5 and dehydrating condensation agent is 1:1.
4. the synthetic method of procymidone haptens as claimed in claim 2, which is characterized in that in the step S1, it is described in
Mesosome A's the preparation method comprises the following steps:
It is reacted by methyl methacrylate and methyl α-chloroacrylate, obtains compound 1, the compound 1 has knot
Structure formula；
(2) reaction is hydrolyzed in the compound 1, obtains compound 2, the compound 2 has structural formula；
(3) compound 2 is subjected to condensation reaction, obtains intermediate A.
5. the synthetic method of procymidone haptens as claimed in claim 4, which is characterized in that the preparation side of the compound 1
Method are as follows: reaction dissolvent is added in the reaction vessel, under the conditions of ice-water bath, sodium hydride is added, stirring is slowly dripped after a certain period of time
Add methyl α-chloroacrylate and methyl methacrylate, reacts at room temperature overnight after being added dropwise, after completion of the reaction, separated
Purifying, obtains the compound 1；Wherein the reaction dissolvent is one of toluene, methylene chloride and DMF or a variety of；It is described
The ratio between amount of substance of sodium hydride, methyl α-chloroacrylate and methyl methacrylate is (2-5): 1:(1-1.3).
6. the synthetic method of procymidone haptens as claimed in claim 4, which is characterized in that the preparation side of the compound 2
Method are as follows: Weigh Compound 1 after being dissolved with ethyl alcohol, is added alkaline solution, is heated to reflux, reaction is hydrolyzed；Subtract after reaction
Ethyl alcohol is distilled off in pressure, and water is added, is extracted with ethyl acetate, and separates water-yielding stratum, water layer adjusts pH to 2-3 with dilute hydrochloric acid, then uses
Ethyl acetate extracts, and obtains the compound 2 after washed, dry, revolving；Wherein, the alkaline solution uses mass concentration
It is 10% or more sodium hydroxide or potassium hydroxide solution.
7. the synthetic method of procymidone haptens as claimed in claim 4, which is characterized in that condensation is anti-in the step (3)
The step of answering are as follows: acetic anhydride is added in the compound 2, acetic anhydride, residue is distilled off after reaction in back flow reaction
The intermediate A is obtained through oil pump vacuum distillation, crystallization；Wherein, the time of the back flow reaction is 2-5h；Acetic acid is distilled off
The temperature of acid anhydride is 160-180 DEG C.
8. the synthetic method of procymidone haptens as claimed in claim 2, which is characterized in that in the step S2, it is described in
Mesosome B's the preparation method comprises the following steps:
(1) the chloro- 5- nitrophenol of 3- and compound 3 are reacted, obtains compound 4, the compound 3 has structural formula, wherein the X in compound 3 is halogen, and the compound 4 has structural formula；
(2) compound 4 is subjected to nitro-reduction reaction, obtains intermediate B.
9. the synthetic method of procymidone haptens as claimed in claim 8, which is characterized in that the preparation side of the compound 4
Method are as follows: the chloro- 5- nitrophenol of 3- and the compound 3 are dissolved in DMF, Anhydrous potassium carbonate is added, is carried out at 50-110 DEG C
Reaction is added distilled water, is extracted with ethyl acetate after reaction, collects ethyl acetate layer, washed, dry, revolving
After processing, the compound 4 is obtained using column chromatography for separation；Wherein, the chloro- 5- nitrophenol of the 3-, compound 3 and nothing
The ratio between amount of substance of aqueous carbonate potassium is 1:(1-3): (1.5-10).
10. application of the procymidone haptens in the analysis of procymidone immune detection, which is characterized in that the procymidone haptens is adopted
With procymidone haptens described in claim 1.
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| CN111943882A (en) * | 2020-07-22 | 2020-11-17 | 北京勤邦生物技术有限公司 | Pythium hapten, artificial antigen and antibody as well as preparation method and application thereof |
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