CN103951704A - Preparation method of chemiluminescent compound AMPPD for immunoassay - Google Patents
Preparation method of chemiluminescent compound AMPPD for immunoassay Download PDFInfo
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- CN103951704A CN103951704A CN201310662461.0A CN201310662461A CN103951704A CN 103951704 A CN103951704 A CN 103951704A CN 201310662461 A CN201310662461 A CN 201310662461A CN 103951704 A CN103951704 A CN 103951704A
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Abstract
The invention relates to a preparation method of a chemiluminescent compound for immunoassay, and especially a preparation method of a chemiluminescent compound 4-methoxy-4-(3-phosphate acyl phenyl) spiro (1,2-dioxane-3,2'-adamantane), disodium salt AMPPD for immunoassay. According to the method, adamantane-2-aldehyde and substituted phenyl Grignard reagent are subjected to a Grignard reaction and oxidation to prepare a substituted phenyl adamantane-2-ketone intermediate, the intermediate is subjected to oxygen-alkylation to construct a carbon-carbon double bond, and phenolic hydroxyl group deprotection to obtain a key intermediate substituted phenyl methoxy methylene adamantane, and a routine photooxidation reaction is carried out to obtain the AMPPD, which can be widely used as an illuminant for immunochemiluminometry.
Description
Technical field
The present invention relates to a kind of immunoassay chemiluminescent substance AMPPD is 4-methoxyl group-4-(3-phosphoric acid acyl phenyl) spiral shell [1,2-dioxane-3,2 '-diamantane], the preparation method of disodium salt (hereinafter to be referred as AMPPD).
Background technology
AMPPD is the crucial chemical luminous substrate using on Full-automatic chemiluminescence immunoassay analysis meter device, can send the optical signal that intensity is very high, be widely used as at present chemiluminescence immune assay, there is the highest detection sensitivity, detected result stable, reproducible, medically promoting the use of.But because its price is higher, limit its large-scale development and used.The major cause that price is high is comparatively difficulty of this material preparation, synthesis technique complexity.
Chinese patent (patent No. CN200510021054) has been reported with 3-dimethyl tertiary butyl siloxy-1-(1 '-p diethylaminobenzoic acid ester group)-benzyl methyl ether and 2-diamantane ketone is that raw material is prepared AMPPD(formula one); United States Patent (USP) (the patent No. 4956477; United States Patent (USP), the patent No. 6417380; United States Patent (USP), the patent No. 6124478) be that raw material is prepared AMPPD(formula two with the prussiate of diamantane and the grignard reagent of substituted benzene).These two kinds of method yields are lower, add that intermediate preparation will carry out polystep reaction, and separation and purification is difficulty comparatively, and cost is higher, uses hypertoxic prussiate in United States Patent (USP) report method.
European patent (patent No. EP0518387) is that raw material is prepared the firm alkane of key intermediate substituted-phenyl methoxyl group methene fund by 2-diamantane ketone and 3-methyl hydroxybenzoate at metallic compound, then prepare AMPPD(formula three by conventional photoxidation), this reaction method flow process is short, yield is high, to complete reaction with the catalyst system of Li-Al hydrogen/anhydrous titanous chloride composition but prepare key intermediate crucial catalyzer used, but anhydrous titanous chloride performance is active, belong to spontaneously combustible, meet air and water burning or blast, tool strong and stimulating, to mucous membrane, the upper respiratory tract, eye and skin have strong impulse.After suction, can be because of larynx and bronchial spasm, inflammation, oedema, chemical pneumonitis or pulmonary edema and lethal.After contact, cause burning sensation, cough, pant, laryngitis, breathe hard, have a headache, feel sick, vomiting etc., have not yet to see large-scale production, also there is no the supply of commodities of mass-producing.
Summary of the invention
The preparation method who the object of this invention is to provide a kind of immunoassay chemiluminescent substance AMPPD is the new synthetic method of AMPPD.Obtain the firm alkane of key intermediate substituted-phenyl methoxyl group methene fund by new synthetic route, then obtain AMPPD by conventional photooxidation reaction.The AMPPD that the present invention makes can be widely used as the shiner of immunochemiluminometry.Can be used for the fields such as reproductive hormone, Triiodothyronine, anaemia, cardiovascular systems, suprarenal gland/hypophysis, tumor markers, infectious diseases, diabetes, anaphylactic disease, Therapeutic Drug Monitoring, bone metabolism, blood disease HIV.
The present invention is that phenyl grignard reagent taking diamantane-2-aldehyde and the protection of 3 phenolic hydroxyl groups is as starting raw material; through grignard reaction; benzyl position hydroxyl oxidize obtains substituted-phenyl adamantan-2-one intermediate; this intermediate is again through oxygen-alkylation structure carbon-to-carbon double bond; phenolic hydroxyl group deprotection two-step reaction obtains the firm alkane of key intermediate substituted-phenyl methoxyl group methene fund, then obtains AMPPD(by conventional photooxidation reaction and sees formula four):
The Grignard reagent of substituted-phenyl of the present invention; refer to the phenolic hydroxyl group of 3 existence of phenyl grignard reagent with protecting group; phenolic hydroxyl group protecting group is characterised in that under grignard reaction condition stable, includes but not limited to methoxyl methyl (MOM), t-Butyldimethylsilyl (TBS), methyl, benzyl, allyl group etc.The mol ratio of the Grignard reagent of this step reaction diamantane-2-aldehyde and substituted-phenyl is 1:1.1-5.0, is preferably 1:1. 1-3.
Oxygenant of the present invention is characterised in that benzyl position hydroxyl is had to good oxidation effectiveness, includes but not limited to activated manganese dioxide, pyridinium chlorochromate drone salt, clorox, swern oxygenant etc.The mol ratio of this step reaction substrate and oxygenant is 1:1.1-10.0, is preferably 1:1. 1-5.
Embodiment
Embodiments of the invention are as follows:
One, [(3-methoxy methoxy base) phenyl] adamantan-2-one
dpreparation
In dry round-bottomed flask, add magnesium powder (2.99 g, 124.4 mmol) and 10 mL dry THF, air in argon replaces bottle, by bromo-3-methoxyl methyl ether
b(18.0 g, 82.9 mmol) are dissolved in 30 mL dry THF, and disposable 5 mL that first add are in magnesium powder bottle, and question response dropwise adds after causing, and maintains THF slight boiling condition, dropwises in 60
oc stirs 2 h.Diamantane-2-aldehyde
a(4.08 g, 24.9 mmol) are dissolved in 20 mL dry THF, 0
ounder C, the Grignard reagent crosspointer making is proceeded in diamantane-2-aldehyde solution, room temperature reaction 5 h, saturated ammonium chloride solution cancellation reaction, reaction solution adds ethyl acetate (100 mL) and water (50 mL), separates organic layer, water layer extracts (3 X 30 mL) by ethyl acetate, merge organic layer, saturated common salt water washing, anhydrous sodium sulfate drying, concentrating under reduced pressure solvent, obtains intermediate
c(5.87 g, 78% yield).
1H NMR (CDCl
3, 400 MHz) 7.35-7.28 (m, 3H), 7.12-7.02 (m, 1H), 6.02 (s, 2H), 4.89-4.86 (d,
J = 1.6 Hz, 1H), 3.31 (s, 3H), 2.33 (br.s, 1H), 2.10-2.08 (m, 1H), 1.93-1.86 (m, 5H), 1.81-1.21 (m, 9H);
13C NMR (100 MHz, CDCl
3): δ = 160.5, 144.1, 128.4, 127.7, 126.8, 93.8, 55.8, 51.6, 39.1, 38.9, 38.2, 32.2, 31.8, 28.9, 28.2, 28.0, 27.8.
In pyridinium chlorochromate (PCC, 975.2 mg, 4.5 mmol) and sodium-acetate (98.4 mg, 1.2 mmol), add 20 mL methylene dichloride, intermediate
c(907.2 mg, 3 mmol) be dissolved in 5 mL methylene dichloride, disposablely add above-mentioned PCC solution, room temperature reaction 2 h, add 10 mL ether, stir and pour out organic layer a moment, ether washing (3 X 10 mL) for insoluble solid, merge organic layer, concentrating under reduced pressure solvent, gained resistates obtains intermediate through silica gel by chromatography purification (petrol ether/ethyl acetate=10:1)
d(856.1 mg, 95% yield).
1H NMR (CDCl
3, 400 MHz) 7.92-7.77 (m, 3H), 7.63-7.55 (m, 1H), 6.09 (s, 2H), 3.50-3.40 (m, 1H), 3.30 (s, 3H), 2.30 (m, 2H), 2.10-1.40 (m, 12 H);
13C NMR (CDCl
3, 100 MHz) 204.0, 160.8, 137.3, 132.1, 128.4, 128.0, 95.1, 55.6, 52.1, 38.8, 37.4, 32.7, 30.3, 27.9, 27.5.
Two, [(3-hydroxy phenyl) methoxyl group methene alkane] diamantane
fpreparation
Potassium tert.-butoxide (1.54 g, 13.75 mmol) is dissolved in the dry DMSO of 15 mL, air in argon replaces bottle, 50
ounder C, add intermediate
d(2.04 g, 6.79 mmol), splash into methyl-sulfate (1.3 mL after stirring 0.5 h, 13.75 mmol) TLC monitors to feedstock conversion complete, be cooled to room temperature, add ethyl acetate and water (each 50 mL) extraction, separate organic layer, water layer extracts (3 X 20 mL) by ethyl acetate, merge organic layer, saturated common salt water washing, anhydrous sodium sulfate drying, concentrating under reduced pressure solvent, gained resistates obtains intermediate through silica gel by chromatography purification (petrol ether/ethyl acetate=15:1)
e(1.35 g, 63% yield).
1H NMR (400 MHz, CDCl
3): 7.52-7.48 (m, 1H), 7.44-7.31 (m, 3H), 6.07 (s, 2H), 3.37 (s, 3H), 3.28 (s, 3H), 3.30-3.27 (m, 1H), 2.68-2.63 (m, 1H), 1.99-1.60 (m, 12H);
13C NMR (100 MHz, CDCl
3): 160.6, 147.4, 138.5, 136.3, 135.1, 129.6, 94.9, 64.7, 55.6, 46.0, 39.3, 37.3, 32.5, 31.1, 30.5, 28.7.
Intermediate
e(628.8 mg, 2 mmol) are dissolved in 20 mL methyl alcohol, add 1 mL concentrated hydrochloric acid, 2 h that reflux, TLC monitors reaction, feedstock conversion completely after stopped reaction, add ethyl acetate (40 mL) and water (30 mL) extraction, separate organic layer, water layer extracts (3 X 20 mL) by ethyl acetate, merges organic layer, saturated common salt water washing, anhydrous sodium sulfate drying, concentrating under reduced pressure solvent, gained resistates obtains intermediate through silica gel by chromatography purification (petrol ether/ethyl acetate=5:1)
f(497.5 mg, 92% yield).
1H NMR (400MHz, CDCl
3) 7.49-7.45 (m, 1H), 7.38-7.22 (m, 3H), 5.35 (br.s, 1H), 3.32 (s, 3H), 3.24 (s, 1H), 2.65 (s,1H), 1.64-1.96 (m, 12H);
13C NMR (100 MHz, CDCl
3): 158.2, 147.3, 137.7, 135.3, 133.8, 128.9, 55.9, 39.3, 37.3, 32.5, 31.1, 30.5, 28.7.
Three, 3-(2-spiral diamantane)-4-methoxyl group-4-(3-phosphorus oxygen acyl)-phenyl-1, the preparation of 2-dioxane disodium salt (AMPPD) salt
By intermediate
f(500 g, 1.58 mol) are dissolved in 5 mL anhydrous pyridines (dry with alkali alumina), and this solution is slowly added to the cooling mixture by phosphorus oxychloride (1 L, 10.7 mol) and 5 L pyridines composition, and its feed rate should maintain 5 by temperature of reaction
ounder C; after 30 minutes; termination reaction, and dichlor-phosphoryl product is poured on the mixture being made up of 20 kg ice and 1 L sodium hydroxide (10N), this mixture is moved in separating funnel; wash with 5 × 30 L methylene dichloride; in refrigerator, spend the night cooling after, from the aqueous solution, be settled out product, first use 3 × 10 L cold water washing solid products; then by this white solid drying under reduced pressure, obtain intermediate
g(360 g, 58% yield).
1H NMR (400MHz, CDCl
3) 7.2-6.7 (m, 4H), 3.19 (s, 3H), 3.04 (s, 1H), 2.50 (s, 1H), 1.67-1.83 (m,12H).
Adopt the high-pressure mercury lamp of 1000 watts at 300 mL H
2in O/P-diox (1:1, V/V), in 10
oc is by intermediate
g(20 g, 50.7 mmol) dissolve, and logical oxygen carries out photooxidation reaction, steam solvent and obtain head product after having reacted, and obtain product A MPPD (16 g, 74% yield) after recrystallization.
1H NMR (400MHz, CDCl
3) 7.26-7.00 (m, 4H), 3.07 (s, 3H), 2.80 (s, 1H), 2.08 (s, 1H), 1.70-0.91 (m, 12H).
Claims (3)
1. chemiluminescent substance 4-methoxyl group-4-(3-phosphoric acid acyl phenyl for immunoassay) spiral shell [1; 2-dioxane-3; 2 '-diamantane]; the preparation method of disodium salt AMPPD; it is characterized in that: the method is prepared substituted-phenyl adamantan-2-one intermediate with the Grignard reagent of diamantane-2-aldehyde and substituted-phenyl through grignard reaction, oxidation; this intermediate is again through oxygen-alkylation structure carbon-to-carbon double bond, and phenolic hydroxyl group deprotection two-step reaction obtains the firm alkane of key intermediate substituted-phenyl methoxyl group methene fund.
2. the Grignard reagent of substituted-phenyl as claimed in claim 1; refer to the phenolic hydroxyl group of 3 existence of phenyl grignard reagent with protecting group; comprise methoxyl methyl (MOM), t-Butyldimethylsilyl (TBS), methyl, benzyl, allyl group etc., the mol ratio of the Grignard reagent of this step reaction diamantane-2-aldehyde and substituted-phenyl is 1:1.1-3.
3. oxygenant as claimed in claim 1 is characterised in that oxygenant is Manganse Dioxide, pyridinium chlorochromate drone salt, clorox etc., and the mol ratio of this step reaction substrate and oxygenant is 1:1.1-10.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105445452A (en) * | 2015-11-17 | 2016-03-30 | 苏州浩欧博生物医药有限公司 | Anti-gp210 antibody test kit and testing method thereof |
Citations (5)
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|---|---|---|---|---|
| US5777135A (en) * | 1995-07-31 | 1998-07-07 | Lumigen, Inc. | Di-substituted 1,2-dioxetane compounds having increased water solubility and assay compositions |
| US6124478A (en) * | 1987-12-31 | 2000-09-26 | Tropix, Inc. | Methods of using 1,2-dioxetanes and kits therefore |
| CN1876663A (en) * | 2005-06-09 | 2006-12-13 | 四川琢新生物材料研究有限公司 | Chemiluminescent reagent 4-methox-4-(3-phosphorylphenyl)spiro[1,2- dioxycyclohexane-3,2'- adamantane], disodium salt synthesis method |
| WO2012006351A1 (en) * | 2010-07-08 | 2012-01-12 | Life Technologies Corporation | In situ chemiluminescent substrates and assays |
| CN102875600A (en) * | 2012-09-28 | 2013-01-16 | 深圳市宝凯仑科技有限公司 | Synthetic method of 1,2-dioxetane compound |
-
2013
- 2013-12-10 CN CN201310662461.0A patent/CN103951704A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6124478A (en) * | 1987-12-31 | 2000-09-26 | Tropix, Inc. | Methods of using 1,2-dioxetanes and kits therefore |
| US5777135A (en) * | 1995-07-31 | 1998-07-07 | Lumigen, Inc. | Di-substituted 1,2-dioxetane compounds having increased water solubility and assay compositions |
| CN1876663A (en) * | 2005-06-09 | 2006-12-13 | 四川琢新生物材料研究有限公司 | Chemiluminescent reagent 4-methox-4-(3-phosphorylphenyl)spiro[1,2- dioxycyclohexane-3,2'- adamantane], disodium salt synthesis method |
| WO2012006351A1 (en) * | 2010-07-08 | 2012-01-12 | Life Technologies Corporation | In situ chemiluminescent substrates and assays |
| CN102875600A (en) * | 2012-09-28 | 2013-01-16 | 深圳市宝凯仑科技有限公司 | Synthetic method of 1,2-dioxetane compound |
Non-Patent Citations (1)
| Title |
|---|
| EDWARDS. B. ET AL,: ""Naphthyl Dioxetane Phosphates: Synthesis of Novel Substrates for Enzymatic Chemiluminescent Assays"", 《J. ORG. CHEM》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105445452A (en) * | 2015-11-17 | 2016-03-30 | 苏州浩欧博生物医药有限公司 | Anti-gp210 antibody test kit and testing method thereof |
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Application publication date: 20140730 |