CN101193888A - Synthesis of pyrroloquinoline quinone (PQQ) - Google Patents
Synthesis of pyrroloquinoline quinone (PQQ) Download PDFInfo
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- CN101193888A CN101193888A CNA2006800137648A CN200680013764A CN101193888A CN 101193888 A CN101193888 A CN 101193888A CN A2006800137648 A CNA2006800137648 A CN A2006800137648A CN 200680013764 A CN200680013764 A CN 200680013764A CN 101193888 A CN101193888 A CN 101193888A
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Abstract
The invention relates to a novel nine step process for synthesizing PQQ (methoxatin). This process is efficient and reliably provides PQQ in excellent purity and high yield.
Description
Invention field
The present invention relates to a kind of three acid sylvite Synthetic 2s that utilize, 7, the method for 9-three carboxyls-pyrroles-quinoline-quinone (" PQQ "), described 2,7,9-three carboxyls-pyrroles-quinoline-quinone (" PQQ ") are a kind of oxydo-reductase prothetic groups, and a kind of method for the treatment of multiple disease can be provided.
The background technology of invention
PQQ is also referred to as methoxatin (perhaps 2,7,9-three carboxyls-1 hydrogen-pyrroles (2,3f) quinoline-4,5-diketone, perhaps 4,5-dioxy-4,5-dihydro-1 hydrogen-pyrroles [2,3-f] quinoline-2,7, the 9-tricarboxylic acid), obtain (article of (nature) delivering among the 280:843-844 at " nature " in 1979 referring to people such as Salsibury) from methylotrophic bacteria, separating first in 1979.Pyrroloquinoline quinone (PQQ) (1) has following structure, and can be reduced to semiquinone by reversible, perhaps is reduced to Resorcinol PQQH fully
2(2):
Pyrroloquinoline quinone (PQQ) (1) can be used as the oxydo-reductase prothetic group of a lot of bacterial enzymes, in Mammals, show the characteristic of VITAMIN, and known it can suppress the activity (article of delivering in " Helv.Chem.Acta " 76:1667 in 1993 referring to people such as Martin) of aldose reductase and reversed transcriptive enzyme (comprising HIV-1 (human immunodeficiency virus)).More particularly, in following reaction process, all include pyrroloquinoline quinone (PQQ), for example: 1) be directed to cataractous anti-oxidation protection reaction by glucocorticoid inducible, simultaneous is kept the glutathione level (article of delivering in " life science " (Life Sci.) 45:593-598 in 1989 referring to people such as Nishigori) of reduction; 2) be directed to by the protective reaction of the hepatotoxin inductive liver injury (article of (Curr.Therap.Res.) delivering among the 44:896-901 in " contemporary therapeutics research " in 1988 referring to people such as Watanabe; People's such as Urakami United States Patent (USP) NO.5061711); 3) acceleration of the releasing alcoholism reaction that causes by the expansion (augmented) of oxidation of acetaldehyde reaction (article of (Pharmacology) delivering among the 37:264-267 at " pharmacology " in 1988 referring to people such as Hobara); 4) be directed to by the anti-inflammatory response of carrageenin inductive rat paw edema (article of delivering in " J.Pharmacol.Exp.Therap. " 255:980-985 in nineteen ninety referring to people such as Hamagishi); 5) to regulation and control (referring to people's such as Aizenman United States Patent (USP) NO.5091391) by the neuronal damage of NMDA (N-methyl-D-aspartate) mediation; And 6) to the formation of osteoclast and the inhibited reaction of bone resorption (referring to people's such as Hauschka United States Patent (USP) NO.5616576).People also thought deeply pyrroloquinoline quinone (PQQ) (1) may be able to effectively be used for the treatment of disease, for example: IJD, hemolytic anemia, neurologic defect, hepatopathy, and osteoporosis (referring to people's such as Gallop United States Patent (USP) NO.5460819).
As a kind of crude substance, pyrroloquinoline quinone (PQQ) (1) can be prepared by biological production (referring to people's such as Urakami United States Patent (USP) NO.5061711; People's such as Narutomi United States Patent (USP) NO.4898870; And people's such as Ameyama United States Patent (USP) NO.4994382).Yet, similar with other crude substance, be difficulty and expensive by biological method production with separating pyrroloquinoline quinone (PQQ) (1).Selectable, pyrroloquinoline quinone (PQQ) (1) can be carried out the chemosynthesis (article of delivering in " J.Am.Chem.Soc. " 103:5599 in 1981 referring to people such as Corey by the method for describing in the diagram 1; The article that people such as Martin delivered in " Helv.Chem.Acta " 76:1667 in 1993).
Diagram 1
Corey and Tramontano utilize ten step synthesis methods to produce the pyrroloquinoline quinone (PQQ) (1) (article of delivering in " J.Am.Chem.Soc. " 103:5599 in 1981 referring to people such as Corey) of 50 milligrams of scales first.After this, Martin utilizes the path of preparing pyrroloquinoline quinone (PQQ) (1) similar to Corey, but this synthetic method is to be suitable for method large-scale, half pilot plant's scale output.Martin improves in the final step of building-up reactions, and the total quantity of synthesis step is reduced to nine steps (article of delivering among " Helv.Chem.Acta " 76:1667 in 1993 referring to people such as Martin) from ten steps.Also reported other synthetic methods (referring to people's such as Freeman patent WO.9401142) that are used to prepare pyrroloquinoline quinone (PQQ) (1) in the prior art.
When need be with the macro preparation pyrroloquinoline quinone (PQQ) (1) of many grams (multi-gram scale), the method for Martin and Corey all shows defective.The method that Corey is used for preparing pyrroloquinoline quinone (PQQ) (1) can only provide the product of (50 milligrams) on a small scale, and than the method for Martin, it also needs to carry out an extra synthesis step.Yet although the method for Martin can be used for preparing the pyrroloquinoline quinone (PQQ) (1) of multigram-scale, this method need be carried out a separating reaction tediously long, two stages in the final step of building-up reactions.Therefore, need provide a kind of method of synthetic pyrroloquinoline quinone (PQQ) (1) of extensive growth, this method can more effectively make product possess high output and outstanding purity.
Summary of the invention
Present invention relates in general to the synthetic method of a kind of improved pyrroloquinoline quinone (PQQ) (1).More specifically, the present invention relates to the method for a kind of synthetic pyrroloquinoline quinone (PQQ) (1) and its intermediate, derivative and analogue, described method can be reliably and the product with high yield and highly purified multigram-scale is provided efficiently.Described reaction generates a kind of trihydric salt 11, and it comprises the potassium metal, shown in structural formula I:
M wherein
1Represent hydrogen or potassium; M
2Represent hydrogen or potassium; And M
3Represent hydrogen or potassium; In one embodiment, M
1, M
2, and M
3Not all be hydrogen.In second embodiment, M
1, M
2, and M
3In two be not hydrogen.In another embodiment, M
1, M
2, and M
3All be potassium.
The present invention has instructed the method for a kind of synthetic pyrroloquinoline quinone (PQQ), this method is used for last step of building-up reactions, comprise: at low temperatures, (base) (for example in alkaliferous tetrahydrofuran (THF), lithium hydroxide) handles 4,5-dioxy-4,5-dihydro-1 hydrogen-pyrroles [2,3-f] quinoline-2,7,9-tricarboxylic acid 2-ethyl ester 7,9-dimethyl ester (10), on this basis,, under the pH environment of being controlled, generate the trihydric salt of the pyrroloquinoline quinone that comprises single metallic element (for example potassium) to wherein adding salt and hydrochloric acid.The trihydric salt that obtains is dissolved in the sulfuric acid, and the solution that obtains is joined in the cold water output pyrroloquinoline quinone (PQQ) (1).
Different with the method for the Martin of aforementioned report, in the method that Martin uses, pyrroloquinoline quinone (PQQ) (1) is by 4,5-dioxy-4,5-dihydro-1 hydrogen-pyrroles [2,3-f] quinoline-2,7,9-tricarboxylic acid 2-ethyl ester 7,9-dimethyl ester (10) preparation and come, and hydrolysis at room temperature, it is handled to use two kinds of salt pairs respectively, and use two-step reaction that they are separated with the form (sodium salt and cesium salt) of two kinds of different trihydric salts, this production method of the present invention has been eliminated complicated dual-step type reactions steps.The present invention uses a kind of easy, reliable method, the pyrroloquinoline quinone that will comprise single metallic element is separated with the form of trihydric salt, described pyrroloquinoline quinone has outstanding purity, and this reaction is to carry out under the temperature of strictness control and pH condition.
In one embodiment, the method of synthetic pyrroloquinoline quinone (PQQ) comprising: at low temperatures, (for example, lithium hydroxide) handles 4 in alkaliferous tetrahydrofuran (THF), 5-dioxy-4,5-dihydro-1 hydrogen-pyrroles [2,3-f] quinoline-2,7,9-tricarboxylic acid 2-ethyl ester 7,9-dimethyl ester (10) is afterwards to wherein adding salt and hydrochloric acid.One preferred embodiment in, halide salts---Repone K is used in the preparation of described trihydric salt.In another embodiment, lithium hydroxide and a kind of carbonate are used in the preparation of described trihydric salt.In a further embodiment, carbonate---salt of wormwood is used in the preparation of described trihydric salt.In another embodiment, a kind of amine salt is used in the preparation of described trihydric salt.In a further embodiment, amine salt---ammonium chloride is used in the preparation of described trihydric salt.
In the present invention, the temperature of reaction mixture remains on 17 ℃ or be lower than 17 ℃.One preferred embodiment in, the temperature of reaction mixture remains on 16 to 17 ℃.The pH value of reaction mixture is adjusted and remains on 6 or be lower than 6 by strict.One preferred embodiment in, described pH is adjusted and remains on 5.3.In one embodiment, the order of adding all ingredients in the reaction flask to is: 4, and 5-dioxy-4,5-dihydro-1 hydrogen-pyrroles [2,3-f] quinoline-2,7,9-tricarboxylic acid 2-ethyl ester 7,9-dimethyl ester, tetrahydrofuran (THF), lithium hydroxide, Repone K, and hydrochloric acid.One preferred embodiment in, described compound comprises single metallic element potassium.Be dissolved in the sulfuric acid and by the salt that will obtain and be added to the water, thereby make trihydric salt be converted into pyrroloquinoline quinone (PQQ) acid solution.
In one embodiment, the method for synthetic pyrroloquinoline quinone (PQQ) comprises the steps: step a, in the presence of acetic acid and water, uses formic acid to handle 2-methoxyl group-5-N-methyl-p-nitroaniline, generates N-(2-methoxyl group-5-nitrophenyl) methane amide; Step b in the presence of metallic palladium and dimethyl formamide, uses hydrogen treat N-(2-methoxyl group-5-nitrophenyl) methane amide, generates N-(5-amino-2-p-methoxy-phenyl) methane amide; Step c, in the presence of water and alcoholic acid, use Sodium Nitrite and fluoroboric acid to handle N-(5-amino-2-p-methoxy-phenyl) methane amide, in the presence of water, add 2-methyl-acetoacetic ester (ethyl 2-methylacetoacetate) and sodium-acetate then, generate the 2-[(3-formamido group)-4-methoxyl group aryl] hydrazono-]-ethyl propionate; Steps d uses formic acid to handle the 2-[(3-formamido group)-4-methoxyl group aryl] hydrazono-]-ethyl propionate, generate 6-formamido group-5-methoxyl group-1 hydrogen-Ethyl indole-2-carboxylate; Step e in the presence of hydrochloric acid and water, uses acetone treatment 6-formamido group-5-methoxyl group-1 hydrogen-Ethyl indole-2-carboxylate, generates 6-amino-5-methoxyl group-1 hydrogen-Ethyl indole-2-carboxylate; Step f, in the presence of methylene dichloride, use 2-oxidation propene dicarboxylic acid dimethyl ester (dimethyl 2-oxoglutaconate) to handle 6-amino-5-methoxyl group-1 hydrogen-Ethyl indole-2-carboxylate, generate 9-hydroxy-5-methyl oxygen base-6,7,8,9-tetrahydrochysene-1 hydrogen-pyrroles [2,3-f] quinoline-2,7,9-tricarboxylic acid 2-ethyl ester-7, the 9-dimethyl ester; Step g in the presence of methylene dichloride and hydrogenchloride, uses a hydration cupric acetate (II) to handle 9-hydroxy-5-methyl oxygen base-6,7,8,9-tetrahydrochysene-1 hydrogen-pyrroles [2,3-f] quinoline-2,7,9-tricarboxylic acid 2-ethyl ester-7, the 9-dimethyl ester, generate 5-methoxyl group-1 hydrogen-pyrroles [2,3-f] quinoline-2,7,9-tricarboxylic acid 2-ethyl ester-7, the 9-dimethyl ester; Step h in the presence of acetonitrile and water, uses ammonium ceric nitrate (cerric ammonium nitrate) to handle 5-methoxyl group-1 hydrogen-pyrroles [2,3-f] quinoline-2,7,9-tricarboxylic acid 2-ethyl ester-7, the 9-dimethyl ester, generate 4,5-dioxy-4,5-dihydro-1 hydrogen-pyrroles [2,3-f] quinoline-2,7,9-tricarboxylic acid 2-ethyl ester 7,9-dimethyl ester; Step I at low temperatures, in tetrahydrofuran (THF), uses lithium hydroxide to handle 4,5-dioxy-4,5-dihydro-1 hydrogen-pyrroles [2,3-f] quinoline-2,7,9-tricarboxylic acid 2-ethyl ester 7, the 9-dimethyl ester adds halide salts, adds hydrochloric acid afterwards, generates the trihydric salt that comprises single metallic element; Step j is dissolved in described trihydric salt in the sulfuric acid, and the acid solution that forms is added in the entry, generates 4,5-dioxy-4,5-dihydro-1 hydrogen-pyrroles [2,3-f] quinoline-2,7,9-tricarboxylic acid (PQQ).
One preferred embodiment in, the temperature of reaction of step I remains on 17 ℃ or be lower than 17 ℃.In a preferred embodiment, the temperature of reaction of step I remains on 16 to 17 ℃.
In one embodiment, the pH value of step I remains on less than 6.One preferred embodiment in, described pH remains on 5.3.
One preferred embodiment in, the interpolation of reagent is in proper order in step I: 4,5-dioxy-4,5-dihydro-1 hydrogen-pyrroles [2,3-f] quinoline-2,7,9-tricarboxylic acid 2-ethyl ester 7,9-dimethyl ester, tetrahydrofuran (THF), lithium hydroxide, Repone K, and hydrochloric acid.
One preferred embodiment in, the trihydric salt that generates in step I has the constitutional features of structural formula I:
Wherein, M
1Represent hydrogen or potassium; M
2Represent hydrogen or potassium; And M
3Represent hydrogen or potassium; In one case, M
1, M
2, and M
3Have at least one not to be hydrogen.In another embodiment, M
1, M
2, and M
3In have at least two not to be hydrogen.In another embodiment, M
1, M
2, and M
3All be potassium.
One preferred embodiment in, described trihydric salt makes by following step: step a, in the presence of acetic acid and water, use formic acid to handle 2-methoxyl group-5-N-methyl-p-nitroaniline, generate N-(2-methoxyl group-5-nitrophenyl) methane amide; Step b in the presence of metallic palladium and dimethyl formamide, uses hydrogen to handle N-(2-methoxyl group-5-nitrophenyl) methane amide, generates N-(5-amino-2-p-methoxy-phenyl) methane amide; Step c, in the presence of water and alcoholic acid, use Sodium Nitrite and fluoroboric acid to handle N-(5-amino-2-p-methoxy-phenyl) methane amide, in the presence of water, add ethyl 2-methyl-acetoacetic ester (ethyl 2-methylacetoacetate) and sodium-acetate then, generate the 2-[(3-formamido group)-4-methoxyl group aryl] hydrazono-]-ethyl propionate; Steps d uses formic acid to handle the 2-[(3-formamido group)-4-methoxyl group aryl] hydrazono-]-ethyl propionate, generate 6-formamido group-5-methoxyl group-1 hydrogen-Ethyl indole-2-carboxylate; Step e in the presence of hydrochloric acid and water, uses acetone treatment 6-formamido group-5-methoxyl group-1 hydrogen-Ethyl indole-2-carboxylate, generates 6-amino-5-methoxyl group-1 hydrogen-Ethyl indole-2-carboxylate; Step f, in the presence of methylene dichloride, use 2-oxidation propene dicarboxylic acid dimethyl ester (dimethyl 2-oxoglutaconate) to handle 6-amino-5-methoxyl group-1 hydrogen-Ethyl indole-2-carboxylate, generate 9-hydroxy-5-methyl oxygen base-6,7,8,9-tetrahydrochysene-1 hydrogen-pyrroles [2,3-f] quinoline-2,7,9-tricarboxylic acid 2-ethyl ester-7, the 9-dimethyl ester; Step g in the presence of methylene dichloride and hydrogenchloride, uses a hydration cupric acetate (II) to handle 9-hydroxy-5-methyl oxygen base-6,7,8,9-tetrahydrochysene-1 hydrogen-pyrroles [2,3-f] quinoline-2,7,9-tricarboxylic acid 2-ethyl ester-7, the 9-dimethyl ester, generate 5-methoxyl group-1 hydrogen-pyrroles [2,3-f] quinoline-2,7,9-tricarboxylic acid 2-ethyl ester-7, the 9-dimethyl ester; Step h in the presence of acetonitrile and water, uses ammonium ceric nitrate (cerric ammonium nitrate) to handle 5-methoxyl group-1 hydrogen-pyrroles [2,3-f] quinoline-2,7,9-tricarboxylic acid 2-ethyl ester-7, the 9-dimethyl ester, generate 4,5-dioxy-4,5-dihydro-1 hydrogen-pyrroles [2,3-f] quinoline-2,7,9-tricarboxylic acid 2-ethyl ester 7,9-dimethyl ester; Step I at low temperatures, in tetrahydrofuran (THF), uses lithium hydroxide to handle 4,5-dioxy-4, and 5-dihydro-1 hydrogen-pyrroles [2,3-f] quinoline-2,7,9-tricarboxylic acid 2-ethyl ester 7, the 9-dimethyl ester adds excessive salt, adds hydrochloric acid afterwards.In a preferred embodiment, the salt that adds in the step I is Repone K.
Above content is the general description of carrying out at key character of the present invention, and purpose is in order to make it to be understood easily by detailed description, and the contribution that the present invention is done prior art can better be accepted.By ensuing detailed description and accompanying drawing associated therewith, the other objects and features of the invention are also become apparent.Yet the purpose of design that is appreciated that accompanying drawing only is to be used for setting forth, and can not be construed as limiting the invention, and it plays the effect of a reference for appended claim.
Brief description of drawings
Accompanying drawing 1 is a synoptic diagram, has roughly described the entire synthesis process of production pyrroloquinoline quinone (PQQ) (1);
Accompanying drawing 2 is synoptic diagram, has roughly described the building-up process of the present invention in order to last step in production pyrroloquinoline quinone (PQQ) (1) process;
Accompanying drawing 3A is the mass spectrum of nuclear magnetic resonance of pyrroloquinoline quinone (PQQ) (1);
Accompanying drawing 3B is the carbon-13 nmr spectra of pyrroloquinoline quinone (PQQ) (1), and
Accompanying drawing 3C is the high performance liquid chromatography (HPLC) in the pyrroloquinoline quinone of 255 nanometers (PQQ) (1) (98.9%).
Detailed Description Of The Invention
PQQ (PQQ) (1) be the people such as Salisbury in 1979 " nature " (nature) in the article delivered of 280:843-844 by reported first. The synthetic method of PQQ of the present invention (PQQ) (1) comprises one nine step linear synthetic method (accompanying drawing 1). Use this synthetic method can obtain for example 17 gram PQQs (PQQ) (1), account for 14% of total output.
Described in accompanying drawing 2, in last step of the method that the present invention uses, make at low temperatures 4,5-dioxy-4,5-dihydro-1 hydrogen-pyrroles [2,3-f] quinoline-2,7,9-tricarboxylic acids 2-ethyl ester 7,9-dimethyl ester (10) and lithium hydroxide (LiOH) (THF-H in oxolane-aqueous solution2O) react, (step a) to generate three thick acid lithium salts in reactant liquor. Be different from the synthetic method that Martin uses, the adding of halide salts---potassium chloride (KCL) was carried out before regulating the pH value, re-uses after this hydrochloric acid (HCL) the pH value of reaction mixture is adjusted to 5.3 (step b). Under the condition of strict control pH value (5.3), the three acid sylvite 11 that reaction is obtained are precipitated out from solution and are collected by the vacuum filtration method, and make water and solvent wash (step c) to it. By being dissolved in concentrated vitriol (H with 112SO
4) in method make three acid sylvite change into PQQ (PQQ), and gained acid solution is fallen on ice, to obtain end product PQQ (PQQ) (1) (steps d and step e).
Identify product and checked for impurities with following these technology: thin-layer chromatography (TLC), NMR spectrum (NMR), high performance liquid chromatography (HPLC), and elementary analysis. Use sulfuric acid to carry out extra process and can be further purified PQQ (PQQ) and remove impurity, filter to isolate PQQ (PQQ), then under vacuum, carry out drying.
The all processes of synthetic PQQ (PQQ) (1) starts from two-step method and prepares 2-oxidation glutaconate dimethyl ester (12). The reagent of described diketone 12 synthetic PQQ (PQQ) (1) of conduct in step g (the 7th step) subsequently is as shown in accompanying drawing 1. A preferred method of preparation diketone 12 is the method used according to Corey (articles of delivering in " J.Am.Chem.Soc. " 103:5599 in 1981 referring to people such as Corey). With 2-oxidation glutaconate dimethyl ester and carrene (CH2CL
2) mix, and add hot reflux, after this to wherein adding bromine solutions and carrene, and under stirring, refluxed 3.5 hours. The cooling reaction mixture, and use rotary evaporator in a vacuum it to be concentrated. Residue is dissolved in diethyl etherate (Et2O), and to wherein slowly adding triethylamine (Et3N), to maintain the temperature at below 35 ℃. After stirring 45 minutes, the accessory substance tribromo ethamine that obtains is removed by filter, and in filtrate, passed into nitrogen 2 hours. Filtrate is carried out vacuum filtration by silicon dioxide colloid, use afterwards diethyl etherate (Et2O) washing silicon dioxide colloid. Use rotary evaporator that the ethereal solution that mixes is carried out Vacuum Concentration, to obtain 2-oxidation glutaconate dimethyl ester (12).
As shown in Figure 1, linear PQQ (PQQ) synthetic method starts to the acetic anhydride (Ac of cooling2O) add commercially available 2-methoxyl group-5-nitroaniline (3) (Aldrich, Milwaukee, WI) in the solution, and to wherein adding formic acid, to form a kind of thick slurry, its stirring is spent the night. In aforementioned reaction mixture, add water, and stirred again two days. Collect the pale product of color by the vacuum filtration method, water washs, until the pH value of water lotion is neutral. (house vacuum) carries out after the drying under the vacuum condition of mansion, and the first step of synthetic reaction has just been finished, and generates N-(2-methoxyl group-5-nitrobenzophenone) formamide (4).
In step b, to ParrTMPack in the pressure reactor N-(2-methoxyl group-5-nitrobenzophenone) formamide (4), dimethyl formamide (DMF) and the palladium that is present in 5% on the active carbon. Use nitrogen that reactor is pressurizeed, under the temperature levels that raises, hydrogenization occurs. Because this reaction is heat release, therefore need to cool off. Reaction mixture is passed through CeliteTMDevice carries out vacuum filtration, removes catalyst, and under vacuum concentrated filtrate. Residue after filtering is dissolved in the methyl alcohol (MeOH), and stirs and spend the night. The slurries that obtain cool off in ice bath and stir, and collect product by filtering, and use ether (diethyl etherate Et2O) washed product obtains N-(5-amino-2-methoxyphenyl) formamide (5).
Next in step c, pack in the reactor concentrating hydrochloric acid (HCL) and water, and be cooled to-26 ℃. In sour mixed liquor, add N-(5-amino-2-methoxyphenyl) formamide (5), afterwards again to wherein adding ethanol. Next, add liquid natrium nitrosum (NaNO2), the temperature with reaction mixture remains on-20 ℃ to-25 ℃ after this. Add the ethanol be cooled to 0 ℃, and continuous stirring 20 minutes. Add fluoboric acid (HBF4), in the process that adds, keep low temperature. Again add ethanol, and continuous stirring 30 minutes. Rise again reaction mixture (warm) 30 minutes. Collect the diazonium tetrafluoroborate by filter method, and use cold ethanol that it is washed. The ethanol slurries that will contain salt stir at low temperatures, under the state that keeps low temperature, to wherein adding 2-methyl-acetoacetic ester (CH3C(O)CH(CH
3)CO
2Et) (Aldrich, Milwaukee, WI), sodium acetate (NaOAc) and water (steps d). Remove cooling bath, and continuous stirring is spent the night. In mixed liquor, pass into nitrogen atmosphere overnight, use the vacuum filtration method to collect product, and use ethanol/water mixed liquor washed product. In this reaction, initial substitution product carries out spontaneous acetyl and the double bond migration of taking off, and this process is called as the reaction of Japp-Klingemann Reaction (the graceful reaction of Yao Pu-Florian Kringe) exactly. The solid by filtration device that reaction obtains carries out vacuum drying and spends the night, and uses extra ethanol and isopropyl alcohol to wash, and obtains the 2-[(3-formamido group)-4-methoxyl group aryl] hydrazono-]-ethyl propionate (6).
After step e, with the 2-[(3-formamido group)-4-methoxyl group aryl] hydrazono-]-ethyl propionate (6) and formic acid place a reaction vessel, and spend the night 80 ℃ of lower stirrings. Cool off described reaction mixture, and to wherein adding ethanol. The bright green slurries that obtain are cooled to 0 ℃ and stirring. Collect product by the vacuum filtration method, use the ethanol washed product, place filter dry, and 80 ℃ of lower vacuum drying, by the Fishcher indole synthesis to obtain desired indoles---6-formamido group-5-methoxyl group-1 hydrogen-indole-2-carboxylic ethyl ester (7).
In the solution that contains acetone, concentrating hydrochloric acid and water, add 6-formamido group-5-methoxyl group-1 hydrogen-indole-2-carboxylic ethyl ester (7). The reaction mixture that obtains refluxed 4 hours, was cooled to afterwards 0 ℃ (step f). Collect the solid obtain by the vacuum filtration method, and be placed in the filter vacuum drying and spend the night. Dried substance dissolves in 1.5N liquified hydrogen sodium oxide molybdena (NaOH), and was stirred 45 minutes. Product is collected in vacuum filtration, and washed product, obtains 6-amino-5-methoxyl group-1 hydrogen-indole-2-carboxylic ethyl ester (8).
To 6-amino-5-methoxyl group-1 hydrogen-indole-2-carboxylic ethyl ester (8) and carrene (CH is housed2CL
2) reaction vessel in add the dichloromethane solution (CH of 2-oxidation glutaconate dimethyl ester (12)2CL
2) (step g). After adding, the reaction mixture that obtains is delivered in second reaction vessel, in cold water, stir, and vacuum filtration. Collect product, with carrene-heptane wash product, and carry out vacuum drying, obtain 9-hydroxy-5-methyl oxygen base-6,7,8,9-tetrahydrochysene-1 hydrogen-pyrroles [2,3-f] quinoline-2,7,9-tricarboxylic acids 2-ethyl ester-7, the 9-dimethyl ester, and with itself and a hydration cupric acetate (Cu (OAc2)·H
2O) and carrene mix (step h). Pass into air stream in the reaction mixture that obtains and stir, and under the state that keeps room temperature to wherein passing into anhydrous hydrogen chloride gas. Venting process was carried out 6 hours continuously, only passed into continuously air after this and spent the night. In reaction mixture, add liquid sodium carbonate (Na2CO
3) solution is with cessation reaction. Stirring reaction mixed liquor 2 hours, and vacuum filtration. The filtrate of using the further dilute filtration of carrene to obtain. Mix liquid, can be dissolved in the solution fully to guarantee product. Isolate dichloromethane layer, and use carrene that the liquid phase layer is extracted. Mix organic layer, wash with water, use dried over sodium sulfate, and filter and concentrate, obtain a kind of solid, use diethyl etherate (Et2O) described solid is stirred spend the night, in ice bath, cool off, filter, use diethyl etherate (Et2O) washing, and in high vacuum environment, under 50 ℃, carry out drying, obtain 5-methoxyl group-1 hydrogen-pyrroles [2,3-f] quinoline-2,7,9-tricarboxylic acids 2-ethyl ester-7,9-dimethyl ester (9).
In step I, with 5-methoxyl group-1 hydrogen-pyrroles [2,3-f] quinoline-2,7,9-tricarboxylic acids 2-ethyl ester-7,9-dimethyl ester (9) and acetonitrile together place reaction vessel, and cooling is also stirred. In the suspension that obtains, add liquid ammonium ceric nitrate solution ((NH4)
2Ce(NO
3)
6Perhaps CAN), obtains bright orange solution, then this solution is poured in the frozen water. Collect cesium salt under vacuum condition, filtrate uses carrene to extract. Use magnesium sulfate that organic extract is carried out drying and concentrated. The residue that obtains is dissolved in toluene-ethyl acetate (EtOAc) solution, filter and collect the crystal that obtains, use ethyl acetate-heptane wash crystal, and it is dissolved in the carrene, use the silicon dioxide colloid stirring reaction 1 hour, and then passed through CeliteTMFilter. Remove solvent, obtain 4,5-dioxy-4,5-dihydro-1 hydrogen-pyrroles [2,3-f] quinoline-2,7,9-tricarboxylic acids 2-ethyl ester 7,9-dimethyl ester (10).
Shown in step j last in the accompanying drawing 1 and step k, also as the more detailed description in the accompanying drawing 2, in reaction vessel, mix 4,5-dioxy-4,5-dihydro-1 hydrogen-pyrroles [2,3-f] quinoline-2,7,9-tricarboxylic acids 2-ethyl ester 7,9-dimethyl ester (10) and oxolane (THF), and to the liquid solution that wherein adds alkali, lithium hydroxide for example, NaOH, potassium hydroxide or cesium hydroxide, and keep the temperature of reaction mixture to be lower than 10 ℃. One preferred embodiment in, use lithium hydroxide, to generate three acid lithium salts (for example, corresponding lithium atom of each acid group). (step a) in 30.5 hours at 16 ℃ of-17 ℃ of lower stirring reaction mixed liquors. Carry out small-scale hydrolysis, determine whether the change of temperature can affect the generation quantity of impurity. We find that when reaction temperature raise, the percentage composition of impurity also increased thereupon. Best reaction temperature is confirmed as 16 ℃-17 ℃.
In step b, after stirring, add excessive salt in a large number in the reaction mixture, potassium chloride for example, ammonium chloride, then ammonium carbonate or potash cool off mixed liquor in ice bath. One preferred embodiment in, the salt of adding is potassium chloride. For example use that inorganic acid or the formic acid of hydrochloric acid, sulfuric acid and so on carry out acidifying to reaction mixture, be approximately 6 until the pH value of reaction mixture adjusted to. One preferred embodiment in, use the salt acid for adjusting pH value. After this, the hydrochloric acid that uses 2N adjusts to 5.3 with the pH value is more accurate. Strict control to the pH value can make tracid salt be separated from each batch reaction with consistent output and outstanding purity. One preferred embodiment in, described tracid salt comprises single counter ion counterionsl gegenions (counterion) (potassium (K for example+)). In another embodiment, described tracid salt comprises a kind of in the following metal: potassium, caesium, ammonium, perhaps sodium. One preferred embodiment in, described metal makes potassium, and then produces tracid salt 11 (as follows). Make in this way and produce sylvite in the situation of strict control pH value, most of organic impurities remains in the solution, and therefore isolated tracid salt has minimum impurity. Do not estimate the pH value in the prior art research for the impact of the quantity of the purity of isolated tracid salt or reaction impurities. Referring to the research such as people such as Martin.
M wherein1Represent hydrogen or potassium; M2Represent hydrogen or potassium; And M3Represent hydrogen or potassium; And M1、M
2, and M3All not hydrogen.
Add after the acid, the cooling reaction mixture is collected the solid that reaction obtains by the vacuum filtration method afterwards, uses frozen water and acetonitrile to wash, and dry (step c). The three acid sylvite 11 that obtain are dissolved in concentrated vitriol (H2SO
4) in, and stir 2.5 hours (steps d). The acid solution that obtains is poured in the ice, and the black solid suspension that obtains is stirred. Filter and collect the solid product that obtains, with the frozen water washing, vacuum drying under nitrogen obtains a few gram PQQs (PQQ) (1), and described PQQ (PQQ) (1) has the amount of impurities that can only detect.
The PQQ (PQQ) that obtains is carried out large batch of being further purified, with a few gram materials (for example, 1-100 gram, for example 50-100 gram, 75-100 gram for example) be dissolved in the concentrated vitriol, and at room temperature or be lower than under the room temperature and stir. Sulfuric acid has the ability of unique dissolving PQQ (PQQ), is the solvent that is fit to that can be used for purification reaction. Acid solution is dropwise joined in 5 premium on currency, keep temperature less than 33 ℃. By at room temperature stirring, desired product PQQ (PQQ) is precipitated out from solution, filter and collect product, wash with water, and vacuum drying.
All publications and patent documentation that the present invention quotes are hereby incorporated by, and the particular content of each piece publication and document all is introduced among the present invention respectively. Do not think that quoting of publication and patent documentation is the license of relevant prior art, do not consist of any license about its content and date yet. The present invention is that the mode that is described in detail is described, those skilled in the art can find, the present invention can implement by various embodiment, the aforesaid specific embodiment and following embodiment are the purposes that elaborates in order to reach, and do not consist of the restriction to subsequently claim.
Embodiment
Embodiment 1: pyrroloquinoline quinone (PQQ) (1) synthetic
A.2-oxidation propene dicarboxylic acid dimethyl ester (12)
2-propene dicarboxylic acid dimethyl ester (87.0 gram, 0.50 rubs) and 328 milliliters of methylene dichloride joined in one 1 liter the three-necked bottle, described three-necked bottle has also loaded a mechanical stirrer, an extra funnel and a heating mantles.Stir the solution in the three-necked bottle, and it is delivered to backflow.After 45 minutes, to wherein adding the bromine solutions (77.0 grams, 0.48 rubs) that is dissolved in 165 milliliters of methylene dichloride.Reaction mixture is stirred, and refluxed 3.5 hours.
Under vacuum condition, reaction mixture is concentrated, obtain a kind of orange oil.To wherein adding ether (328 milliliters), be removed in a vacuum then.Add ether (328 milliliters) once more, stir gained solution, and, in solution, slowly add triethylamine (65.6 milliliters, 0.44 rubs) temperature being controlled at when being lower than 35 ℃.The underflow that obtains is stirred 45 minutes, removes by filter the triethylamine Hydrogen bromide afterwards, and uses ether that it is washed.In filtrate, fed nitrogen 2 hours.Obtain about 700 milliliters of bolarious opaque solution.Above-mentioned solution is filtered by 70 gram silicon dioxide colloids.Use the silicon dioxide colloid behind 3.5 liters the ether washing and filtering.Under vacuum condition the blended ethereal solution is concentrated, obtain a kind of glassy yellow solid, described solid at room temperature carries out drying, obtains 71.5 gram (83.1%) 2-oxidation propene dicarboxylic acid dimethyl ester compounds.
B.N-(2-methoxyl group-5-nitrophenyl) methane amide (4)
To one 5 liters, loaded a mechanical stirrer and a temperature sensor and three-necked bottle that place ice bath adds acetic anhydride (367 grams, 9.72 rub).Then to wherein adding formic acid (367 gram, 6.01 rub), and stir while adding cooling.The speed that adds will guarantee that temperature is no more than 32 ℃.Continuously stirring is 1 hour at ambient temperature.Cooling once more, and gradation adds 2-methoxyl group-5-N-methyl-p-nitroaniline (3) (415 grams, 2.47 rub) in 1.5 hour time.In the process that adds, keep temperature to be lower than 32 ℃.Thick xanchromatic slurries are stirred spend the night.
Add entry (3 liters), and once more mixed solution was stirred 48 hours.Filter and collect the xanchromatic product, wash product with water, be neutral until the pH of liquid water washing lotion value.The total amount of the water that uses is 9.2 liters.Under mansion vacuum (house vacuum) condition,, obtain 480 gram (99.1%) N-(2-methoxyl group-5-nitrophenyl) benzamide compounds in 60 ℃ of desciccates.
C.N-(5-amino-2-p-methoxy-phenyl) methane amide (5)
In this reaction, use one 2 liters Parr pressure reactor.Described pressure reactor has loaded a mechanical stirrer, a temperature sensor, a spiral coil cooling tube and a heating mantles.In reactor, add N-(2-methoxyl group-5-nitrophenyl) methane amide (4) (170.0 gram, 0.867 rubs), 1100 milliliters of dimethyl formamides (DMF) and 7.75 grams and be loaded into the palladium of 5% on the gac.Use nitrogen that reactor is forced into 60psig, blow away the air in the reactor, and air is discharged.Then, use hydrogen that reactor is forced into 60psig, and divide three discharges.
Hydrogenation carries out under 70 ℃ and 60psig hydrogen.Need the frequent hydrogen that charges into, when pressure is reduced to 30psig, begin to charge into.This reaction is the height heat release, therefore current is cooled off through spiral coil cooling tube, till reaction is nearly finished.After three hours, the absorption of hydrogen is obviously slack-off, and reaction finishes.
Reaction mixture is passed through Celite
TM521 filter, and remove the palladium catalyst on the gac of being loaded into wherein.Remove in a vacuum and desolvate.With the residue that obtains and with above-mentioned reacting phase with the not purified products that generate of other two reactions mix, wherein each reaction all is to carry out with the scales of 170 grams (sums of 3 reactions), uses 300 ml methanol that its stirring is spent the night.In ice bath, the dark-brown slurries are cooled off, and stir 3 hours once more.Filter and collect product, and use the ether washed product.The filtrate that washing for the first time obtains is green.Continue to use ether to wash, till the filtrate color shoals.The output of the N-that obtains (5-amino-2-p-methoxy-phenyl) methane amide is 387.9 grams (89.7%).
D.2-[(3-formamido group)-and 4-methoxyl group aryl] hydrazono-]-ethyl propionate (6)
To one 12 liters, loaded a mechanical stirrer, a temperature sensor and placed the reactor of the dry ice bath to add 560 milliliters of concentrated hydrochloric acids and 105 ml waters.Stir gained solution, and it is cooled to-26 ℃.And gradation adds N-(5-amino-2-p-methoxy-phenyl) methane amide (5) (368.0 grams, 2.21 rub) in 15 fens clock times.Add ethanol (81 milliliters).After 25 minutes, add 257 milliliters of Sodium Nitrites (171.3 grams, 2.48 rub) aqueous solution.In the process that adds, the temperature that keeps reaction mixture is at-20 ℃ to-25 ℃.Continuously stirring was also cooled off 15 minutes, added 1165 milliliters of freezing ethanol afterwards.Remain on-5 ℃ in temperature under-10 ℃ condition, continue to stir 20 minutes.
After 10 minutes, and the adding fluoroboric acid (370 milliliters, aq.50%, 2.96 rub).In the process that adds, keep temperature to be lower than-3 ℃.Add ethanol (739 milliliters), and under-5 ℃ of conditions, continue to stir 30 minutes, afterwards, reaction mixture was risen again 5 ℃ at 30 minutes within the clock time.After mixed solution warmed, the slurries of the bright orange wingceltis look color that becomes was darker.Filter and collect the diazonium a tetrafluoro borate, and use cold ethanol to wash, till the elutriant color shoals.
The diazonium a tetrafluoro borate is sent back in the reactor again, and use 2030 milliliters cold ethanol that it is stirred.Reaction mixture is cooled to-8 ℃.After 12 minutes, to the solution that wherein adds 2-methyl-acetoacetic ester (314.5 grams, 2.18 rub), sodium-acetate (602.4 grams, 7.34 rub), and 1770 milliliters water.In the process that adds, make temperature remain below-6 ℃.Remove cooling bath, and continuously stirring 22 hours.
In reaction mixture, feed nitrogen atmosphere overnight.Filter and collect product, and use ethanol/water solution and 5.5 liters of frozen water washed product of 740 milliliter 10%.Use strainer that wet cake is carried out dried overnight, use 800 milliliters of cold ethanol that it is washed afterwards.Under 50 ℃, the solid that obtains is being carried out drying under the vacuum state of mansion, is obtaining 407.5 gram (65.9%) 2-[(3-formamido groups)-4-methoxyl group aryl] hydrazono-]-the ethyl propionate compound.
Further mother liquor is concentrated,, obtain more solid material, and collect these solid materials by filter method by in mother liquor, feeding nitrogen 2 days.Use ethanol/water solution, 2 liters of cold water and 500 milliliters of Virahols of 500 milliliter 10% to wash these extra solid materials that obtain, obtain 13.3 extra gram (3.6%) 2-[(3-formamido groups)-4-methoxyl group aryl] hydrazono-]-the ethyl propionate compound.The ultimate production of above-claimed cpd is 420.8 grams (69.5%).
E.6-formamido group-5-methoxyl group-1 hydrogen-Ethyl indole-2-carboxylate (7)
The 2-[(3-formamido group of in one 3 liters three-necked bottle, packing into)-and 4-methoxyl group aryl] hydrazono-]-ethyl propionate (6) (425.0 grams, 1.53 rub) and 1530 milliliters of formic acid, described three-necked bottle has also loaded a mechanical stirrer, a temperature sensor, a condenser and a heating mantles.Stirring described reaction mixture down at 80 ℃ spends the night.
Make the reaction mixture cooling, and to wherein adding 770 milliliters of ethanol.The bright green slurries that obtain are cooled to 0 ℃, stirred 2 hours.Filter and collect product, and use 700 milliliters of washing with alcohol products.In strainer, the brown-green product that obtains is carried out drying, and dry under 80 ℃ under the vacuum condition of mansion, obtain 300.1 gram (75.2%) 6-formamido group-5-methoxyl group-1 hydrogen-Ethyl indole-2-carboxylate's compounds.
F.6-amino-5-methoxyl group-1 hydrogen-Ethyl indole-2-carboxylate (8)
In three-necked bottle one 12 liters, that loaded a mechanical stirrer, a temperature sensor, a heating mantles and a condenser, pack into 7.70 liters of acetone and be diluted with water to 365 milliliters of concentrated sulfuric acid solutions of 765 milliliters.At room temperature stir described solution, and to wherein adding 6-formamido group-5-methoxyl group-1 hydrogen-Ethyl indole-2-carboxylate (7), to obtain a kind of wingceltis green (tan-green) slurries.Reaction mixture was refluxed 4 hours, in ice bath, it is cooled to 0 ℃ afterwards.Filter and collect the wingceltis look product that obtains, dried overnight in strainer.Use the liquid hydrogen sodium oxide of 1.70 liters of 1.5N to decompose desciccate, and stirred 45 minutes.Filter and collect product, and use 2 liters frozen water that it is washed 6 times.Washing reaction carried out 5.5 hours, and last elutriant has neutral pH value, obtained 214.0 gram (79.8%) 6-amino-5-methoxyl group-1 hydrogen-Ethyl indole-2-carboxylate's compounds.
G.5-methoxyl group-1 hydrogen-pyrroles [2,3-f] quinoline-2,7,9-tricarboxylic acid 2-ethyl ester-7,9-dimethyl ester (9)
In three-necked bottle one 3 liters, that loaded a mechanical stirrer, an extra funnel, a temperature sensor, a heating mantles and a condenser that under static pressure, is connected, add 6-amino-5-methoxyl group-1 hydrogen-Ethyl indole-2-carboxylate (8) and 1325 milliliters of methylene dichloride with nitrogen tube.Under nitrogen environment, the green suspension that obtains is stirred, after 10 minutes, to 700 milliliters of dichloromethane solutions that wherein add 2-oxidation propene dicarboxylic acid dimethyl ester (12) (175.0 grams, 1.02 rub).Reusing 100 milliliters of methylene dichloride all washes 2-oxidation propene dicarboxylic acid dimethyl ester (12) to reactor.Last in adition process, reaction mixture almost becomes black.Temperature no longer raises, and uses conventional method that reaction mixture is stirred and spends the night.
Reaction mixture delivered in single neck bottle of one 3 liters, and under vacuum condition, it is concentrated into about 1/4th of original volume.Afterwards, reaction mixture is stirred, and in cold water, cooled off 1 hour.Last temperature reaches 7 ℃.Filter and collect product, and use 350 milliliters of 1: 5 methylene dichloride-n-heptane solution washed product.In strainer, the amber product that obtains is carried out the part drying, under high vacuum state, under 50 ℃, carry out drying afterwards, obtain 270.8 gram (73.1%) 9-hydroxy-5-methyl oxygen bases-6,7,8,9-tetrahydrochysene-1 hydrogen-pyrroles [2,3-f] quinoline-2,7,9-tricarboxylic acid 2-ethyl ester-7,9-dimethyl ester.
The 9-hydroxy-5-methyl oxygen base-6 of in jacketed reactor one 22 liters, that loaded a mechanical stirrer, a bottom titration valve (bottom-drop valve), extra funnel, a temperature sensor, a heating mantles, a dip tube and a condenser, packing into, 7,8,9-tetrahydrochysene-1 hydrogen-pyrroles [2,3-f] quinoline-2,7,9-tricarboxylic acid 2-ethyl ester-7,9-dimethyl ester (270.8 grams, 0.666 rub), the methylene dichloride of a hydration cupric acetate (146.3 gram, 0.733 rubs) and 11.50 liters.The stirring reaction mixed solution, and to stream of bubbling air wherein and anhydrous hydrogen chloride gas.Because this reaction is thermopositive reaction, thereby the temperature of reaction chuck is set to 10 ℃, is about 20 ℃ to keep temperature of reaction.After about 30 minutes, the reaction mixture heat release reduces, and this moment, jacket temperature was set to 20 ℃.Continue to be blown into air and anhydrous hydrogen chloride 6 hours, continue in reaction mixture, to be blown into air and spend the night.
Add in 30 fens clock times that 550 gram sodium bicarbonates are dissolved in 6.0 premium on currency and the solution that forms makes reaction terminating.Find that reaction transforms out very a spot of carbonic acid gas.Obtain very dark dark blue solution of a kind of color and blue-greenish colour solid.The boundary of organic phase and liquid phase is not obvious.Mixed solution is stirred filtration afterwards in 2 hours.Filtering reaction slowly carried out 7 hours.Because the evaporation of methylene dichloride, make black solid stay in the strainer and the exit of strainer.Reaction is fast finish in, with the slurries shape, all be that liquid mixed solution is poured in the clean strainer and filtered in essence.Two strainers have all been filled methylene dichloride, rely on action of gravity to discharge, and reaction is spent the night.Use the further washing and filtering device of methylene dichloride, and the filtrate that obtains is mixed, return in the reaction vessel again.Add several methylene dichloride that rise, make liquid volume reach roughly the same with original volume.
The two-phase mixed solution was stirred 5 hours, guarantee that product is dissolved among the solution fully.The separate dichloromethane layer.The blue-greenish colour solid adrift on the top of dichloromethane layer.The upper strata of dichloromethane layer is collected separately, remove solid by filter method.Use 2 liters of dichloromethane extraction liquid phase layers.Use the washing of 4 premium on currency to mix the dichloromethane solution that obtains, and use 750 gram sodium sulfate to stir and drying.The solution that filtration obtains peels off out the black semisolid with citrine light.Using 1 liter ether that the raw material that obtains is stirred spends the night.Slurries are cooled off in ice, and filter and collect product.Afterwards, use 700 milliliters of ether washed product, under 50 ℃, carrying out drying under the vacuum condition of mansion, obtain brassiness product 5-methoxyl group-1 hydrogen-pyrroles [2,3-f] quinoline-2,7 of 221.7 grams (86.1%), 9-tricarboxylic acid 2-ethyl ester-7,9-dimethyl ester.
I.4,5-dioxy-4,5-dihydro-1 hydrogen-pyrroles [2,3-f] quinoline-2,7,9-tricarboxylic acid 2-ethyl ester 7,9-dimethyl ester (10)
One 3 liters, loaded a mechanical stirrer, a temperature sensor, an extra funnel and placed the three-necked bottle of dry ice/acetone batch, add 5-methoxyl group-1 hydrogen-pyrroles [2,3-f] quinoline-2,7,9-tricarboxylic acid 2-ethyl ester-7,9-dimethyl ester (9) (12.09 grams, 0.0313 rubs) and 600 milliliters of acetonitriles.The suspension that stirring obtains, and it is cooled to-5 ℃.Within 5 minutes, ammonium ceric nitrate (84.87 grams, the 0.155 rubs) solution that is dissolved in 120 ml waters is joined in the suspension.Obtain a kind of bright orange solution.Continuously stirring 1.5 hours, and-5 ℃ of lasting down coolings.
Pour into reaction mixture in 1380 milliliters of cold water and carry out strong stirring, stir and continue 0.5 hour.Remove by filter cesium salt, the methylene dichloride that uses 300 milliliters is to filtrate extraction 3 times.Use 75 gram sal epsom that dichloromethane solution is carried out drying, go down to desolventize, obtain 10.15 gram solids in vacuum.The solid that obtains is stirred in 10 milliliters of toluene and 10 milliliters of ethyl acetate solutions, filter and collect the bright orange red catalyzer that obtains, and the ethyl acetate-n-heptane solution that used 3: 1 washs it.In 360 milliliters methylene dichloride, and use 5 gram silicon dioxide colloids to stir 1 hour catalyst dissolution.Solution is passed through Celite
TM521 filtration units filter, and peel off out the orange red solid 4 of (stripped) 7.50 grams (62.0%), 5-dioxy-4,5-dihydro-1 hydrogen-pyrroles [2,3-f] quinoline-2,7,9-tricarboxylic acid 2-ethyl ester 7,9-dimethyl ester.
J.4,5-dioxy-4,5-dihydro-1 hydrogen-pyrroles [2,3-f] quinoline-2,7,9-tricarboxylic acid (PQQ) (1)
One 1 liter, loaded a mechanical stirrer, a temperature sensor, an extra funnel, a purging with nitrogen gas system and placed three-necked bottle among the ice bath, add 4,5-dioxy-4,5-dihydro-1 hydrogen-pyrroles [2,3-f] quinoline-2,7,9-tricarboxylic acid 2-ethyl ester 7,9-dimethyl ester (10) (21.45 grams, 0.0555 rubs) and 215 milliliters of tetrahydrofuran (THF)s (THF).Under nitrogen environment, stir the heterogeneous solution obtain, and in 1 hour time to wherein adding a hydronium(ion) oxidation lithium (11.58 grams, the 0.276 rubs) solution that is dissolved in 515 ml waters.In the process that adds, keep temperature to be lower than 10 ℃.16-17 ℃ of following stirring reaction mixed solution 30.5 hours.
Add Repone K (15 grams, 1.98 rub).Reaction flask is placed ice bath, and use 10.0 milliliters concentrated hydrochloric acid that the pH of reaction mixture is adjusted to be approximately 6.By adding the hydrochloric acid of 2N, the pH value further is adjusted to 5.3.Need to add acid continuously and keep stable p H value.The hydrochloric acid that adds 16.0 milliliters of 2N altogether.Continuously stirring 1 hour, and continue cooling.Filter and collect the reddish-brown solid that forms, use a small amount of frozen water and 100 milliliters of acetonitrile washing solids.In nitrogen gas stream, use strainer that the reddish-brown trihydric salt 11 that obtains is carried out drying.
Exsiccant 11 (21.21 gram) is dissolved in 330 milliliters of concentrated vitriols, and stirred 2.5 hours.Pour acid solution into 1400 grams on ice, obtain a kind of blood red solids suspension.Under cooling conditions, suspension was stirred 1 hour.Filter and collect product, and use frozen water that it is washed.Under nitrogen gas stream, use strainer that product is carried out drying, under 40 ℃, carrying out drying under the vacuum condition of mansion then, obtain 17.85 grams (97.4%) 4,5-dioxy-4,5-dihydro-1 hydrogen-pyrroles [2,3-f] quinoline-2,7,9-tricarboxylic acid (PQQ).NMR (Nuclear Magnetic Resonance) spectrum shows and wherein still has moisture, even product has been dried to the almost state of constant weight.Thermogravimetric analysis shows and contains 7.8% impurity (ash) in the product.Described thermogravimetric analysis is included in the step of heated sample under the Oxygen Flow.Still any impurity that is present among the sample after heating shows that all this impurity is metal (inorganic) impurity.
Embodiment 2:4,5-dioxy-4,5-dihydro-1 hydrogen-pyrroles [2,3-
F] quinoline-2,7, the purifying in batches of 9-tricarboxylic acid (PQQ) (1)
By with 4,5-dioxy-4,5-dihydro-1 hydrogen-pyrroles [2,3-f] quinoline-2,7,9-tricarboxylic acid (PQQ) (1) (75.49 gram) is dissolved in 100 milliliters of methods in the concentrated vitriol, make large batch of 4,5-dioxy-4,5-dihydro-1 hydrogen-pyrroles [2,3-f] quinoline-2,7,9-tricarboxylic acid (PQQ) (1) (75.49 gram) is purified to remove any residual impurity.At room temperature suspension was stirred 2 hours.In 40 minutes time, acid solution is dropwise joined in the water that 5 liters of quilts stir effectively slowly, keep temperature to be lower than 33 ℃ simultaneously.From solution, be settled out desired product, and at ambient temperature suspension stirred 1 hour.Filter and collect product, and use 1 premium on currency washed product.Under high vacuum condition, under 40 ℃, product is carried out drying.The yield that obtains is 63.0 grams (83.5%).After having calculated purity and twice extra purifying, 4,5-dioxy-4,5-dihydro-1 hydrogen-pyrroles [2,3-f] quinoline-2,7, the productive rate of 9-tricarboxylic acid (PQQ) is 71.6%.
Embodiment 3: to 4, and 5-dioxy-4,5-dihydro-1 hydrogen-pyrroles [2,3
-f] quinoline-2,7, the evaluation of 9-tricarboxylic acid (PQQ) (1)
Use conventional method to 4,5-dioxy-4,5-dihydro-1 hydrogen-pyrroles [2,3-f] quinoline-2,7,9-tricarboxylic acid (PQQ) (1) (for example carries out purity check and evaluation, NMR (Nuclear Magnetic Resonance) spectrum (NMR), high performance liquid chromatography (HPLC), karl Fischer titration (Karl Fisher titration), ultimate analysis).It is 4 of 62.7 gram scales that table 1 has been expressed for a collection of output, 5-dioxy-4,5-dihydro-1 hydrogen-pyrroles [2,3-f] quinoline-2,7, the concise and to the point analytical results of 9-tricarboxylic acid (PQQ).In the literature Bao Dao mass spectrum of nuclear magnetic resonance (
1HNMR) data that data are used to obtain with the present invention compare (seeing Table 2).In high performance liquid chromatography (HPLC) is analyzed, used to comprise damping fluid (20mM potassium primary phosphate (pH2.1)) and acetonitrile (CH
3CN) different solvents gradient.Described gradient is as follows: be under the situation of 0.7 ml/min at flow velocity, and during T=0,2: 98 acetonitrile/damping fluids; In the time of T=6 minute, 90: 10 acetonitrile/damping fluids; In the time of T=10 minute, 90: 10 acetonitrile/damping fluids; In the time of T=16 minute, 2: 98 acetonitrile/damping fluids; In the time of T=25 minute, 2: 98 acetonitrile/damping fluids.Sample is by with 4, and 5-dioxy-4,5-dihydro-1 hydrogen-pyrroles [2,3-f] quinoline-2,7,9-tricarboxylic acid (PQQ) are dissolved in and make in the dimethyl sulfoxide (DMSO) (DMSO).The post that reaction is used is Waters Atlantis
TMC18 (SN W40541), 3 microns, 3.0 * 100 millimeters, volumetric injection is 5 microlitres.In liquid chromatography biomass spectroscopic analysis (liquid chromatography mass spectral analysis), at flow velocity is under the condition of 0.3 ml/min, uses the no gradient solvent that comprises 50% acetonitrile and 50% damping fluid (5mM ammonium acetate aqueous solution).Reaction times is 12 minutes.Sample is by with 1.25 milligram 4, and 5-dioxy-4,5-dihydro-1 hydrogen-pyrroles [2,3-f] quinoline-2,7,9-tricarboxylic acid (PQQ) are dissolved in and make in 10 milliliters of dimethyl sulfoxide (DMSO) (DMSO).The solution that obtains further uses dimethyl sulfoxide (DMSO) to dilute with 1: 50 ratio.Volumetric injection is 10 microlitres.
Table 1:4,5-dioxy-4,5-dihydro-1 hydrogen-pyrroles [2,3-F] quinoline-2,7, the brief analysis result of 9-tricarboxylic acid (PQQ)
| Test | Test method | Test-results |
| Identification (Identity) | Mass spectrum of nuclear magnetic resonance ( 1HNMR) | Consistent |
| Identification (Identity) | Carbon-13 nmr spectra ( 13CNMR) | Consistent |
| Purity | High performance liquid chromatography color development system (HPLC TAN) | 98.9％ |
| Impurity Distribution | High performance liquid chromatography color development system (HPLC TAN) | The highest impurity number: 0.2% |
| Water content | Karl Fischer titration (Karl Fisher titration) | 6.4％ |
| Form (comprising water and impurity in theory) | Ultimate analysis | In theory: 47.36% carbon, 2.42% hydrogen, 7.89% nitrogen in fact: 46.85% carbon, 2.43% hydrogen, 7.79% nitrogen |
| Loss on heating | TGA | 0.6% impurity |
| Specify purity (Assigned Purity): 91.9% | ||
Table 2:4,5-dioxy-4,5-dihydro-1 hydrogen-pyrroles [2,3-f] quinoline-2,7, the mass spectrum of nuclear magnetic resonance data of 9-tricarboxylic acid (PQQ)
| Method of the present invention 1HNMR(300MHz，(D 6)DMSO)：7.22(q，∫＝1&2，H-C(3))；8.60(d，J＝1，H-C(8))；14.40(br，s，NH) | Reference value (coming from Martin) 1HNMR(250MHz，(D 6)DMSO)：7.21(d，J＝2，H-C(3))；8.60(s，H-C(8))；13.25(br，s，NH) |
Equivalence transformation
Though the present invention is described and describes in detail the new feature as basis of the present invention with preferred embodiment form, but those skilled in the art can understand, under the situation that does not deviate from essence of the present invention, to content disclosed by the invention do any in form and omission and replacement on the details, all fall within the scope of the present invention.Therefore, scope of the present invention is to be limited by subsequently claim.
Claims (23)
1. the method for a synthetic PQQ comprises: under cold condition, handle 4 in alkaliferous tetrahydrofuran (THF), 5-dioxy-4,5-dihydro-1 hydrogen-pyrroles [2,3-f] quinoline-2,7,9-tricarboxylic acid 2-ethyl ester 7, the 9-dimethyl ester, and, add hydrochloric acid subsequently to wherein adding salt.
2. method according to claim 1 wherein adds lithium hydroxide and halide salts to generate a kind of trihydric salt that comprises single metallic element.
3. method according to claim 1 wherein adds lithium hydroxide and carbonate to generate a kind of trihydric salt that comprises single metallic element.
4. method according to claim 1, wherein the temperature of reaction mixture remains on 17 ℃ or be lower than 17 ℃.
5. method according to claim 1, wherein the temperature of reaction mixture remains on 16-17 ℃.
6. method according to claim 2, wherein said halide salts is a Repone K.
7. method according to claim 2, wherein said metallic element is a potassium.
8. method according to claim 1, the order of the reagent that wherein adds in reaction flask is: 4,5-dioxy-4,5-dihydro-1 hydrogen-pyrroles [2,3-f] quinoline-2,7,9-tricarboxylic acid 2-ethyl ester 7,9-dimethyl ester, tetrahydrofuran (THF), lithium hydroxide, Repone K, and hydrochloric acid.
9. method according to claim 1 wherein remains on the pH value 6 or be lower than 6.
10. method according to claim 1 wherein remains on 5.3 with the pH value.
11. method according to claim 2 wherein further comprises trihydric salt being dissolved in the sulfuric acid and acid solution being added to the water to generate the step of PQQ.
12. the method for a synthetic PQQ comprises the following steps:
Step a in the presence of acetic acid and water, uses formic acid to handle 2-methoxyl group-5-N-methyl-p-nitroaniline, generates N-(2-methoxyl group-5-nitrophenyl) methane amide;
Step b in the presence of metallic palladium and dimethyl formamide, uses hydrogen treat N-(2-methoxyl group-5-nitrophenyl) methane amide, generates N-(5-amino-2-p-methoxy-phenyl) methane amide;
Step c, in the presence of water and alcoholic acid, use Sodium Nitrite and fluoroboric acid to handle N-(5-amino-2-p-methoxy-phenyl) methane amide, in the presence of water, add ethyl 2-methyl-acetoacetic ester and sodium-acetate then, generate the 2-[(3-formamido group)-4-methoxyl group aryl] hydrazono-]-ethyl propionate;
Steps d uses formic acid to handle ethyl 2-[(3-formamido group)-4-methoxyl group aryl] hydrazono-]-ethyl propionate, generate 6-formamido group-5-methoxyl group-1 hydrogen-Ethyl indole-2-carboxylate;
Step e in the presence of hydrochloric acid and water, uses acetone treatment 6-formamido group-5-methoxyl group-1 hydrogen-Ethyl indole-2-carboxylate, generates 6-amino-5-methoxyl group-1 hydrogen-Ethyl indole-2-carboxylate;
Step f in the presence of methylene dichloride, uses 2-oxidation propene dicarboxylic acid dimethyl ester to handle 6-amino-5-methoxyl group-1 hydrogen-Ethyl indole-2-carboxylate, generates 9-hydroxy-5-methyl oxygen base-6,7,8,9-tetrahydrochysene-1 hydrogen-pyrroles [2,3-f] quinoline-2,7,9-tricarboxylic acid 2-ethyl ester-7,9-dimethyl ester;
Step g in the presence of methylene dichloride and hydrogenchloride, uses a hydration cupric acetate (II) to handle 9-hydroxy-5-methyl oxygen base-6,7,8,9-tetrahydrochysene-1 hydrogen-pyrroles [2,3-f] quinoline-2,7,9-tricarboxylic acid 2-ethyl ester-7, the 9-dimethyl ester, generate 5-methoxyl group-1 hydrogen-pyrroles [2,3-f] quinoline-2,7,9-tricarboxylic acid 2-ethyl ester-7, the 9-dimethyl ester;
Step h in the presence of acetonitrile and water, uses ammonium ceric nitrate to handle 5-methoxyl group-1 hydrogen-pyrroles [2,3-f] quinoline-2,7,9-tricarboxylic acid 2-ethyl ester-7, the 9-dimethyl ester generates 4,5-dioxy-4,5-dihydro-1 hydrogen-pyrroles [2,3-f] quinoline-2,7,9-tricarboxylic acid 2-ethyl ester 7,9-dimethyl ester;
Step I at low temperatures, in tetrahydrofuran (THF), uses lithium hydroxide to handle 4,5-dioxy-4,5-dihydro-1 hydrogen-pyrroles [2,3-f] quinoline-2,7,9-tricarboxylic acid 2-ethyl ester 7, the 9-dimethyl ester adds halide salts, adds hydrochloric acid afterwards, generates the trihydric salt that comprises single metallic element;
Step j is dissolved in the trihydric salt that obtains in the step I in the sulfuric acid, and the acid solution that forms is added in the entry, generates 4,5-dioxy-4,5-dihydro-1 hydrogen-pyrroles [2,3-f] quinoline-2,7,9-tricarboxylic acid (PQQ).
13. method according to claim 12, wherein the temperature of step I remains on 17 ℃ or be lower than 17 ℃.
14. method according to claim 12, wherein the temperature of step I remains on 16-17 ℃.
15. method according to claim 12, wherein the pH value with step I remains below 6.
16. method according to claim 12, wherein the pH value with step I remains on 5.3.
17. method according to claim 12, the order of the reagent that wherein adds in step I is: 4, and 5-dioxy-4,5-dihydro-1 hydrogen-pyrroles [2,3-f] quinoline-2,7,9-tricarboxylic acid 2-ethyl ester 7,9-dimethyl ester, tetrahydrofuran (THF), lithium hydroxide, Repone K, and hydrochloric acid.
18. method according to claim 12, the trihydric salt that wherein generates in step I has the structure of following structural formula I:
Structural formula 1
Wherein, M
1Represent hydrogen or potassium; M
2Represent hydrogen or potassium; And M
3Represent hydrogen or potassium; M wherein
1, M
2, M
3In have at least one not to be hydrogen.
19. method according to claim 18, wherein M
1, M
2, M
3In have at least two not to be hydrogen.
20. method according to claim 18, wherein M
1, M
2, M
3All be potassium.
21. trihydric salt with structure of structural formula I:
Structural formula 1
Wherein, M
1Represent hydrogen or potassium; M
2Represent hydrogen or potassium; And M
3Represent hydrogen or potassium; M wherein
1, M
2, M
3In have at least one not to be hydrogen.
22. method according to claim 21, wherein M
1, M
2, M
3In have at least two not to be hydrogen.
23. method according to claim 22, wherein M
1, M
2, M
3All be potassium.
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| US66498905P | 2005-03-24 | 2005-03-24 | |
| US60/664,989 | 2005-03-24 | ||
| US11/387,014 | 2006-03-21 |
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