CN1942430A - 5-aminolevulinic acid phosphate, method for producing the same and use thereof - Google Patents
5-aminolevulinic acid phosphate, method for producing the same and use thereof Download PDFInfo
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- CN1942430A CN1942430A CNA2005800107065A CN200580010706A CN1942430A CN 1942430 A CN1942430 A CN 1942430A CN A2005800107065 A CNA2005800107065 A CN A2005800107065A CN 200580010706 A CN200580010706 A CN 200580010706A CN 1942430 A CN1942430 A CN 1942430A
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Abstract
A 5-aminolevulinic acid salt which is useful in the fields of microorganisms, fermentation, animals, medical science, plants and so on; a process for producing the same; a medicinal composition containing the same; and a plant vitalizer composition containing the same.
Description
Technical field
The present invention relates to: the 5-aminolevulinic acid, it can be used in the fields such as microorganism, fermentation, animal, medicine, plant; The method for preparing this salt; The pharmaceutical composition that contains this salt; And the plant activator composition that contains this salt.
Background technology
Be known that the 5-amino-laevulic acid in the microbial fermentation field, can be used for VB
12With preparation of the preparation of hemachrome enzyme, microorganism culturing, porphyrin etc.; In the treatment of animals field, can be used for treatment (referring to non-patent literature 1), sterilization, diagnosis (referring to non-patent literature 5), animal cell culture, UV excision, protoheme metabolism research, the hair-care of infectious diseases, to by the diagnosis of the porphyria of heavy metal poisoning initiation, exsanguine prevention etc. in the diagnosis of the disease that causes by influenzae, the starting material, depilation, rheumatism treatment (referring to non-patent literature 2), cancer therapy (referring to non-patent literature 3), thrombus treatment (referring to non-patent literature 4), cancer operation as derivative; In plant field, can be used as agricultural chemicals etc.
On the other hand, in the 5-amino-laevulic acid, the preparation method who has only its hydrochloride is known, and people have reported use urobenzoic acid (referring to patent documentation 1), monomester succinate acyl chlorides (referring to patent documentation 2), chaff amine (referring to patent documentation 3), hydroxymethylfurfural (referring to patent documentation 4), oxopentanoic acid methyl esters (referring to patent documentation 5) or succinyl oxide (referring to the patent documentation 6) method as raw material.
Yet, because the 5-aminolevulinic acid hydrochloride contains hydrochloric acid, so must consider in production process and preparation and branch process of assembling, therefore corrosion that device is caused by evaporated hydrogen chloride and the stimulation that causes preferably take some countermeasures and prevent above-mentioned these situations.
In addition, with 5-aminolevulinic acid hydrochloride oral administration or when being applied to people's skin, tongue or skin can be experienced scorching hot stimulation.Therefore, as the 5-amino-laevulic acid that uses in field of medicaments, what people paid close attention to is the pungency 5-aminolevulinic acid littler than 5-aminolevulinic acid hydrochloride.
In addition, because the 5-aminolevulinic acid hydrochloride has the character of partly decomposing and decomposing fully in the time of 156 ℃ in the time of 130-156 ℃, so exist the problem that is difficult to bear the heat sterilising treatment.
The radiation exposure method for disinfection is known to be a kind of method that addresses this problem (referring to patent documentation 7), but this method needs the radiation exposure device.
Therefore, for by routine, stassanization comes sterilization easily, just is necessary to improve the thermotolerance of 5-amino-laevulic acid.
In addition, although the 5-aminolevulinic acid hydrochloride is used in the plant field (referring to patent documentation 8), but when itself and plant microbiocidal composition commonly used when for example Silver Nitrate etc. mixes use, in some cases, react and generation silver nitride precipitation thing by 5-aminolevulinic acid hydrochloride and Silver Nitrate, the possibility that can not spray medicine because the nozzle that exists atomizer is blocked is so very careful in operation.In addition, in the aqueous solution with the 5-aminolevulinic acid hydrochloride was applied directly to situation on the fruit, when having chlorion, it is bright-coloured inadequately that the color of fruit becomes sometimes.
In addition, although people have proposed to contain the aqueous solution of 5-amino-laevulic acid ion and nitrate ion, also do not isolate 5-amino-laevulic acid nitrate (referring to non-patent literature 6) at present.
Patent documentation 1:JP-A-48-92328
Patent documentation 2:JP-A-62-111954
Patent documentation 3:JP-A-2-76841
Patent documentation 4:JP-A-6-172281
Patent documentation 5:JP-A-7-188133
Patent documentation 6:JP-A-9-316041
Patent documentation 7:JP-T-2001-514243
Patent documentation 8:JP-A-4-338305
People such as non-patent literature 1:Peter W., J.Am.Acad.Dermatol.,
31, 678-680 (1994)
Non-patent literature 2:Kenneth T., United States Patent (USP) 5,368,841 (1994)
People such as non-patent literature 3:Hillemanns P., Int.J.Cancer,
85, 649-653 (2000)
Non-patent literature 4: people such as hillside plot one youth, Japan forms Society of Surgery's main idea collection (1988)
People such as non-patent literature 5:Kamasaki N., Journal of Japan Society for LaserMedicine,
22, 255-262 (2001)
People such as non-patent literature 6:Baxter C.S., Toxicology And AppliedPharmacology,
47, 477-482 (1979)
Summary of the invention
The problem to be solved in the present invention
In this case, the invention provides: a kind of novel 5-aminolevulinic acid, it has low irritant or can bear the heat sterilising treatment; The method for preparing this salt; Use contains the medical composition of this salt; And the plant activator composition that contains this salt.
The method of dealing with problems
By considering above-mentioned practical situation, the inventor has carried out deep research, found that a kind of 5-aminolevulinic acid that satisfies above-mentioned requirements, this salt can obtain by the following method: wash-out is attracted to the 5-amino-laevulic acid on the Zeo-karb, and elutriant is mixed with phosphoric acid, nitric acid or sulfonic acid.
That is, the present invention relates to following (1) to (23):
(1) a kind of 5-aminolevulinic acid that belongs to the aminolevulinic acid class, wherein this salt is to be selected from least a in phosphoric acid salt, nitrate and the sulfonate.
(2) according to above-mentioned (1) described 5-aminolevulinic acid, this salt is the amino-laevulic acid phosphoric acid salt by following formula (I) expression:
HOCOCH
2CH
2COCH
2NH
2·HOP(O)(OR
1)
n(OH)
2-n (I)
Wherein, R
1Expression hydrogen atom, alkyl, alkenyl, aralkyl or phenyl with 7-26 carbon atom with 2-18 carbon atom with 1-18 carbon atom; N is the integer of 0-2; And wherein when n is 2, two R
1Be identical or different.
(3) according to above-mentioned (2) described 5-aminolevulinic acid, wherein R
1Be hydrogen atom, methyl, ethyl, normal-butyl, hexadecyl, 2-ethylhexyl, oleyl, benzyl or phenyl.
(4) according to above-mentioned (2) or (3) described 5-aminolevulinic acid, this salt is aqueous solution form.
(5) according to above-mentioned (2) or (3) described 5-aminolevulinic acid, this salt is solid form.
(6) according to above-mentioned (1) described 5-aminolevulinic acid, this salt is 5-amino-laevulic acid nitrate.
(7) according to above-mentioned (6) described 5-aminolevulinic acid, this salt is solid.
(8) according to above-mentioned (1) described 5-aminolevulinic acid, this salt is the 5-amino-laevulic acid sulfonate by following formula (II) expression:
HOCOCH
2CH
2COCH
2NH
2·HOSO
2R
2 (II)
Wherein, R
2The phenyl that expression is replaced by low alkyl group.
(9) according to above-mentioned (8) described 5-aminolevulinic acid, wherein substituted phenyl is a 4-aminomethyl phenyl, 2,4-3,5-dimethylphenyl or 2,5-3,5-dimethylphenyl.
(10) according to above-mentioned (8) or (9) described 5-aminolevulinic acid, this salt is aqueous solution form.
(11) according to above-mentioned (8) or (9) described 5-aminolevulinic acid, this salt is solid form.
(12) a kind of preparation is according to the method for any described 5-aminolevulinic acid in above-mentioned (2) to (5), and this method comprises that wash-out is attracted to the 5-amino-laevulic acid on the Zeo-karb, and elutriant is mixed with phosphoric acid class material.
(13) according to above-mentioned (12) described method, wherein said 5-amino-laevulic acid ammoniacal liquor wash-out.
(14) a kind of preparation is according to the method for above-mentioned (6) or (7) described 5-aminolevulinic acid, and this method comprises that wash-out is attracted to the 5-amino-laevulic acid on the Zeo-karb, and elutriant is mixed with nitric acid.
(15) according to above-mentioned (14) described method, wherein said 5-amino-laevulic acid ammoniacal liquor wash-out.
(16) a kind of preparation is according to the method for the 5-amino-laevulic acid sulfonate of above-mentioned (8) or (9), and this method comprises that wash-out is attracted to the 5-amino-laevulic acid on the Zeo-karb, and elutriant is mixed with sulfonic acid substance.
(17) according to above-mentioned (16) described method, wherein said 5-amino-laevulic acid ammoniacal liquor wash-out.
(18) a kind of composition that is used for optical dynamic therapy or light power diagnosis, said composition contain any described 5-aminolevulinic acid in good grounds above-mentioned (1) to (11).
(19) a kind of plant activator composition that comprises according to any described 5-aminolevulinic acid in above-mentioned (1) to (11).
(20) according to the application of any described 5-aminolevulinic acid in above-mentioned (1) to (11) in preparation optical dynamic therapy agent or light power diagnosis reagent.
(21) according to the application of any described 5-aminolevulinic acid in above-mentioned (1) to (11) as plant activator.
Effect of the present invention
5-aminolevulinic acid of the present invention is a kind of material of easy handling, and this is because it can not send niff or irritating smell.And this salt pair skin and tongue show low irritant, and skin etc. is had excellent permeation, and therefore, the composition that contains this salt can be used as optical dynamic therapy or the used reagent of light power diagnosis.And, with the 5-aminolevulinic acid hydrochloride Comparatively speaking, described 5-aminolevulinic acid has high decomposition temperature and excellent thermotolerance.Preparation in accordance with the present invention can be made 5-aminolevulinic acid of the present invention easily and effectively.In addition, because 5-aminolevulinic acid of the present invention has low chlorine ion concentration when forming the aqueous solution, therefore the infringement that is caused by chlorine can take place hardly when being applied to plant.
Description of drawings
Fig. 1 is the figure that the concentration and the relation between the pH value of the 5-aminolevulinic acid aqueous solution are shown.
Fig. 2 is the synoptic diagram of unit with dialysis.
Fig. 3 is the figure that the pigskin skin permeability test result of the phosphoric acid salt of 5-amino-laevulic acid and hydrochloride is shown.
Fig. 4 is the figure that the onion epidermis permeability test result of the phosphoric acid salt of 5-amino-laevulic acid and hydrochloride is shown.
Implement best mode of the present invention
In above-mentioned formula (I), by R
1The alkyl with 1-18 carbon atom of expression can be straight chain, side chain or cyclic.The straight or branched alkyl for example comprises: methyl, ethyl, n-propyl, sec.-propyl, normal-butyl, isobutyl-, the tertiary butyl, n-pentyl, isopentyl, neo-pentyl, tert-pentyl, the 2-methyl butyl, n-hexyl, isohexyl, the 3-methyl amyl, ethyl-butyl, n-heptyl, 2-methyl hexyl, n-octyl, iso-octyl, uncle's octyl group, the 2-ethylhexyl, the 3-methylheptyl, n-nonyl, different nonyl, the 1-Methyl Octyl, the ethyl heptyl, positive decyl, 1-methyl nonyl, the n-undecane base, 1,1-dimethyl nonyl, dodecyl, the n-tridecane base, the n-tetradecane base, the Pentadecane base, n-hexadecyl, the n-heptadecane base, Octadecane base etc.Cycloalkyl or the alkyl that contains cyclic group for example comprise: cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, suberyl, the ring octyl group, 2-cyclopropyl ethyl, 2-cyclobutyl ethyl, 2-cyclopentyl ethyl, cyclohexyl methyl, 2-cyclohexyl ethyl, the suberyl methyl, 2-ring octyl group ethyl, the 3-methylcyclohexyl, the 4-methylcyclohexyl, 4-ethyl cyclohexyl, 2-methyl ring octyl group, 3-(3-methylcyclohexyl) propyl group, 2-(4-methylcyclohexyl) ethyl, 2-(4-ethyl cyclohexyl) ethyl, 2-(2-methyl ring octyl group) ethyl etc.As above-mentioned alkyl, preferably has the alkyl of 1-16 carbon atom, special preferable methyl, ethyl, normal-butyl, n-hexadecyl or 2-ethylhexyl with 1-18 carbon atom.
Alkenyl with 2-18 carbon atom for example comprises: vinyl, allyl group, pseudoallyl, crotyl, the 2-methacrylic, 1, the 1-dimethyl-allyl, 3-methyl-2-butene base, 3-methyl-3-butenyl, the 4-pentenyl, hexenyl, octenyl, the nonene base, the decene base, cyclopropenyl radical, the cyclobutene base, cyclopentenyl, cyclohexenyl, cycloheptenyl, the cyclooctene base, 4-methyl cyclohexane thiazolinyl, 4-ethyl-cyclohexene base, 2-cyclopentenyl ethyl, the cyclohexenyl methyl, the cycloheptenyl methyl, 2-cyclobutene base ethyl, 2-cyclooctene base ethyl, 3-(4-methyl cyclohexane thiazolinyl) propyl group, 5-(4-ethyl-cyclohexene base) amyl group, oleyl, 11-octadecylene base (vaccenyl), 9,12-18 dialkylenes (linoleyl), 9,12,15-octadecylene base (linolenyl) etc., the preferred oil thiazolinyl.
Aralkyl with 7-26 carbon atom is preferably: by alkyl with 1-6 carbon atom and the group that aryl constituted with 6-20 carbon atom.Alkyl with 1-6 carbon atom for example comprises: methyl, ethyl, n-propyl, sec.-propyl, normal-butyl, isobutyl-, the tertiary butyl, n-pentyl, n-hexyl, cyclopropyl, cyclobutyl, cyclohexyl etc., the aryl with 6-20 carbon atom for example comprises: phenyl, naphthyl etc.In aralkyl with 7-26 carbon atom, preferred benzyl or styroyl, preferred especially benzyl.Aryl in the aralkyl can be replaced by 1-3 substituting group, described substituting group for example: above-mentioned alkyl with 1-6 carbon atom; Alkoxyl group with 1-6 carbon atom, for example methoxyl group, oxyethyl group, positive propoxy, n-butoxy, isobutoxy and tert.-butoxy; Hydroxyl; Amino; Nitro; Cyano group; Halogen, for example fluorine, chlorine, bromine and iodine; Carboxyl; Or the like.
In formula (II), replace by R
2The low alkyl group of the phenyl of expression is the alkyl with 1-6 carbon atom.Described low alkyl group can be straight chain, side chain or cyclic.The straight or branched alkyl for example comprises: methyl, ethyl, n-propyl, sec.-propyl, normal-butyl, isobutyl-, the tertiary butyl, n-pentyl, isopentyl, neo-pentyl, tert-pentyl, 2-methyl butyl, n-hexyl, isohexyl, 3-methyl amyl, ethyl-butyl etc., preferable methyl, ethyl or n-propyl, special preferable methyl.The alkyl that contains closed chain for example comprises: cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, 2-cyclopropyl ethyl, 2-cyclobutyl ethyl etc.The position of substitution and substituent quantity to low alkyl group are not particularly limited, but the quantity of low alkyl group is preferably 1-3, and more preferably 1 or 2.
The phenyl that is replaced by low alkyl group for example comprises: the phenyl with alkyl replacement of 1-6 carbon atom, as the 2-aminomethyl phenyl, the 3-aminomethyl phenyl, the 4-aminomethyl phenyl, 2, the 3-3,5-dimethylphenyl, 2, the 4-3,5-dimethylphenyl, 2, the 5-3,5-dimethylphenyl, 2, the 6-3,5-dimethylphenyl, 3, the 4-3,5-dimethylphenyl, 3, the 5-3,5-dimethylphenyl, 2,4, the 6-trimethylphenyl, 3,4, the 5-trimethylphenyl, the 2-ethylphenyl, tert-butyl-phenyl, the amyl group phenyl, neo-pentyl phenyl and hexyl phenyl, preferred especially 4-aminomethyl phenyl, 2,4-3,5-dimethylphenyl or 2, the 5-3,5-dimethylphenyl.
5-aminolevulinic acid of the present invention can be solid or solution.Solid is meant crystal, but also can be hydrate.Solution is meant the dissolving of this salt or is dispersed in state in the solvent that comprises water, adjustings such as the available pH regulator agent of pH value of solution.In addition, can use comprising that in the solvent of water two or more mix.The pH regulator agent for example comprises: the damping fluid or Good ' the s damping fluid that use phosphoric acid, boric acid, phthalic acid, citric acid, succsinic acid, Tris, acetate, lactic acid, tartrate, phthalic acid, toxilic acid and salt thereof.
Preferably with the solution form of the aqueous solution as the 5-aminolevulinic acid.The concentration of 5-aminolevulinic acid in the aqueous solution is preferably 0.01 ppm by weight to 10 weight %, more preferably 0.1 ppm by weight to 5 weight %, most preferably 1 ppm by weight to 1 weight %.In addition, the preferred 3-7 of the pH of the aqueous solution, more preferably 3.5-7, most preferably 4-7.In addition, can also contain the salt except 5-aminolevulinic acid of the present invention in the described aqueous solution, in the case, the concentration of chlorion is preferably 50mol% or lower, and more preferably 10mol% or lower most preferably is 3mol% or lower.In this connection, term " not chloride ion-containing " is meant that the concentration of chlorion is essentially 0mol%, that is, preferably, this concentration is equal to or less than the limit of detection (0.1ppm) when for example measuring with ion chromatography.
5-aminolevulinic acid of the present invention can be prepared by the following method: be attracted to 5-amino-laevulic acid on the Zeo-karb with containing the ion solution wash-out, and elutriant is mixed with phosphoric acid, nitric acid or sulfonic acid.In addition, can be by making it crystallization in the mixed solution that poor solvent is joined above-mentioned 5-aminolevulinic acid, thus obtain solid-state 5-aminolevulinic acid.The 5-amino-laevulic acid that is attracted on the Zeo-karb is not particularly limited, its purity etc. also is not particularly limited.That is, can use according to disclosed method preparation among patent documentation JP-A-48-92328, JP-A-62-111954, JP-A-2-76841, JP-A-6-172281, JP-A-7-188133 etc. and the JP-A-11-42083 those, chemical reaction solution and fermented liquid and commercial goods before the purifying.In this connection, preferably use the 5-aminolevulinic acid hydrochloride.
Described Zeo-karb can be a storng-acid cation exchange resin, also can be weakly acidic cation-exchange resin.In addition, resin also is suitable for using.Wherein, preferred storng-acid cation exchange resin.As the kind of storng-acid cation exchange resin, the preferred polystyrene resins of those and sulfonic group bonding.
By the following method the 5-amino-laevulic acid is adsorbed on the Zeo-karb: the 5-amino-laevulic acid is dissolved in the suitable solvent and makes solution, this solution is flow through from Zeo-karb.Described solvent is not particularly limited, as long as it can dissolve the 5-amino-laevulic acid, the example of solvent comprises water; Dimethyl sulfoxide (DMSO); Alcohol, for example methyl alcohol, ethanol, propyl alcohol, Virahol, butanols and isopropylcarbinol; Acid amides, N for example, dinethylformamide and N,N-dimethylacetamide; Pyridine; Or the like.Preferably water, dimethyl sulfoxide (DMSO), methyl alcohol or ethanol particularly preferably are water, methyl alcohol or ethanol.In addition, two or more solvent can be used.In addition, when chemical reaction solution before using purifying or fermented liquid, can remove reaction solvent or dilute with suitable solvent.In this connection, the chemical reaction solution before above-mentioned solvent and the purifying or the pH of fermented liquid can use above-mentioned pH regulator agent to regulate.
Though the ionic aqueous solution that contains used in the eluant is not particularly limited, but phosphoric acid class material preferably, nitric acid, sulfonic acid substance, the oxyhydroxide of basic metal or alkaline-earth metal or carbonate, ammonia, amine and contain amino compound dissolution in water and the solution that forms, lithium hydroxide more preferably, sodium hydroxide, magnesium hydroxide, potassium hydroxide, calcium hydroxide, cesium hydroxide, hydrated barta, volatile salt, bicarbonate of ammonia, yellow soda ash, sodium bicarbonate, salt of wormwood, sodium potassium carbonate, saleratus, ammonia, methylamine, dimethyl amine, Trimethylamine, ethylamine, diethylamide or triethylamine are dissolved in the water and the solution that forms, particularly preferably are that ammonia is dissolved in the water and the solution that forms.These aqueous solution can two or more be used in combination.The concentration of ammoniacal liquor is preferably 0.01N to 10N, more preferably 0.1N to 3N.
As with 5-amino-laevulic acid elutriant blended phosphoric acid class material, can use compound by formula (III) expression:
HOP(O)(OR
1)
n(OH)
2-n (III)
R wherein
1With n as mentioned above.
This phosphoric acid class material for example comprises: phosphoric acid; Phosphate monoester is as methyl orthophosphoric acid, etherophosphoric acid, phosphate n-butyl, phosphoric acid 2-ethylhexyl, phosphoric acid cetyl ester, benzyl phosphate ester, phosphoric acid oleyl ester and phenyl-phosphate; And phosphodiester, as dimethyl phosphate, diethyl phosphoric acid, phosphoric acid di-n-butyl, di(2-ethylhexyl)phosphate (2-ethylhexyl) ester, di(2-ethylhexyl)phosphate (hexadecyl) ester, phosphate dibenzyl ester, di(2-ethylhexyl)phosphate oleyl ester and diphenyl phosphate.Special preferably phosphoric acid methyl esters, etherophosphoric acid, phosphoric acid oleyl ester, phenyl-phosphate, dimethyl phosphate, diethyl phosphoric acid, phosphoric acid di-n-butyl, di(2-ethylhexyl)phosphate (2-ethylhexyl) ester, di(2-ethylhexyl)phosphate (hexadecyl) ester, phosphate dibenzyl ester, di(2-ethylhexyl)phosphate oleyl ester or diphenyl phosphate.In addition, Hypophosporous Acid, 50 or phosphorous acid also are suitable for using.
Phosphoric acid class material can be its hydrate or salt, dissolves or be dispersed in the appropriate solvent those also to be suitable for using.Based on the elution amount by the 5-amino-laevulic acid of being inferred by the amount of 5-amino-laevulic acid of absorption, the combined amount of phosphoric acid class material is preferably 1-5000 times of molar weight, and more preferably 1-500 times of molar weight most preferably is 1-50 times of molar weight.In this connection, the elution amount of the 5-amino-laevulic acid of being inferred by the amount of 5-amino-laevulic acid of absorption changes along with the flow of the kind of Zeo-karb and elutriant and elutriant, but is generally the 90-100% of the adsorptive capacity of 5-amino-laevulic acid.
With 5-amino-laevulic acid elutriant blended nitric acid can be its salt, be dissolved in the appropriate solvent those and also be suitable for using.The combined amount of nitric acid is identical with the combined amount of phosphoric acid class material mentioned above.
For example comprise with 5-amino-laevulic acid elutriant blended sulfonic acid substance: tosic acid, 2,4-acid dimethyl, 2,5-acid dimethyl, 3,5-acid dimethyl, 2,4,6-tri-methyl p-toluenesulfonate etc., preferred especially tosic acid, 2,4-acid dimethyl or 2, the 5-acid dimethyl.This sulfonic acid substance can be its hydrate or salt, dissolves or be dispersed in the appropriate solvent those also to be suitable for using.The combined amount of this sulfonic acid substance is identical with the combined amount of phosphoric acid class material mentioned above.
Solvent comprises water; Dimethyl sulfoxide (DMSO); Alcohol, for example methyl alcohol, ethanol, propyl alcohol, Virahol, propyl carbinol and isopropylcarbinol; Acid amides, N for example, dinethylformamide and N,N-dimethylacetamide; Pyridine etc.Preferably water, dimethyl sulfoxide (DMSO), methyl alcohol or ethanol, special preferably water, methyl alcohol or ethanol.Equally, two or more solvent can be used.
Poor solvent mentioned above is not particularly limited, as long as can separate out solid therein, the example of described solvent comprises: alcohol, as methyl alcohol, ethanol, propyl alcohol, Virahol, propyl carbinol and isopropylcarbinol; Ether is as ether, Di Iso Propyl Ether, two alkane, tetrahydrofuran (THF) and glycol dimethyl ether; Ester is as methyl acetate, ethyl acetate, propyl acetate, isopropyl acetate and gamma-butyrolactone; Ketone is as acetone and methylethylketone; Nitrile, as acetonitrile and cyanobenzene etc., preferred methyl acetate, ethyl acetate, gamma-butyrolactone, acetone or acetonitrile.Preferred especially methyl acetate, gamma-butyrolactone, acetone or acetonitrile.Equally, two or more solvent can be used.
With containing the eluting temperature that the ionic aqueous solution carries out wash-out, and phosphoric acid class material, nitric acid or sulfonic acid substance and elutriant blended mixing temperature all are preferably-20 ℃ to 60 ℃, more preferably-10 ℃ to 30 ℃, under such condition, elutriant and phosphoric acid, nitric acid or sulfonic acid can not become solid.
Can prepare 5-aminolevulinic acid of the present invention by such 5-amino-laevulic acid; in described 5-amino-laevulic acid; amino is protected with hydrolyzable protecting group (for example acyl group); perhaps be connected with on the amino and can form 1; 3-dioxo-1, the protecting group of 3-xylylenimine-such molecular skeleton of 2-base.In addition; also can prepare 5-aminolevulinic acid of the present invention by being different from preparation method of the present invention; promptly; use required phosphoric acid, nitric acid or sulfonic acid to make the method for 2-phenyl-4-(β-alkoxy carbonyl propionyl) azoles quinoline-5-ketone generation hydrolysis, perhaps make the salt (for example 5-aminolevulinic acid hydrochloride) that is different from phosphoric acid salt, nitrate and sulfonate in solvent, produce the method that contacts with required phosphoric acid.Can use above as the represented compound of the described formula of phosphoric acid (III), and use those materials of describing as nitric acid, sulfonic acid substance and reaction solvent hereinbefore.
Shown in embodiment hereinafter, with the 5-aminolevulinic acid hydrochloride Comparatively speaking, 5-aminolevulinic acid of the present invention can not send niff, particularly, 5-amino-laevulic acid phosphoric acid salt is all little to the pungency of skin and tongue, and does not find that it has mutagenicity.In addition, 5-aminolevulinic acid of the present invention all has excellent permeation to animal skin and plant epidermis.Therefore, similar to the situation of 5-aminolevulinic acid hydrochloride, 5-aminolevulinic acid of the present invention (preferred 5-amino-laevulic acid phosphoric acid salt) can be used as animal (comprising the people) is carried out optical dynamic therapy or the used reagent of light power diagnosis.As the reagent that is used for optical dynamic therapy or light power diagnosis, can be exemplified as the reagent that is used for cancer, infectious diseases, rheumatosis, thrombus, papule etc. are carried out optical dynamic therapy or diagnosis.
5-aminolevulinic acid of the present invention can be used for optical dynamic therapy or diagnosis with known condition, more particularly, can use according to disclosed prescription and method among patent documentation JP-T-2001-501970 (WO98/30242), JP-T-4-500770 (WO 91/01727), JP-T-2005-501050 (WO2003/011265), JP-T-2004-506005 (WO2002/013788), JP-T-2001-518498 (WO 99/17764) and the JP-T-8-507755 (WO 94/17797).
Specifically, by with the 5-aminolevulinic acid of the present invention of significant quantity to animal (comprising the mankind) administration, and carry out rayed, can carry out optical dynamic therapy to disease.In addition, can carry out light power diagnosis to disease by the fluorescence that detects diseased region.
The composition (it contains 5-aminolevulinic acid of the present invention) that is used for optical dynamic therapy or light power diagnosis of the present invention can be made into the agent shape such as preparation for external application to skin, injection, oral preparations and suppository.When making these agent shapes, can use pharmaceutically useful carrier.As this carrier, can make water, binding agent, disintegrating agent, solubilizing agent, lubricant, weighting agent, filler etc.
Dosage changed along with age, body weight, symptom, curative effect, medication, treatment time etc., but usually, dosage is everyone each per kilogram of body weight 10mg to 10g of adult, and 100mg to 1g more preferably was administered once or repeatedly in one day.
In addition, when 5-aminolevulinic acid of the present invention being used for for example plant application, can also add normally used fertilizer components etc.The as fertilizer sources component can be exemplified as disclosed material among the patent documentation JP-A-4-338305 (United States Patent (USP) 5,298,482, EP-A-0514776).
5-aminolevulinic acid of the present invention also can be used as plant activator.When the plant activator, can use this salt with known condition, more particularly, can use that disclosed method is used for plant with this salt among the patent documentation JP-A-4-338305 (United States Patent (USP) 5,298,482, EP-A-0514776).
More particularly, as plant activator, can be exemplified as cauline leaf treatment agent, processing soil treatment agent etc.In addition, this reagent is absorbed in the plant before can or transplanting the plantation plant, perhaps is added to the water when water planting.
When 5-aminolevulinic acid of the present invention is used as the cauline leaf treatment agent, the concentration of the 5-aminolevulinic acid that contains in this treatment agent is preferably 1-1,000ppm, particularly 10-500ppm, and use this treatment agent with the amount that per 10 ares of 10-100 liters, particularly 50-300 rise.
When 5-aminolevulinic acid of the present invention was used as processing soil treatment agent, preferably with per 10 ares of 1-1, the amount of 000g, particularly 10-500g was used the 5-aminolevulinic acid.
When before plantation, using 5-aminolevulinic acid of the present invention as the cauline leaf treatment agent, the concentration of the 5-aminolevulinic acid that contains in this treatment agent is preferably 1-1,000ppm, 10-500ppm particularly, and use this treatment agent with the amount that per 10 ares of 10-100 liters, particularly 50-300 rise.In this connection, also preferably use the amount that almost equates therewith during water planting.
As processed plant, can be exemplified as cereal, vegetables, fruit tree, flower and ornamental plant, trees, beans, potato, Wales onion, herbage etc.
Example
Below based on example the present invention is described in more detail, but the present invention is not limited thereto.
Embodiment 1
The phosphatic preparation of 5-amino-laevulic acid
In the strong-acid ion exchange resin of 180ml (AMBERLITE IR 120B Na is produced by the Japanese Organo Co., Ltd.) post of packing into.Used ion exchange resin is to have passed through the salt acid treatment and the ion exchange resin that changes into the hydrogen ion type from sodium-ion type.Then, the 5-aminolevulinic acid hydrochloride of 20.00g (119mmol) is dissolved in the ion exchanged water of 1000ml, and this solution is crossed post, then the ion exchanged water of 1000ml is crossed post.Then, with the slow post of crossing of the ammoniacal liquor of 1N, collect 346ml xanchromatic elutriant.With the elutriant adding 16ml that collects, 85% phosphoric acid (H
3PO
4, 238mmol) in, and use vaporizer to concentrate.In concentrated solution, add the acetone of 400ml, and use the agitator vigorous stirring, under 4 ℃, left standstill 16 hours then.Suction strainer reclaims the solid separated out, and with the washing with acetone of 500ml.With gained solid drying under reduced pressure 12 hours, obtain the target substance of 23.04g (101mmol).Its physical performance data is as follows.
Fusing point: 108-109 ℃
1H-NMR(D
2O，400MHz)δppm：2.67(t，2H，CH
2)，2.86(t，2H，CH
2)，4.08(s，2H，CH
2)
13C-NMR(D
2O，100MHz)δppm：30(CH
2)，37(CH
2)，50(CH
2)，180(CO)，207(COO)
C
5H
9NO
3H
3PO
4The ultimate analysis data:
Calculated value: C 26.21%; H 5.28%; N 6.11%
Measured value: C 25.6%; H 5.2%; N 6.1%
The PO that obtains by ion chromatography
4 3-Content
Calculated value: 41.45%
Measured value: 43%
Ion chromatography condition: separator column: the IonPacAS12A that makes by Japanese Dionex Co., Ltd.; Eluant: contain Na
2CO
3And NaHCO
3(Na
2CO
3: 3.0mmol/l, NaHCO
3: aqueous solution 0.5mmol/l), flow velocity: 1.5ml/ minute, sample size: 25 μ l, column temperature: 35 ℃, detector: electrical conductivity detector.
The preparation of 5-amino-laevulic acid (phosphoric acid di-n-butyl) salt
In the strong-acid ion exchange resin of 180ml (AMBERLITE IR 120B Na is produced by the Japanese Organo Co., Ltd.) post of packing into.Used ion exchange resin is to have passed through the salt acid treatment and the ion exchange resin that changes into the hydrogen ion type from sodium-ion type.Then, the 5-aminolevulinic acid hydrochloride of 20.00g (119mmol) is dissolved in the ion exchanged water of 1000ml, and this solution is crossed post, then the ion exchanged water of 1000ml is crossed post.Then, with the slow post of crossing of the ammoniacal liquor of 1N, collect 321ml xanchromatic elutriant.The elutriant of collecting is added in the phosphoric acid di-n-butyl of 50.00g (238mmol), and use vaporizer to concentrate.In concentrated solution, add the acetone of 400ml, and use the agitator vigorous stirring, then mixture was left standstill under-25 ℃ 16 hours.Suction strainer reclaims the solid of being separated out.With gained solid drying under reduced pressure 12 hours, obtain the target substance of 14.67g (43mmol).Its physical performance data is as follows.
1H-NMR(D
2O，400MHz)δppm：0.75(6H，CH
3)，1.23(4H，CH
2)，1.41(4H，CH
2)，2.46(2H，CH
2)，2.59(2H，CH
2)，3.66(4H，CH
2)，3.80(2H，CH
2)
13C-NMR(D
2O，100MHz)δppm：14(CH
3)，20(CH
2)，29(CH
2)，34.2(CH
2)，34.3(CH
2)，36(CH
2)，67(CH
2O)，176(COO)，204(CO)
Embodiment 3
The phosphatic measurement of odor of 5-amino-laevulic acid
5 experimenters directly hear the phosphatic aqueous solution of 5-amino-laevulic acid (mixed solution of post elutriant and phosphoric acid) and the solid smell thereof of embodiment 1 preparation, and estimate their smell according to following standard.The results are shown in the table 1.
Judgement criteria:
0: do not have smell.
1: scent of but not unpleasant.
2: niff.
Comparative example 1
Except the aqueous solution and solid thereof that use the 5-aminolevulinic acid hydrochloride, estimate smell in the mode identical with embodiment 3.In this connection, the aqueous solution of 5-aminolevulinic acid hydrochloride is to use 5-aminolevulinic acid hydrochloride solid, hydrochloric acid and ion exchanged water preparation, wherein, make 5-amino-laevulic acid and chlorion volumetric molar concentration respectively with embodiment 1 in 5-amino-laevulic acid aqueous phosphatic in the 5-amino-laevulic acid and the volumetric molar concentration of phosphate anion identical.The results are shown in the table 1.
Table 1
| Embodiment 3 | The | 0 | 0 | 0 | 0 | 0 |
| Solid | 0 | 0 | 0 | 0 | 0 | |
| Comparative example 1 | The | 2 | 2 | 2 | 2 | 2 |
| Solid | 1 | 1 | 1 | 1 | 1 |
Make in the 1ml water the aqueous solution except being dissolved in, estimate smell in the mode identical with embodiment 3 by 5-amino-laevulic acid phosphoric acid salt with 0.5g.The results are shown in the table 2.
Comparative example 2
Make in the 1ml water the aqueous solution except being dissolved in, estimate smell in the mode identical with embodiment 3 by 5-aminolevulinic acid hydrochloride with 0.5g.The results are shown in the table 2.
Table 2
| The experimenter | A | B | C | | E |
| Embodiment | |||||
| 4 | 0 | 0 | 0 | 0 | 0 |
| Comparative example 2 | 1 | 0 | 1 | 1 | 0 |
Based on table 1 and 2, compare with the aqueous solution of 5-aminolevulinic acid hydrochloride, not smelling in the phosphatic aqueous solution of 5-amino-laevulic acid has any smell.Because the preparation process of 5-amino-laevulic acid aqueous phosphatic has been simplified the measure of necessary deodorization measure and non-corrosibility gas in the preparation process of the 5-aminolevulinic acid hydrochloride aqueous solution, so the former operation is more easy.In addition, compare with the solid of 5-aminolevulinic acid hydrochloride, the phosphatic solid of 5-amino-laevulic acid does not send any smell yet, therefore such as weigh and packing operation more convenient.
Embodiment 5
The acidity measurement of 5-amino-laevulic acid aqueous phosphatic:
Preparing concentration respectively is the phosphatic aqueous solution of 5-amino-laevulic acid of 1mM to 1000mM and the aqueous solution of 5-aminolevulinic acid hydrochloride, measures acidity at 25 ℃ of following use pH meters.The results are shown in Fig. 1.Obviously as can be seen, under same concentration conditions, the acidity of the phosphatic aqueous solution of 5-amino-laevulic acid is lower than the acidity of the aqueous solution of 5-aminolevulinic acid hydrochloride from Fig. 1.
The phosphatic irritation test of 5-amino-laevulic acid:
5 experimenters are placed directly in the 5-amino-laevulic acid phosphoric acid salt solid of 5mg on the tongue respectively and according to following standard, the phosphatic taste of 5-amino-laevulic acid that obtains among the embodiment 1 are estimated.The results are shown in the table 3.
Judgement criteria:
0: do not feel pungency.
1: pungency is arranged but pungency a little less than.
2: strong impulse is arranged.
Comparative example 3
Except the 5-aminolevulinic acid hydrochloride solid that uses 5mg, estimate the sense of taste in the mode identical with embodiment 6.The results are shown in the table 3.
Table 3
| | 1 | 1 | 1 | 1 | 1 |
| Comparative example 3 | 2 | 2 | 2 | 2 | 2 |
As shown in table 3, compare with the 5-aminolevulinic acid hydrochloride, do not feel that 5-amino-laevulic acid phosphoric acid salt has strong impulse.
Embodiment 7
The mutagenicity test (reverse mutation test) of microorganism (bacterium):
Test according to " Fine bacterium The becomes Iso Try test suddenly with い Ru Complex Kaesa " in " the microorganism The becomes Iso originality Try test base Quasi with the い Ru " (work in 1988 economizes No. the 77th, bulletin) (work in 1997 economize part revised edition of No. 67 promulgation of bulletin) and To Department Ru Try test method To つ い て such as " " new ruleization thing Quality (No. 1121002, the development of medicine food of promulgation on November 21st, 2003, No. the 2nd, 2003.11.13 System office, look forward to No. the 031121002nd, development) to reach the Ring guarantor.The 5-amino-laevulic acid phosphoric acid salt of 5% (weight/volume) is dissolved in the distilled water (being produced by Wako Pure Chemical Industries, Ltd.) makes solution, the 0.1M sodium phosphate buffer (pH 7.4) (S9mix that in the metabolic activity test, adds 0.5ml) that in this solution of 0.1ml, adds 0.5ml, the suspension that adds each test strain of 0.1ml again (uses the Salmonellatyphimurium TA 100 of histidine defect type, TA 98, the Escherichia coli WP2 uvrA (being provided by Japanese bioanalysis research centre) of TA 1535 and TA 1537 and tryptophane defective type is totally 5 strain bacteriums), 37 ℃ of pre-down cultivations 20 minutes, vibrate simultaneously then.After cultivating end, in nutrient solution, add 2.0ml top-layer agar (in advance it being incubated down at 45 ℃), and at the dull and stereotyped minimum medium of agar glucose upper berth layer.In the case, the sample with every kind of dosage is provided with 2 flat boards.But, the solvent control sample is provided with 3 flat boards (negative control).Cultivated 48 hours down at 37 ℃, whether the growth of observing each test strain then under stereoscopic microscope is suppressed, and the quantity of the bacterium colony of reverse mutation appears in counting.In measurement, use automatic bacterium colony analyser (CA-11: by System Science Co., Ltd. make) on the flat board of the diameter (internal diameter 84mm) of 86mm, the area in the diameter of about 80mm to be carried out count measurement, and use Personal Computer by carrying out area and proofread and correct and the counting loss correction calculating above-mentioned colony number.Yet when colony number is 1500 or more for a long time, the reliability of bacterium colony analyser can reduce automatically, therefore, under stereoscopic microscope 5 points on the flat board is carried out manual measurement, proofreaies and correct thereby mean value is carried out area.Carry out the dosage setting test as follows: use 5, the dosage of 000 μ g/ flat board is as the sample of maximal dose (this is the maximal dose of stipulating in the test guide), with common ratio 4 it is diluted the sample that obtains 7 kinds of dosage, these 7 kinds of samples are carried out the dosage setting test.The result is as follows: compare with the solvent control sample, no matter whether have S9mix in the dosage setting test sample, do not find all that in each bacterial strain recovery mutation colony number increases by 2 times or more situation; The growth of not finding bacterial strain is subjected to the situation that this analyte of 5-amino-laevulic acid phosphoric acid salt suppresses; Do not find to occur in the analyte sedimentary situation yet.
Use the sample of the dosage of 5,000 μ g/ flat boards, it is diluted the sample that obtains 5 kinds of dosage, also carry out this test with common ratio 2 as maximal dose (this is the maximal dose of stipulating in the above-mentioned guide).The result is as follows: compare with the solvent control sample, no matter whether the dosage setting test sample has metabolic activity, do not find all that in each bacterial strain recovery mutation colony number increases by 2 times or more situation (table 4).Confirm that thus 5-amino-laevulic acid phosphoric acid salt does not have sudden change and brings out ability.
Table 4
| Have or not the metabolism activation system | The phosphatic dosage of 5-amino-laevulic acid (μ g/ flat board) | Reverse mutation number (colony number/flat board) | |||||
| The base pair replacement type | Frame shift type | ||||||
| TA100 | TA1535 | WP2uvrA | TA98 | TA1537 | |||
| S9Mix(-) | Solvent control | 92 108 83 (94) | 77 12 (9) | 28 26 23 (26) | 23 24 18 (22) | 4 6 5 (5) | |
| 313 | 87 82 (85) | 10 10 (10) | 25 36 (31) | 9 7 (8) | 3 1 (2) | ||
| 625 | 84 95 (90) | 13 6 (10) | 27 28 (28) | 5 12 (9) | 1 5 (3) | ||
| 1250 | 84 114 (99) | 8 11 (10) | 28 33 (31) | 8 3 (6) | 4 2 (3) | ||
| 2500 | 76 82 (79) | 6 5 (6) | 25 28 (27) | 15 17 (16) | 2 2 (2) | ||
| 5000 | 115 99 (107) | 11 3 (7) | 25 36 (31) | 9 7 (8) | 4 1 (3) | ||
| S9Mix(+) | Solvent control | 112 143 90 (115) | 12 10 11 (11) | 29 30 27 (29) | 30 33 23 (29) | 8 8 9 (8) | |
| 313 | 146 122 (134) | 11 10 (11) | 31 17 (24) | 13 11 (12) | 2 7 (5) | ||
| 625 | 107 96 (102) | 13 6 (10) | 40 24 (32) | 9 9 (9) | 3 9 (6) | ||
| 1250 | 128 129 (129) | 10 14 (12) | 34 29 (32) | 14 27 (21) | 6 6 (6) | ||
| 2500 | 109 102 (106) | 8 8 (8) | 23 27 (25) | 7 15 (11) | 5 3 (4) | ||
| 5000 | 130 136 (133) | 6 8 (7) | 25 30 (28) | 20 21 (21) | 6 2 (4) | ||
| Positive control | Needn't use S9Mix | Title | AF-2 | NaN 3 | AF-2 | AF-2 | ICR-191 |
| Dosage (μ g/ flat board) | 0.01 | 0.5 | 0.01 | 0.1 | 1.0 | ||
| Colony number/flat board | 564 580 (572) | 203 216 (210) | 155 149 (152) | 575 560 (568) | 4068 3934(4001) | ||
| The essential S9Mix that uses | Title | AF-2 | NaN 3 | AF-2 | AF-2 | ICR-191 | |
| Dosage (μ g/ flat board) | 1.0 | 2.0 | 10.0 | 0.5 | 2.0 | ||
| Colony number/flat board | 1275 1184 (1230) | 137 138 (138) | 1230 1304 (1267) | 661 668 (665) | 257 267 (262) | ||
Remarks) positive control substance
AF-2:2-(2-furyl)-3-(5-nitro-2-furyl) acrylamide; NaN
3: sodiumazide; ICR-191:6-chloro-9-[3-(2-chloroethyl amino)-propyl group amino]-2-methoxyl group acridine dihydrochloride: the 2-AA:2-anthranylamine
Embodiment 8
The acute oral toxicity test:
This experimental evidence OECD guide No.423 " Grade method such as acute The mouth toxicity-acute toxicity " (employing on December 17 calendar year 2001) carries out.With 5-amino-laevulic acid phosphoric acid salt with the dosage of 300mg/kg body weight, the female rats (Sprague-Dawley CD species) of one group of 3 fasting is handled.In addition, with the dosage of 2000mg/kg body weight the female rats of other two or more sets fasting is handled.Carry out the continuous observation in 2 weeks after the administration.Found that: all rats all do not have death (table 5), the symptom that does not have whole body to poison, and generally speaking, the body weight of all rats all increases (table 6) to some extent, and by inference, acute oral 50% lethal quantity (LD50) is greater than 2, the 500mg/kg body weight.
Table 5
| Dosage mg/kg | Animal number | Per hour dead size of animal after the administration | The size of animal of death every day after the administration | ||||||||||||||||
| Female | 0.5 | 1 | 2 | 4 | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | 13 | 14 | |
| 300 | 1-0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| 1-1 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | |
| 1-2 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | |
| 2000 | 2-0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| 2-1 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | |
| 2-2 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | |
| 3-0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | |
| 3-1 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | |
| 3-2 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | |
Table 6
| Dosage mg/kg | Animal number | Body weight (g)/ | ||
| Female | ||||
| 0 | 7 | 14 | ||
| 300 | 1-0 | 205 | 242 | 263 |
| 1-1 | 214 | 262 | 287 | |
| 1-2 | 221 | 250 | 289 | |
| 2000 | 2-0 | 210 | 240 | 257 |
| 2-1 | 221 | 258 | 274 | |
| 2-2 | 180 | 221 | 247 | |
| 3-0 | 208 | 244 | 260 | |
| 3-1 | 222 | 259 | 275 | |
| 3-2 | 214 | 252 | 271 | |
Embodiment 9
The acute skin irritation test:
Test method according to OECD guide No.404 " urgency skin Skin pungency/corruption property touched Try test " (employing on July 17th, 1992) and EU council instruction 92/69/EEC B4 method acute toxicity (skin irritation) carry out this test.The 5-amino-laevulic acid phosphoric acid salt of 0.5g is dissolved in makes solution (pH 3.1) in the 0.5ml distilled water, use three New Zealand white rabbit (male) to test, with described solution the 2.5cm on every animal skin, unhairing but do not have on the square region of wound and be coated with 4 hours, in 1 hour, 24 hours, 48 hours and 72 hours, observe then.The result observed very slight erythema in 24 hours, but reached normal (table 7 and 8) after 48 hours.In addition, with solution (the 5-amino-laevulic acid phosphoric acid salt of 0.5g is dissolved in the 0.5ml distilled water and make) (pH 3.1) the 2.5cm on a New Zealand white rabbit (male) skin, unhairing but do not have on the square region of wound and be coated with 3 minutes or 1 hour, in 1 hour, 24 hours, 48 hours and 72 hours, observe then, also do not observe skin irritation (table 7 and 8) in this case.Because P.I.I. value (primary cutaneous stimulation index) is 0.5, confirm thus not belong to pungent outside the pungency classification of this salt in the GHS that existing United Nations is recommended.At this on the one hand, the 5-aminolevulinic acid hydrochloride of 0.5g is dissolved in the 0.5ml distilled water, makes comparative solution, and judge corrodibility, because the pH of this solution is 2.0 or lower, so do not test according to the OECD guide.
Table 7
| The skin reaction symptom | Observation period (hour) | The formation number of white rabbit numbering/described symptom | Amount to | ||
| No. 33 | No. 67 | No. 68 | |||
| Form erythema and dried scab | 1 | 1 | 1 | 1 | 3 |
| 24 | 1 | 1 | 1 | 3 | |
| 48 | 0 | 0 | 0 | 0 | |
| 72 | 0 | 0 | 0 | 0 | |
| Form oedema and edema | 1 | 0 | 0 | 0 | 0 |
| 24 | 0 | 0 | 0 | 0 | |
| 48 | 0 | 0 | 0 | 0 | |
| 72 | 0 | 0 | 0 | 0 | |
| Total with above-mentioned symptom after 72 hours after 24 hours forms number: 3 | |||||
| Primary stimulation index: 3/6=0.5 | |||||
Table 8
| The skin reaction symptom | Observation period (hour) | The formation number of white rabbit numbering/described symptom | |
| No. 33 | |||
| Be coated with 3 minutes | Be coated with 1 hour | ||
| Form erythema and dried scab | 1 | 0 | 0 |
| 24 | 0 | 0 | |
| 48 | 0 | 0 | |
| 72 | 0 | 0 | |
| Form oedema and edema | 1 | 0 | 0 |
| 24 | 0 | 0 | |
| 48 | 0 | 0 | |
| 72 | 0 | 0 | |
The animal cuticle permeability test:
Unit with dialysis (useful area 1.13m is used in this test
2, as shown in Figure 2), be that 6.8 physiological saline stirs and remain on 37 ℃ to the pH of 17ml in the receptive layers.To put into film filter through pretreated pigskin holostrome (epidermis+dermis), and it will be arranged on the unit with dialysis.The 5-amino-laevulic acid aqueous phosphatic 0.5ml that in donor layer, adds 1mM.In receptive layers, after the preset time section collects the solution of 0.2ml, replenish this layer with fresh physiological saline.Then, collected specimens or the reference liquid of 0.05ml are mixed with the liquid A (mixing solutions that contains methyl ethyl diketone/ethanol/water=15/10/75 (volume/volume/volume) of 4g sodium-chlor in 1 liter) of 3.5ml and the liquid B (water is diluted to 1 liter of prepared solution with the formalin of 85ml) of 0.45ml, and to mixed solution heat treated 30 minutes, after 30 minutes, use water cooling.Subsequently, (analysis condition is: flow velocity 1.0ml/ minute with HPLC, 25 ℃ of temperature, use excitation wavelength to be 473nm and wavelength of fluorescence fluorimetric detector as 363nm, the mixing solutions that uses methyl alcohol/2.5% acetic acid aqueous solution=40/60 (volume/volume) is as eluant, and use Wakosil-II5C18HG, 4.6m φ * 150mm are as post) measure.Obtain the concentration of each sample by the peak area of reference liquid.
Then, use onion epidermis to replace pigskin to carry out same test, but change the concentration of 5-amino-laevulic acid aqueous phosphatic into 0.1mM.The result is shown in Fig. 3 and 4.Be appreciated that by Fig. 3 and 4 5-aminolevulinic acid hydrochloride and 5-amino-laevulic acid phosphoric acid salt show similar perviousness in pigskin and onion epidermis.
Comparative example 4
Except replace 5-amino-laevulic acid phosphoric acid salt with the 5-aminolevulinic acid hydrochloride, measure perviousness in the mode identical with embodiment 10.
Can confirm by above-mentioned test, though when the 5-aminolevulinic acid hydrochloride directly imposes on skin, can produce pungency, but 5-amino-laevulic acid phosphoric acid salt does not produce skin irritation (shown in embodiment 9), and they have identical perviousness to skin, show thus: 5-amino-laevulic acid phosphoric acid salt is a kind of in some medical treatment (optical dynamic therapy and light power diagnosis) and plant application facet, than the more useful salt of 5-aminolevulinic acid hydrochloride.
Embodiment 11
Produce the test of silver nitride precipitation thing:
In the ion exchanged water of 10ml, 5-amino-laevulic acid phosphoric acid salt and the 0.5g Silver Nitrate of dissolving 0.5g leave standstill mixture 5 minutes, and observe the state of liquid.Do not find to produce throw out.In this connection, 5-aminolevulinic acid hydrochloride and the 0.5g Silver Nitrate of 0.5g is dissolved in the ion exchanged water of 10ml, mixture was left standstill 5 minutes, and observe the state of liquid.Discovery has produced throw out.
The test that apple is painted
Predetermined concentration shown in the 5-amino-laevulic acid phosphoric acid salt following table that obtains among the embodiment 1 is dissolved in the ion exchanged water.(by " the ア プ ロ-チ BI " of ball and the manufacturing of produce Co., Ltd.) joins in the aforesaid liquid with the concentration of 0.1 weight % with spreader.Regulate pH with phosphoric acid.
Except above-mentioned 5-amino-laevulic acid phosphoric acid salt is replaced to the 5-aminolevulinic acid hydrochloride, and the phosphoric acid that will regulate pH replace with the hydrochloric acid, prepare the aqueous solution of 5-aminolevulinic acid hydrochloride in an identical manner.
The liquid of preparation like this is sprayed onto on 3 major branches with 2 liters/ratio, and wherein the young fruit of " Fuji " apple has been born fruit but has not also been become redness (September 15).After about 2 months (November 6), the results apple is also checked their colourity.The colourimeter CR-200 that makes with MINOLTA CAMERA Co., Ltd. measures colourity.The results are shown in the table 9.
Table 9
| Scheme | Colourity (L, a, b value) | |||
| L | a | b | ||
| 5-amino-laevulic acid phosphoric acid salt | 100ppm(pH 5.0) | 42.37 | 27.45 | 14.54 |
| 200ppm(pH 5.4) | 42.43 | 31.06 | 14.63 | |
| 200ppm(pH 2.0) | - | - | - | |
| The 5-aminolevulinic acid hydrochloride | 100ppm(pH 5.0) | 42.28 | 25.96 | 14.72 |
| 200ppm(pH 4.8) | 42.34 | 30.92 | 14.41 | |
| 200ppm(pH 2.0) | - | - | - | |
| Be untreated | 5-amino-laevulic acid (0) | 42.03 | 25.16 | 14.66 |
"-" expression finds on the fruit a large amount of spots are arranged.
In the Lab value shown in the table 9, L represents brightness, and a represents redness, and b represents yellow.Therefore, the high more redness that means of a value is dense more.Spray red ratio in the 5-amino-laevulic acid phosphoric acid salt situation and spray denseer in the 5-aminolevulinic acid hydrochloride situation.
Embodiment 13
The plant activation effect:
The 600g andosoil is contained in the porcelain jar that internal diameter is 12cm, prepare 12 jars altogether, plant the Herba Commelinae that the long spiderwort to the 15cm height of 1 strain is arranged in each jar, place it under 20 ℃ the isoperibol, use processing with the cauline leaf that following treatment solution carries out once a day.Observe the situation of blade after 21 days.The result is summarised in the table 10.
Table 10
| Concentration (ppm) | 0 | 1 | 2 | |
| 5-amino-laevulic acid phosphoric acid salt is dissolved in the tap water and makes | 1 | 6 strains | 5 strains | 1 |
| 10 | 7 strains | 3 | 2 strains | |
| 100 | 6 | 4 | 2 strains | |
| The 5-aminolevulinic acid hydrochloride is dissolved in the tap water and makes | 1 | 2 strains | 7 strains | 3 |
| 10 | 5 strains | 5 | 2 strains | |
| 100 | 5 | 4 strains | 3 strains | |
| Sodium phosphate is dissolved in the tap water and makes | 1 | 4 strains | 3 strains | 5 |
| 10 | 2 | 4 | 6 strains | |
| 100 | 3 | 2 strains | 7 strains | |
| Tap water | 3 strains | 3 | 6 strains |
Judgement criteria:
0: do not find to have on the leaf surface any unusual
1: the zone of finding to have on the leaf surface flavescence
2: finding has downright bad zone on the leaf surface
Based on the result shown in the table 10, find that the phosphatic plant activation effect of 5-amino-laevulic acid is similar to or greater than the effect of 5-aminolevulinic acid hydrochloride.
The plant growth regulating effect:
Rice paddy seed (Akinishiki) is soaked diel in Benlate (being produced by Sumika Takeda Engei Co., Ltd.) (200 times) aqueous solution, under 30 ℃, dark condition, cultivate then with stratification.Select the seed of broken chest phase, 10 seeds are held under the arm in the groove (groove is cut into cutter knife) of polyethylene foamed plate with tweezers, this plate is swum in the petri diss (the concentration 5-amino-laevulic acid phosphate solution as shown in table 11 respectively of 150ml is housed in this culture dish) of high type, and under the condition of 5,000 lux continuous lights, cultivated 24 hours down at 25 ℃.The repeat number of each concentration is set at 3.Observe after three days, measure the length of the root of the length of first leaf sheath in each scheme and seed, calculate them with the ratio of the scheme that is untreated and calculate mean value.The results are shown in the table 11.
Table 11
| The compound title | Concentration (ppm) | The length of first leaf sheath (%) | The length of the root of seed (%) |
| 5-amino-laevulic acid phosphoric acid salt | 1 10 100 | 102 106 101 | 106 108 101 |
| The 5-aminolevulinic acid hydrochloride | 1 10 100 | 107 101 98 | 103 96 109 |
| Be untreated | 100 | 100 |
5-amino-laevulic acid phosphoric acid salt shows the effect of the promotion plant-growth similar or stronger to the 5-aminolevulinic acid hydrochloride.
Embodiment 15
Salt tolerance is improved effect:
To internal diameter is the dryland soil of filling 600g in the porcelain jar (not having water vent) of 12cm, sowing 7-8 grain cotton seeds (kind: M-5Acala), will cover the thick soil of 1cm on the seed, and seed is grown in the greenhouse.Subsequently, carry out Routine Management, in the time of the tender leaf unfolded, preparation contains the spreader (Neoesterin of testing compound (its concentration is as shown in table 12 respectively) and 0.05% (volume/volume), make by Kumiai chemical industry Co., Ltd.) the salt tolerance activator, and its volumetric usage with per 10 ares 100 liters is applied on the cauline leaf.Every kind of testing compound is configured to the concentration shown in the table 12.After four days, the amount of sodium-chlor with the 0-1.5% that is equivalent to soil weight shown in the table 12 is dissolved in the 30ml water, and is added drop-wise in the soil.Proceed conventional cultivation again, check after 23 days.Carry out described inspection by visual inspection, and estimate according to the result of following 6 ranks to salt damage.The results are shown in the table 12.
Estimate rank:
0: do not observe salt damage fully.
1: observe very slight salt damage.
2: observe slighter salt damage.
3: observe tangible salt damage.
4: observe serious salt damage.
5: because salt damage causes plant materials withered.
Table 12
| Test compound [concentration of treatment (ppm)] | NaCl treatment capacity/soil weight (weight %) | |||||
| 0 | 0.5 | 0.75 | 1 | 1.5 | ||
| Comparative example | Be untreated | 0 | 1 | 2 | 3 | 5 |
| Embodiment | 5-amino-laevulic acid phosphoric acid salt (10) | 0 | 0 | 1 | 2 | 3 |
| 5-amino-laevulic acid phosphoric acid salt (30) | 0 | 0 | 0 | 1 | 2 | |
| 5-amino-laevulic acid phosphoric acid salt (100) | 0 | 1 | 2 | 3 | 4 | |
| 5-amino-laevulic acid phosphoric acid salt (300) | 0 | 1 | 1 | 2 | 3 | |
| Comparative example | 5-aminolevulinic acid hydrochloride (10) | 0 | 1 | 1 | 1 | 2 |
| 5-aminolevulinic acid hydrochloride (30) | 0 | 1 | 2 | 3 | 3 | |
| 5-aminolevulinic acid hydrochloride (100) | 0 | 1 | 1 | 2 | 3 | |
| 5-aminolevulinic acid hydrochloride (300) | 0 | 0 | 1 | 1 | 2 | |
As shown in table 12,5-amino-laevulic acid phosphoric acid salt shows the salt tolerance similar or stronger to the 5-aminolevulinic acid hydrochloride and improves effect.
When adopting ion chromatographic column under the following conditions, when the concentration of the chlorion in the 5-amino-laevulic acid aqueous phosphatic that uses in the foregoing description was measured, the concentration of the chlorion in each sample all was equal to or less than limit of detection (0.1ppm).
Measuring condition is as follows: A: separator column (IonPacAS12A that is made by Japanese Dionex Co., Ltd.), B: guard column (the IonPac AG12A that is made by Japanese Dionex Co., Ltd.), C: eluant (contains Na
2CO
3And NaHCO
3(Na
2CO
3: 3.0mmol/l, NaHCO
3: aqueous solution 0.5mmol/l)), D: flow velocity (1.5ml/ minute), E: suppressor (ASRS (regeneration type, current value are 50mA)), F: sample size (25 μ l), G: constant temperature oven temperature (35 ℃), and H: detector (electrical conductivity detector).
Embodiment 16
The preparation of 5-amino-laevulic acid nitrate
In the strong-acid ion exchange resin of 180ml (AMBERLITE IR 120B Na is produced by the Japanese Organo Co., Ltd.) post of packing into.Used ion exchange resin is to have passed through the salt acid treatment and the ion exchange resin that changes into the hydrogen ion type from sodium-ion type.Then, the 5-aminolevulinic acid hydrochloride of 36.00g (214mmol) is dissolved in the ion exchanged water of 1800ml, and this solution is crossed post, then the ion exchanged water of 1000ml is crossed post.Then, with the slow post of crossing of the ammoniacal liquor of 1N, collect 594ml xanchromatic elutriant.60% the nitric acid (HNO that the elutriant collected is added 33ml
3442mmol), and use vaporizer to concentrate.In concentrated solution, add the acetone of 400ml, and use the agitator vigorous stirring, mixture was left standstill under 4 ℃ 16 hours.Suction strainer reclaims the solid of being separated out, and washs with the methyl acetate of 500ml.With gained solid drying under reduced pressure 12 hours, obtain the target substance of 31.09g (160mmol).Its physical performance data is as follows.
Fusing point: 114 ℃
1H-NMR(D
2O，400MHz)δppm：2.75(t，2H，CH
2)，2.93(t，2H，CH
2)，4.17(s，2H，CH
2)
13C-NMR(D
2O，100MHz)δppm：30(CH
2)，37(CH
2)，50(CH
2)，180(CO)，207(COO)
C
5H
9NO
3HNO
3The ultimate analysis data:
Calculated value: C 30.93%; H 5.19%; N 14.43%
Measured value: C 30.1%; H 5.2%; N 14.7%
The NO that obtains by ion chromatography
3 -Content
Calculated value: 31.94%
Measured value: 31%
Ion chromatography condition: separator column: the IonPacAS12A that makes by Japanese Dionex Co., Ltd.; Eluant: contain Na
2CO
3And NaHCO
3(Na
2CO
3: 3.0mmol/l, NaHCO
3: aqueous solution 0.5mmol), flow velocity: 1.5ml/ minute, sample size: 25 μ l, column temperature: 35 ℃, detector: electrical conductivity detector.
Embodiment 17
The odorimetry of 5-amino-laevulic acid nitrate
5 experimenters directly hear the aqueous solution (mixed solution of post elutriant and nitric acid) and the solid smell thereof of the 5-amino-laevulic acid nitrate of preparation among the embodiment 16, and estimate their smell in the mode identical with embodiment 3.The results are shown in the table 13.
Comparative example 5
Except the aqueous solution and solid thereof that use the 5-aminolevulinic acid hydrochloride, estimate smell in the mode identical with embodiment 17.In this connection, the aqueous solution of 5-aminolevulinic acid hydrochloride is to use 5-aminolevulinic acid hydrochloride solid, hydrochloric acid and ion exchanged water preparation, wherein, make 5-amino-laevulic acid and chlorion volumetric molar concentration respectively with embodiment 16 in 5-amino-laevulic acid nitrate aqueous solution in the 5-amino-laevulic acid and the volumetric molar concentration of nitrate ion identical.The results are shown in the table 13.
Table 13
| The experimenter | A | B | C | D | E | |
| Embodiment 17 | The | 0 | 0 | 0 | 0 | 0 |
| Solid | 0 | 0 | 0 | 0 | 0 | |
| Comparative example 5 | The | 2 | 2 | 2 | 2 | 2 |
| Solid | 1 | 1 | 1 | 1 | 1 | |
Embodiment 18
Make in the 1ml water the aqueous solution except being dissolved in, estimate smell in the mode identical with embodiment 17 by 5-amino-laevulic acid nitrate with 0.5g.The results are shown in the table 14.
Comparative example 6
Make in the 1ml water the aqueous solution except being dissolved in, estimate smell in the mode identical with embodiment 17 by 5-aminolevulinic acid hydrochloride with 0.5g.The results are shown in the table 14.
Table 14
| The experimenter | A | B | C | D | E |
| Embodiment 18 | 0 | 0 | 0 | 0 | 0 |
| Comparative example 6 | 1 | 0 | 1 | 1 | 0 |
Table 13 and 14 explanations are compared with the aqueous solution of 5-aminolevulinic acid hydrochloride, and not smelling in the aqueous solution of 5-amino-laevulic acid nitrate has any smell.Because the preparation process of 5-amino-laevulic acid nitrate aqueous solution has been simplified the measure of necessary deodorization measure and non-corrosibility gas in the preparation process of the 5-aminolevulinic acid hydrochloride aqueous solution, so the former operation is more easy.In addition, compare with the solid of 5-aminolevulinic acid hydrochloride, the solid of 5-amino-laevulic acid nitrate does not send any smell yet, thus such as weigh and packing operation more convenient.
Embodiment 19
Produce the test of silver nitride precipitation thing:
In the ion exchanged water of 10ml, 5-amino-laevulic acid nitrate and the 0.5g Silver Nitrate of dissolving 0.5g leave standstill mixture 5 minutes, and observe the state of liquid.Do not find to produce throw out.
In this connection, 5-aminolevulinic acid hydrochloride and the 0.5g Silver Nitrate of 0.5g is dissolved in the ion exchanged water of 10ml, mixture was left standstill 5 minutes, and observe the state of liquid.Discovery has produced throw out.
The plant activation effect:
The 600g dryland soil is contained in the porcelain jar that internal diameter is 12cm, sows 12 radish seeds then, will cover the thick soil of 5mm on the seed, and seed is grown in the greenhouse.Use processing with the cauline leaf that following treatment liq is carried out once a day.Observe the situation of blade after 21 days.The result is summarised in the table 15.Judgement criteria is identical with embodiment's 13.
Table 15
| Concentration (ppm) | 0 | 1 | 2 | |
| 5-amino-laevulic acid nitrate is dissolved in the tap water and makes | 1 | 5 strains | 5 | 2 |
| 10 | 6 strains | 5 strains | 1 strain | |
| 100 | 4 | 6 | 2 strains | |
| The 5-aminolevulinic acid hydrochloride is dissolved in the tap water and makes | 1 | 4 | 6 | 2 |
| 10 | 4 | 4 | 4 strains | |
| 100 | 3 strains | 5 | 4 strains | |
| SODIUMNITRATE is dissolved in the tap water and makes | 1 | 2 | 6 | 4 |
| 10 | 2 | 4 | 6 strains | |
| 100 | 2 strains | 5 strains | 5 strains | |
| Tap water | 1 | 4 strains | 7 strains |
Found by table 15: the plant activation effect of 5-amino-laevulic acid nitrate is similar or stronger to the 5-aminolevulinic acid hydrochloride.
Embodiment 21
The test that apple is painted
The 5-amino-laevulic acid nitrate that obtains among the embodiment 16 is dissolved in the ion exchanged water with the predetermined concentration shown in the table 16.(by " the ア プ ロ-チ BI " of ball and the manufacturing of produce Co., Ltd.) joins in the aforesaid liquid with the concentration of 0.1 weight % with spreader.Regulate pH with nitric acid.
Except above-mentioned 5-amino-laevulic acid nitrate being replaced to the 5-aminolevulinic acid hydrochloride and nitric acid being replaced with the hydrochloric acid, prepare solution in the same manner as described above.
The liquid of preparation like this is sprayed on 3 major branches with 2 liters/ratio, and wherein the young fruit of " Fuji " apple has been born fruit but has not also been become redness (September 15).After about 2 months (November 6), the results apple is also checked their colourity.The colourimeter CR-200 that makes with MINOLTA CAMERA Co., Ltd. measures colourity.The results are shown in the table 16.
Table 16
| Scheme | Colourity (L, a, b value) | |||
| L | a | b | ||
| 5-amino-laevulic acid nitrate | 100 ppm by weight (pH 5.0) | 42.41 | 26.51 | 14.46 |
| 200 ppm by weight (pH 4.9) | 42.47 | 31.00 | 14.72 | |
| 200 ppm by weight (pH 2.0) | - | - | - | |
| The 5-aminolevulinic acid hydrochloride | 100 ppm by weight (pH 5.0) | 42.28 | 25.96 | 14.72 |
| 200 ppm by weight (pH 4.8) | 42.34 | 30.92 | 14.41 | |
| 200 ppm by weight (pH 2.0) | - | - | - | |
| Be untreated | 5-amino-laevulic acid (0 ppm by weight) | 42.03 | 25.16 | 14.66 |
"-" expression finds on the fruit a large amount of spots are arranged.
In the Lab value shown in the table 16, L represents brightness, and a represents redness, and b represents yellow.Therefore, the high more redness that means of a value is dense more.It is denseer to spray the redness that the red ratio in the 5-amino-laevulic acid nitrate situation sprays in the 5-aminolevulinic acid hydrochloride situation.
Embodiment 22
Planktonic cultivation:
With concentration is that the 5-amino-laevulic acid nitrate of 1mM (194ppm) joins in the sterile medium shown in the table 17 (nutrient solution component) of 100ml, Chlorella sp. is inoculated in this nutrient solution, and under 30 ℃ and aerobic and dark condition, in reciprocating type shaking table, cultivate, measure cell count (OD 660) then.
With concentration is that the 5-aminolevulinic acid hydrochloride of 1mM (168ppm) joins in the sterile medium shown in the table 17 (nutrient solution component) of 100ml, Chlorella sp. is inoculated in this nutrient solution, and under 30 ℃ and aerobic and dark condition, in reciprocating type shaking table, cultivate, measure cell count (OD 660) then.
Chlorella sp. is inoculated in the sterile medium shown in the table 17 (nutrient solution component) of 100ml, and under 30 ℃ and aerobic and dark condition, in reciprocating type shaking table, cultivates, measure cell count (OD 660) then.
Table 17
| The nutrient solution component | mg/l |
| NaNO 3 | 250 |
| CaCl 2·2H 2O | 25 |
| MgSO 4·7H 2O | 75 |
| K 2HPO 4 | 75 |
| KH 2PO 4 | 175 |
| NaCl | 25 |
| NaSiO 2·9H 2O | 50 |
| | 50 |
| FeSO 4·7H 2O | 5 |
| H 3BO4 | 10 |
| ZnSO 4·7H 2O | 10 |
| MnCl 2·4H 2O | 1.5 |
| (NH 4) 6Mo 7O 24·4H 2O | 1 |
| CuSO 4·5H 2O | 1.5 |
| Co(NO 3) 3·6H 2O | 0.5 |
Table 18
Result's (cell count: OD 660nm)
| Incubation time (my god) | ||||
| | 0 | 1 | 2 | 3 |
| 5-amino-laevulic acid nitrate | 1.8 | 6.5 | 12.6 | 15.0 |
| The 5-aminolevulinic acid hydrochloride | 1.8 | 6.3 | 12.5 | 14.8 |
| Do not have | 1.8 | 6.2 | 11.9 | 14.0 |
By the result of table 18 obviously as seen, 5-amino-laevulic acid nitrate shows the effect identical with the 5-aminolevulinic acid hydrochloride.
When adopting ion chromatographic column under the following conditions, when the concentration of the chlorion in the 5-amino-laevulic acid nitrate aqueous solution that uses in the foregoing description was measured, the concentration of the chlorion in each sample all was equal to or less than limit of detection (0.1ppm).
Measuring condition is as follows: A: separator column (IonPacAS12A that is made by Japanese Dionex Co., Ltd.), B: guard column (the IonPac AG12A that is made by Japanese Dionex Co., Ltd.), C: eluant (contains Na
2CO
3And NaHCO
3(Na
2CO
3: 3.0mmol/l, NaHCO
3: aqueous solution 0.5mmol/l)), D: flow velocity (1.5ml/ minute), E: suppressor (ASRS (regeneration type, current value are 50mA)), F: sample size (25 μ l), G: constant temperature oven temperature (35 ℃), and H: detector (electrical conductivity detector).
Embodiment 23
The preparation of 5-amino-laevulic acid tosilate
In the strong-acid ion exchange resin of 180ml (AMBERLITE IR 120B Na is produced by the Japanese Organo Co., Ltd.) post of packing into.Used ion exchange resin is to have passed through the salt acid treatment and the ion exchange resin that changes into the hydrogen ion type from sodium-ion type.Then, the 5-aminolevulinic acid hydrochloride of 36.00g (215mmol) is dissolved in the ion exchanged water of 1800ml, and this solution is crossed post, then the ion exchanged water of 1000ml is crossed post.Then, with the slow post of crossing of the ammoniacal liquor of 1N, collect 555ml xanchromatic elutriant.The elutriant of collecting is mixed with the tosic acid monohydrate of 81.72g (430mmol), and use vaporizer to concentrate.In concentrated solution, add the acetone of 400ml, and use the agitator vigorous stirring, under 4 ℃, left standstill 16 hours then.Suction strainer reclaims the solid separated out, and with the washing with acetone of 400ml.With gained solid drying under reduced pressure 12 hours, obtain the target substance of 47.78g (158mmol).Its physical performance data is as follows.
Fusing point: 186 ℃
1H-NMR (D
2O, 400MHz) δ ppm:2.38 (s, 3H, CH
3), 2.67 (t, 2H, CH
2), 2.84 (t, 2H, CH
2), 4.10 (s, 2H, CH
2) 7.34 (d, 2H, ring H), 7.69 (d, 2H, ring H)
13C-NMR (D
2O, 100MHz) δ ppm:23 (CH
3), 30 (CH
2), 37 (CH
2), 50 (CH
2), 128 (ring C), 132 (ring C), 142 (ring C), 145 (ring C), 180 (CO), 207 (COO)
C
5H
9NO
3C
7H
8SO
3The ultimate analysis data:
Calculated value: C 47.52%; H 5.65%; N 4.62%
Measured value: C 47.4%; H 5.6%; N 4.6%
Embodiment 24
The odorimetry of 5-amino-laevulic acid tosilate
5 experimenters directly hear the aqueous solution (mixed solution of post elutriant and tosic acid) and the solid smell thereof of the 5-amino-laevulic acid tosilate of preparation among the embodiment 23, and estimate their smell in the mode identical with embodiment 3.The results are shown in the table 19.
Comparative example 7
Except the aqueous solution and solid thereof that use the 5-aminolevulinic acid hydrochloride, estimate smell in the mode identical with embodiment 24.In this connection, the aqueous solution of 5-aminolevulinic acid hydrochloride is to use 5-aminolevulinic acid hydrochloride solid, hydrochloric acid and ion exchanged water preparation, wherein, make 5-amino-laevulic acid and chlorion volumetric molar concentration respectively with embodiment 23 in 5-amino-laevulic acid tosic acid salt brine solution in the 5-amino-laevulic acid and the volumetric molar concentration of tosic acid radical ion identical.The results are shown in the table 19.
Table 19
| The experimenter | A | B | C | D | E | |
| Embodiment 24 | The | 0 | 0 | 0 | 0 | 0 |
| Solid | 0 | 0 | 0 | 0 | 0 | |
| Comparative example 7 | The | 2 | 2 | 2 | 2 | 2 |
| Solid | 1 | 1 | 1 | 1 | 1 | |
Embodiment 25
Make in the 1ml water the aqueous solution except being dissolved in, estimate smell in the mode identical with embodiment 24 by 5-amino-laevulic acid tosilate with 0.5g.The results are shown in the table 20.
(comparative example 8)
Make in the 1ml water the aqueous solution except being dissolved in, estimate smell in the mode identical with embodiment 24 by 5-aminolevulinic acid hydrochloride with 0.5g.The results are shown in the table 20.
Table 20
| The experimenter | A | B | C | D | E |
| Embodiment 25 | 0 | 0 | 0 | 0 | 0 |
| Comparative example 8 | 1 | 0 | 1 | 1 | 0 |
Table 19 and 20 explanations are compared with the aqueous solution of 5-aminolevulinic acid hydrochloride, and not smelling in the aqueous solution of 5-amino-laevulic acid tosilate has any smell.Because the preparation process of 5-amino-laevulic acid tosic acid salt brine solution has been simplified the measure of necessary deodorization measure and non-corrosibility gas in the preparation process of the 5-aminolevulinic acid hydrochloride aqueous solution, so the former operation is more easy.In addition, compare with the solid of 5-aminolevulinic acid hydrochloride, the solid of 5-amino-laevulic acid tosilate does not send any smell yet, thus such as weigh and packing operation more convenient.
(embodiment 26)
The thermotolerance of crystalline state:
Use the fusing point instrument to measure fusing point.
Table 21
| Fusing point (℃) | |
| 5-amino-laevulic acid tosilate | 186 |
| The 5-aminolevulinic acid hydrochloride | 156 |
Shown in table 21, the solid-state ability of maintenance of 5-amino-laevulic acid tosilate is better than the 5-aminolevulinic acid hydrochloride.
Embodiment 27
The sterilization decomposition run:
At first, to the 5-amino-laevulic acid tosilate of 50mg or 5-aminolevulinic acid hydrochloride carry out heat sterilization (121 ℃, 20 minutes, 1.5kgf/cm
2).Confirm that the weight before and after the sterilization does not change, adopt the method for describing in the document (Clin.Chem., 36/8,1494 (1990)) then, the resolution of 5-amino-laevulic acid before and after sterilization tested.The results are shown in the table 22.
Table 22
| Resolution (%) | |
| 5-amino-laevulic acid tosilate | 2.7 |
| The 5-aminolevulinic acid hydrochloride | 6.6 |
Shown in table 22, find that the resolution of 5-amino-laevulic acid tosilate is lower than the resolution of 5-aminolevulinic acid hydrochloride through after the heat sterilising treatment.
Embodiment 28
Produce the test of silver nitride precipitation thing:
In the ion exchanged water of 10ml, 5-amino-laevulic acid tosilate and the 0.5g Silver Nitrate of dissolving 0.5g leave standstill mixture 5 minutes, and observe the state of liquid.Do not find to produce throw out.
In this connection, 5-aminolevulinic acid hydrochloride and the 0.5g Silver Nitrate of 0.5g is dissolved in the ion exchanged water of 10ml, mixture was left standstill 5 minutes, and observe the state of liquid.Discovery has produced throw out.
Embodiment 29
The plant activation effect:
The 600g dryland soil is contained in the porcelain jar that internal diameter is 12cm, sows 12 radish seeds then, will cover the thick soil of 5mm on the seed, and seed is grown in the greenhouse.Use processing with the cauline leaf that following treatment liq is carried out once a day.Observe the situation of blade after 21 days.The result is summarised in the table 23.Judgement criteria is identical with embodiment 13.
Table 23
| Concentration (ppm) | 0 | 1 | 2 | |
| 5-amino-laevulic acid tosilate is dissolved in the tap water and makes | 1 | 6 strains | 3 strains | 3 |
| 10 | 5 strains | 5 | 2 strains | |
| 100 | 7 | 4 strains | 1 strain | |
| The 5-aminolevulinic acid hydrochloride is dissolved in the tap water and makes | 1 | 4 | 6 | 2 |
| 10 | 4 | 4 | 4 strains | |
| 100 | 3 strains | 5 | 4 strains | |
| Tosic acid is dissolved in the tap water and makes | 1 | 1 | 2 strains | 9 |
| 10 | 1 | 2 strains | 9 strains | |
| 100 | 0 strain | 3 strains | 9 strains | |
| Tap water | 1 | 4 strains | 7 strains |
Find that by table 23 the plant activation effect of 5-amino-laevulic acid tosilate is similar or stronger to the 5-aminolevulinic acid hydrochloride.
Embodiment 30
The test that apple is painted
The 5-amino-laevulic acid tosilate that obtains among the embodiment 23 is dissolved in the ion exchanged water with the predetermined concentration shown in the table 24.(by " the ア プ ロ-チ BI " of ball and the manufacturing of produce Co., Ltd.) joins in the aforesaid liquid with the concentration of 0.1 weight % with spreader.Regulate pH with tosic acid.
Except above-mentioned 5-amino-laevulic acid tosilate being replaced to the 5-aminolevulinic acid hydrochloride and tosic acid being replaced with the hydrochloric acid, prepare solution in the same manner as described above.
The liquid of preparation like this is sprayed onto on 3 major branches with 2 liters/ratio, and wherein the young fruit of " Fuji " apple has been born fruit but has not also been become redness (September 15).After about 2 months (November 6), the results apple is also checked their colourity.The colourimeter CR-200 that makes with MINOLTA CAMERA Co., Ltd. measures colourity.The results are shown in the table 24.
Table 24
| Scheme | Colourity (L, a, b value) | |||
| L | a | b | ||
| 5-amino-laevulic acid tosilate | 100ppm(pH 5.0) | 42.39 | 26.44 | 14.69 |
| 200ppm(pH 4.9) | 42.36 | 30.93 | 14.34 | |
| 200ppm(pH 2.0) | - | - | - | |
| The 5-aminolevulinic acid hydrochloride | 100ppm(pH 5.0) | 42.28 | 25.96 | 14.72 |
| 200ppm(pH 4.8) | 42.34 | 30.92 | 14.41 | |
| 200ppm(pH 2.0) | - | - | - | |
| Be untreated | 5-amino-laevulic acid (0) | 42.03 | 25.16 | 14.66 |
"-" expression finds on the fruit a large amount of spots are arranged.
In the Lab of table 24 value, L represents brightness, and a represents redness, and b represents yellow.Therefore, the high more redness that means of a value is dense more.Spray red ratio in the situation of 5-amino-laevulic acid tosilate and spray denseer in the 5-aminolevulinic acid hydrochloride situation.
When adopting ion chromatographic column under the following conditions, when the concentration of the chlorion in the 5-amino-laevulic acid tosic acid salt brine solution that uses in the foregoing description was measured, the concentration of the chlorion in each sample all was equal to or less than limit of detection (0.1ppm).
Measuring condition is as follows: A: separator column (IonPacAS12A that is made by Japanese Dionex Co., Ltd.), B: guard column (the IonPac AG12A that is made by Japanese Dionex Co., Ltd.), C: eluant (contains Na
2CO
3And NaHCO
3(Na
2CO
3: 3.0mmol/l, NaHCO
3: aqueous solution 0.5mmol/l)), D: flow velocity (1.5ml/ minute), E: suppressor (ASRS (regeneration type, current value are 50mA)), F: sample size (25 μ l), G: constant temperature oven temperature (35 ℃), and H: detector (electrical conductivity detector).
Though the present invention has been described in detail with reference to embodiment, to those skilled in the art, under the situation that does not deviate from the spirit and scope of the present invention, obviously can make various changes and modifications.
The Japanese patent application (Japanese patent application No. 2004-099670) that the application submitted to based on March 30th, 2004, the Japanese patent application (Japanese patent application No. 2004-099671) that on March 30th, 2004 submitted to, the Japanese patent application (Japanese patent application No. 2004-099672) that on March 30th, 2004 submitted to, the Japanese patent application (Japanese patent application No. 2004-345661) that on November 30th, 2004 submitted to, the Japanese patent application (Japanese patent application No. 2005-051216) that on February 25th, 2005 submitted to, the Japanese patent application (Japanese patent application No. 2005-051217) that on February 25th, 2005 submitted to, with the Japanese patent application (Japanese patent application No. 2005-051218) that on February 25th, 2005 submitted to, the full content of above-mentioned patent application is incorporated this paper into way of reference.
Industrial applicibility
5-ALA salt of the present invention is a kind of material of convenient operation, this is because it can not discharge niff or irritating smell, and, this salt pair skin and tongue show the characteristic of low irritant, it also has excellent permeability to skin etc., and the composition that therefore contains this salt can be used as optical dynamic therapy or the used reagent of light power diagnosis. And, to compare with the 5-ALA hydrochloride, 5-ALA salt of the present invention also has high decomposition temperature and excellent heat resistance. Preparation in accordance with the present invention can be prepared 5-ALA salt of the present invention easily and effectively. In addition, because 5-ALA salt of the present invention has low chlorine ion concentration when forming the aqueous solution, therefore the infringement that is caused by chlorine can appear hardly when being applied to plant.
Claims (21)
1. 5-aminolevulinic acid that belongs to the aminolevulinic acid class, wherein this salt is to be selected from least a in phosphoric acid salt, nitrate and the sulfonate.
2. 5-aminolevulinic acid according to claim 1, this salt are the amino-laevulic acid phosphoric acid salt by following formula (I) expression:
HOCOCH
2CH
2COCH
2NH
2·HOP(O)(OR
1)
n(OH)
2-n (I)
Wherein, R
1Expression hydrogen atom, alkyl, alkenyl, aralkyl or phenyl with 7-26 carbon atom with 2-18 carbon atom with 1-18 carbon atom; N is the integer of 0-2; And wherein when n is 2, two R
1Be identical or different.
3. 5-aminolevulinic acid according to claim 2, wherein R
1Be hydrogen atom, methyl, ethyl, normal-butyl, hexadecyl, 2-ethylhexyl, oleyl, benzyl or phenyl.
4. according to claim 2 or 3 described 5-aminolevulinic acids, this salt is aqueous solution form.
5. according to claim 2 or 3 described 5-aminolevulinic acids, this salt is solid form.
6. 5-aminolevulinic acid according to claim 1, this salt are 5-amino-laevulic acid nitrate.
7. 5-aminolevulinic acid according to claim 6, this salt is solid.
8. 5-aminolevulinic acid according to claim 1, this salt are the 5-amino-laevulic acid sulfonate by following formula (II) expression:
HOCOCH
2CH
2COCH
2NH
2·HOSO
2R
2 (II)
Wherein, R
2The phenyl that expression is replaced by low alkyl group.
9. 5-aminolevulinic acid according to claim 8, wherein substituted phenyl is a 4-aminomethyl phenyl, 2,4-3,5-dimethylphenyl or 2,5-3,5-dimethylphenyl.
10. according to Claim 8 or 9 described 5-aminolevulinic acids, this salt is aqueous solution form.
11. according to Claim 8 or 9 described 5-aminolevulinic acids, this salt is solid form.
12. a method for preparing according to any described 5-aminolevulinic acid in the claim 2 to 5, this method comprise that wash-out is attracted to the 5-amino-laevulic acid on the Zeo-karb, and elutriant is mixed with phosphoric acid class material.
13. method according to claim 12, wherein said 5-amino-laevulic acid ammoniacal liquor wash-out.
14. a method for preparing according to claim 6 or 7 described 5-aminolevulinic acids, this method comprise that wash-out is attracted to the 5-amino-laevulic acid on the Zeo-karb, and elutriant is mixed with nitric acid.
15. method according to claim 14, wherein said 5-amino-laevulic acid ammoniacal liquor wash-out.
16. a method for preparing the 5-amino-laevulic acid sulfonate of claim 8 or 9, this method comprise that wash-out is attracted to the 5-amino-laevulic acid on the Zeo-karb, and elutriant is mixed with sulfonic acid substance.
17. method according to claim 16, wherein said 5-amino-laevulic acid ammoniacal liquor wash-out.
18. a composition that is used for optical dynamic therapy or light power diagnosis, said composition comprise according to any described 5-aminolevulinic acid in the claim 1 to 11.
19. plant activator composition that comprises according to any described 5-aminolevulinic acid in the claim 1 to 11.
20. according to the application of any described 5-aminolevulinic acid in the claim 1 to 11 in preparation optical dynamic therapy agent or light power diagnosis reagent.
21. according to the application of any described 5-aminolevulinic acid in the claim 1 to 11 as plant activator.
Applications Claiming Priority (15)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP099672/2004 | 2004-03-30 | ||
| JP099671/2004 | 2004-03-30 | ||
| JP2004099671 | 2004-03-30 | ||
| JP2004099672 | 2004-03-30 | ||
| JP2004099670 | 2004-03-30 | ||
| JP099670/2004 | 2004-03-30 | ||
| JP345661/2004 | 2004-11-30 | ||
| JP2004345661 | 2004-11-30 | ||
| JP2005051217A JP4417865B2 (en) | 2004-03-30 | 2005-02-25 | 5-Aminolevulinic acid phosphate, method for producing the same, and use thereof |
| JP051216/2005 | 2005-02-25 | ||
| JP2005051218A JP4630087B2 (en) | 2004-03-30 | 2005-02-25 | 5-Aminolevulinic acid nitrate and process for producing the same |
| JP051218/2005 | 2005-02-25 | ||
| JP051217/2005 | 2005-02-25 | ||
| JP2005051216A JP4719483B2 (en) | 2004-03-30 | 2005-02-25 | Process for producing 5-aminolevulinic acid sulfonate |
| PCT/JP2005/005765 WO2005100300A1 (en) | 2004-03-30 | 2005-03-28 | 5-aminolevulinic acid salt, process for producing the same and use thereof |
Related Child Applications (3)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2010102153643A Division CN101885736B (en) | 2004-03-30 | 2005-03-28 | 5-aminolevulinic acid phosphate salt, process for producing the same and use thereof |
| CN2010102154650A Division CN101885692B (en) | 2004-03-30 | 2005-03-28 | 5-aminolevulinic acid phosphate salt, process for producing the same and use thereof |
| CN2010102153906A Division CN101863788B (en) | 2004-03-30 | 2005-03-28 | 5-aminolevulinic acid phosphate salt, process for producing the same and use thereof |
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| Publication Number | Publication Date |
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| CN1942430A true CN1942430A (en) | 2007-04-04 |
| CN1942430B CN1942430B (en) | 2011-04-20 |
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| Application Number | Title | Priority Date | Filing Date |
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| CN2005800107065A Active CN1942430B (en) | 2004-03-30 | 2005-03-28 | 5-aminolevulinic acid phosphate, method for producing the same and use thereof |
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| Country | Link |
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| CN (1) | CN1942430B (en) |
| ES (1) | ES2355978T3 (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103140457A (en) * | 2010-09-30 | 2013-06-05 | 克斯莫石油株式会社 | 5-aminolevulinic acid-containing solid fertilizer and process for producing same |
| CN103265444A (en) * | 2013-04-24 | 2013-08-28 | 浙江大学 | Crystallization method of 5-aminolevulinic acid phosphate |
| CN104661994A (en) * | 2012-08-03 | 2015-05-27 | 光治疗Asa公司 | Compounds |
| CN107094559A (en) * | 2017-07-03 | 2017-08-29 | 中国计量大学 | The method of adjusting and controlling rice heavy metal cadmium accumulation |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5945564A (en) * | 1995-07-12 | 1999-08-31 | Mitsubishi Chemical Corporation | 2,2-dideutero-5-aminolevulinic acid |
-
2005
- 2005-03-28 CN CN2005800107065A patent/CN1942430B/en active Active
- 2005-03-28 ES ES05727585T patent/ES2355978T3/en active Active
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103140457A (en) * | 2010-09-30 | 2013-06-05 | 克斯莫石油株式会社 | 5-aminolevulinic acid-containing solid fertilizer and process for producing same |
| CN103140457B (en) * | 2010-09-30 | 2015-05-13 | 克斯莫石油株式会社 | 5-aminolevulinic acid-containing solid fertilizer and process for producing same |
| CN104661994A (en) * | 2012-08-03 | 2015-05-27 | 光治疗Asa公司 | Compounds |
| CN104661994B (en) * | 2012-08-03 | 2017-08-08 | 光治疗Asa公司 | Compound |
| CN103265444A (en) * | 2013-04-24 | 2013-08-28 | 浙江大学 | Crystallization method of 5-aminolevulinic acid phosphate |
| CN107094559A (en) * | 2017-07-03 | 2017-08-29 | 中国计量大学 | The method of adjusting and controlling rice heavy metal cadmium accumulation |
Also Published As
| Publication number | Publication date |
|---|---|
| ES2355978T3 (en) | 2011-04-01 |
| CN1942430B (en) | 2011-04-20 |
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Effective date of registration: 20160714 Address after: Tokyo, Japan, Japan Patentee after: Maxmow ALA Address before: Tokyo, Japan Patentee before: Cosmo Oil Co., Ltd. |