NO301166B1 - Process for the preparation of folic acid derivatives - Google Patents
Process for the preparation of folic acid derivatives Download PDFInfo
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- NO301166B1 NO301166B1 NO932047A NO932047A NO301166B1 NO 301166 B1 NO301166 B1 NO 301166B1 NO 932047 A NO932047 A NO 932047A NO 932047 A NO932047 A NO 932047A NO 301166 B1 NO301166 B1 NO 301166B1
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- 238000000034 method Methods 0.000 title claims description 35
- 238000002360 preparation method Methods 0.000 title description 4
- 150000002224 folic acids Chemical class 0.000 title 1
- 239000000203 mixture Substances 0.000 claims description 22
- 239000008363 phosphate buffer Substances 0.000 claims description 14
- 239000008366 buffered solution Substances 0.000 claims description 13
- 108010088751 Albumins Proteins 0.000 claims description 12
- 102000009027 Albumins Human genes 0.000 claims description 12
- 238000010828 elution Methods 0.000 claims description 12
- 239000000243 solution Substances 0.000 claims description 12
- 239000007864 aqueous solution Substances 0.000 claims description 10
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 8
- 239000000741 silica gel Substances 0.000 claims description 8
- 229910002027 silica gel Inorganic materials 0.000 claims description 8
- 229910052784 alkaline earth metal Inorganic materials 0.000 claims description 4
- 238000001033 granulometry Methods 0.000 claims description 3
- 238000009776 industrial production Methods 0.000 claims description 3
- 241000283690 Bos taurus Species 0.000 claims description 2
- 241000287828 Gallus gallus Species 0.000 claims description 2
- 101000693916 Gallus gallus Albumin Proteins 0.000 claims description 2
- 241000283973 Oryctolagus cuniculus Species 0.000 claims description 2
- 108010058846 Ovalbumin Proteins 0.000 claims description 2
- 239000003513 alkali Substances 0.000 claims description 2
- 150000001342 alkaline earth metals Chemical class 0.000 claims description 2
- 238000013375 chromatographic separation Methods 0.000 claims 1
- VVIAGPKUTFNRDU-ABLWVSNPSA-N folinic acid Chemical class C1NC=2NC(N)=NC(=O)C=2N(C=O)C1CNC1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 VVIAGPKUTFNRDU-ABLWVSNPSA-N 0.000 claims 1
- 238000005406 washing Methods 0.000 claims 1
- JWUJQDFVADABEY-UHFFFAOYSA-N 2-methyltetrahydrofuran Chemical compound CC1CCCO1 JWUJQDFVADABEY-UHFFFAOYSA-N 0.000 description 10
- 238000000926 separation method Methods 0.000 description 8
- 239000002253 acid Substances 0.000 description 5
- 230000015572 biosynthetic process Effects 0.000 description 4
- 239000011575 calcium Substances 0.000 description 4
- 159000000007 calcium salts Chemical class 0.000 description 4
- OVBPIULPVIDEAO-LBPRGKRZSA-N folic acid Chemical compound C=1N=C2NC(N)=NC(=O)C2=NC=1CNC1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 OVBPIULPVIDEAO-LBPRGKRZSA-N 0.000 description 4
- 238000004128 high performance liquid chromatography Methods 0.000 description 4
- 230000003287 optical effect Effects 0.000 description 4
- 238000003786 synthesis reaction Methods 0.000 description 4
- 230000005526 G1 to G0 transition Effects 0.000 description 3
- 238000005984 hydrogenation reaction Methods 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- FVAUCKIRQBBSSJ-UHFFFAOYSA-M sodium iodide Chemical compound [Na+].[I-] FVAUCKIRQBBSSJ-UHFFFAOYSA-M 0.000 description 3
- 230000000707 stereoselective effect Effects 0.000 description 3
- VVIAGPKUTFNRDU-STQMWFEESA-N (6S)-5-formyltetrahydrofolic acid Chemical class C([C@H]1CNC=2N=C(NC(=O)C=2N1C=O)N)NC1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 VVIAGPKUTFNRDU-STQMWFEESA-N 0.000 description 2
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 2
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 2
- OVBPIULPVIDEAO-UHFFFAOYSA-N N-Pteroyl-L-glutaminsaeure Natural products C=1N=C2NC(N)=NC(=O)C2=NC=1CNC1=CC=C(C(=O)NC(CCC(O)=O)C(O)=O)C=C1 OVBPIULPVIDEAO-UHFFFAOYSA-N 0.000 description 2
- -1 NH 4<+> Inorganic materials 0.000 description 2
- 229910019142 PO4 Inorganic materials 0.000 description 2
- 108010071390 Serum Albumin Proteins 0.000 description 2
- 102000007562 Serum Albumin Human genes 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 2
- 229940098773 bovine serum albumin Drugs 0.000 description 2
- 229910052791 calcium Inorganic materials 0.000 description 2
- VWBBRFHSPXRJQD-ZEDZUCNESA-L calcium-L-methylfolate Chemical compound [Ca+2].C1NC=2NC(N)=NC(=O)C=2N(C)C1CNC1=CC=C(C(=O)N[C@@H](CCC([O-])=O)C([O-])=O)C=C1 VWBBRFHSPXRJQD-ZEDZUCNESA-L 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 235000019152 folic acid Nutrition 0.000 description 2
- 239000011724 folic acid Substances 0.000 description 2
- 229960000304 folic acid Drugs 0.000 description 2
- 238000001640 fractional crystallisation Methods 0.000 description 2
- 159000000003 magnesium salts Chemical class 0.000 description 2
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 2
- 239000010452 phosphate Substances 0.000 description 2
- 150000003839 salts Chemical class 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- 230000001225 therapeutic effect Effects 0.000 description 2
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 description 1
- MSTNYGQPCMXVAQ-KIYNQFGBSA-N 5,6,7,8-tetrahydrofolic acid Chemical class N1C=2C(=O)NC(N)=NC=2NCC1CNC1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 MSTNYGQPCMXVAQ-KIYNQFGBSA-N 0.000 description 1
- OZRNSSUDZOLUSN-UHFFFAOYSA-N 7,8-Dihydrofolic acid Natural products C1NC=2NC(N)=NC(=O)C=2N=C1CNC1=CC=C(C(=O)NC(CCC(O)=O)C(O)=O)C=C1 OZRNSSUDZOLUSN-UHFFFAOYSA-N 0.000 description 1
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 description 1
- 206010016880 Folate deficiency Diseases 0.000 description 1
- MUBZPKHOEPUJKR-UHFFFAOYSA-N Oxalic acid Chemical compound OC(=O)C(O)=O MUBZPKHOEPUJKR-UHFFFAOYSA-N 0.000 description 1
- 108010022394 Threonine synthase Proteins 0.000 description 1
- XJLXINKUBYWONI-DQQFMEOOSA-N [[(2r,3r,4r,5r)-5-(6-aminopurin-9-yl)-3-hydroxy-4-phosphonooxyoxolan-2-yl]methoxy-hydroxyphosphoryl] [(2s,3r,4s,5s)-5-(3-carbamoylpyridin-1-ium-1-yl)-3,4-dihydroxyoxolan-2-yl]methyl phosphate Chemical compound NC(=O)C1=CC=C[N+]([C@@H]2[C@H]([C@@H](O)[C@H](COP([O-])(=O)OP(O)(=O)OC[C@@H]3[C@H]([C@@H](OP(O)(O)=O)[C@@H](O3)N3C4=NC=NC(N)=C4N=C3)O)O2)O)=C1 XJLXINKUBYWONI-DQQFMEOOSA-N 0.000 description 1
- 150000001412 amines Chemical class 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- KVUAALJSMIVURS-QNTKWALQSA-L calcium;(2s)-2-[[4-[[(6s)-2-amino-5-formyl-4-oxo-1,6,7,8-tetrahydropteridin-6-yl]methylamino]benzoyl]amino]pentanedioate Chemical compound [Ca+2].C([C@@H]1N(C=O)C=2C(=O)N=C(NC=2NC1)N)NC1=CC=C(C(=O)N[C@@H](CCC([O-])=O)C([O-])=O)C=C1 KVUAALJSMIVURS-QNTKWALQSA-L 0.000 description 1
- UOSVKQDEUWYYOT-NVGTXZLJSA-L calcium;(4s)-4-[[4-[[(6r)-2-amino-5-formyl-4-oxo-1,6,7,8-tetrahydropteridin-6-yl]methylamino]benzoyl]amino]-5-hydroxy-5-oxopentanoate Chemical compound [Ca+2].C([C@@H]1CNC=2N=C(NC(=O)C=2N1C=O)N)NC1=CC=C(C(=O)N[C@@H](CCC([O-])=O)C(O)=O)C=C1.C([C@@H]1CNC=2N=C(NC(=O)C=2N1C=O)N)NC1=CC=C(C(=O)N[C@@H](CCC([O-])=O)C(O)=O)C=C1 UOSVKQDEUWYYOT-NVGTXZLJSA-L 0.000 description 1
- 125000004432 carbon atom Chemical group C* 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 239000003054 catalyst Substances 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000002425 crystallisation Methods 0.000 description 1
- 230000008025 crystallization Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 102000004419 dihydrofolate reductase Human genes 0.000 description 1
- OZRNSSUDZOLUSN-LBPRGKRZSA-N dihydrofolic acid Chemical compound N=1C=2C(=O)NC(N)=NC=2NCC=1CNC1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 OZRNSSUDZOLUSN-LBPRGKRZSA-N 0.000 description 1
- DNJIEGIFACGWOD-UHFFFAOYSA-N ethyl mercaptane Natural products CCS DNJIEGIFACGWOD-UHFFFAOYSA-N 0.000 description 1
- ZXKXJHAOUFHNAS-UHFFFAOYSA-N fenfluramine hydrochloride Chemical compound [Cl-].CC[NH2+]C(C)CC1=CC=CC(C(F)(F)F)=C1 ZXKXJHAOUFHNAS-UHFFFAOYSA-N 0.000 description 1
- 238000011049 filling Methods 0.000 description 1
- 235000019253 formic acid Nutrition 0.000 description 1
- 125000002485 formyl group Chemical group [H]C(*)=O 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- XNGIFLGASWRNHJ-UHFFFAOYSA-L phthalate(2-) Chemical compound [O-]C(=O)C1=CC=CC=C1C([O-])=O XNGIFLGASWRNHJ-UHFFFAOYSA-L 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 230000001012 protector Effects 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 235000009518 sodium iodide Nutrition 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 1
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- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Description
Foreliggende oppfinnelse angår en fremgangsmåte for The present invention relates to a method for
industriell fremstilling av (6S) folinsyrederivater ved kromatografisk å adskille de diastereoisomere blandinger derav på en kolonne, spesielt angår den en fremgangsmåte for industriell fremstilling av 5-(metyl)-(6S)-tetrahydrofolin-syre og 5-(formyl)-(6S)-tetrahydrofolinsyre, heri referert som "MTHF" og FTHF". MTHF og FTHF er fysiologiske molekyler som sammen med de mindre stabile 6,10-metylentetrahydrofo- industrial production of (6S) folinic acid derivatives by chromatographically separating the diastereoisomeric mixtures thereof on a column, in particular it relates to a process for the industrial production of 5-(methyl)-(6S)-tetrahydrofolinic acid and 5-(formyl)-(6S )-tetrahydrofolinic acid, herein referred to as "MTHF" and FTHF". MTHF and FTHF are physiological molecules which, together with the less stable 6,10-methylenetetrahydrofolic
lin- og tetrahydrofolinsyrer utgjør de in vivo aktive former av folinsyre, vanligvis betegnet som folinsyre ko-faktorer. linoleic and tetrahydrofolic acids constitute the in vivo active forms of folic acid, usually referred to as folic acid co-factors.
Fra det terapeutiske synspunkt finner både MTHF og FTHF anvendelse i alle former for folat-defisienter som generelt leverbeskyttende, og mere nylig, i antisvulstterapier. From the therapeutic point of view, both MTHF and FTHF are used in all forms of folate deficiency as a general liver protector, and more recently, in antitumor therapies.
MTHF og FTHF og de terapeutisk akseptable derivater derav MTHF and FTHF and the therapeutically acceptable derivatives thereof
som vanligvis brukes har den følgende formel: which is commonly used has the following formula:
hvor X = CH3 eller CHO og where X = CH3 or CHO and
Ri og R2 er H, NH4<+>,alkali og jordalkalimetaller, idet R 1 and R 2 are H, NH 4<+>, alkali and alkaline earth metals, in that
R-^ og R2 er like eller forskjellige fra hverandre. R 1 and R 2 are the same or different from each other.
Forbindelsene med formel I inneholder to asymmetriske kar-bonatomer og kan derfor eksistere i fire diastereoisomere former. The compounds of formula I contain two asymmetric carbon atoms and can therefore exist in four diastereoisomeric forms.
Vanligvis er det velkjent at når det eksisterer stereoiso- Generally, it is well known that when stereoiso-
mere farmasøytiske former, er kun én av dem aktiv, idet den andre er inaktiv eller til og med skadelig. I foreliggende tilfelle er det velkjent at de aktive former er de av type more pharmaceutical forms, only one of them is active, the other being inactive or even harmful. In the present case, it is well known that the active forms are those of type
6S. 6S.
Enkelte studier er blitt utført angående industriell fremstilling og/eller isoleringen av de to diastereoisomerer og angående direkte syntese av (6S) formen. Certain studies have been carried out regarding the industrial preparation and/or the isolation of the two diastereoisomers and regarding the direct synthesis of the (6S) form.
Den stereospesifikke syntese av (6S) formen av både MTHF og FTHF, såvel som adskillelsen av de to (6RS) diastereoisomerer, har vist seg å være vanskelig gitt den struktuelle skørhet til sluttproduktene. The stereospecific synthesis of the (6S) form of both MTHF and FTHF, as well as the separation of the two (6RS) diastereoisomers, has proven difficult given the structural fragility of the final products.
Syntesen av de stereoisomere blandinger av MTHF og FTHF er blitt beskrevet i henholdsvis GB-A-1,572,138 patentet og i CH-A-496.012 patentet. The synthesis of the stereoisomeric mixtures of MTHF and FTHF has been described in the GB-A-1,572,138 patent and in the CH-A-496,012 patent, respectively.
For å adskille (6S) diastereoisomerene fra de således erholdte blandinger er det mulig å transformere den stereoisomere blanding i det respektive 5-metyloksykar-bonylderivatet eller i andre derivater med chirale sentra: på en slik måte blir det utnyttet forskjellige oppløselighe-ter i passende oppløsningsmidler, idet det er mulig å adskille de to (6R) og (6S) diastereoisomerer (JCS, CHEM COMM 470, 1987; EP 0 266 042). In order to separate the (6S) diastereoisomers from the mixtures thus obtained, it is possible to transform the stereoisomeric mixture into the respective 5-methyloxycarbonyl derivative or into other derivatives with chiral centers: in such a way, different solubilities in suitable solvents are utilized , it being possible to separate the two (6R) and (6S) diastereoisomers (JCS, CHEM COMM 470, 1987; EP 0 266 042).
Nevnte fremgangsmåte er likevel noe komplisert og utbyttene er ekstremt dårlige. Said method is nevertheless somewhat complicated and the yields are extremely poor.
Andre separasjonsmetoder er basert på multiple krystal-liseringer enten av (6R,S) blandinger (EP 0_367 902; WO 88 08,844) eller av et intermediat derav (EP 348,841), men det oppnås alltid dårlige utbytter. Other separation methods are based on multiple crystallizations either of (6R,S) mixtures (EP 0_367 902; WO 88 08,844) or of an intermediate thereof (EP 348,841), but poor yields are always obtained.
Nyligere har resolusjonen av de stereoisomere blandinger av både'"MTHF og FTHF blitt erholdt (US 5006655) ved fraksjonær krystallisering og eventuell hydrolysering eller redusering av 5,10-metylen-(6RS) tetrahydrofolinintermediatet, men dette er vanskelig å behandle og isoleres fra et skadelig og toksisk oppløsningsmiddel slik som maursyre. More recently, the resolution of the stereoisomeric mixtures of both MTHF and FTHF has been obtained (US 5006655) by fractional crystallization and eventual hydrolysis or reduction of the 5,10-methylene-(6RS) tetrahydrofoline intermediate, but this is difficult to process and is isolated from a harmful and toxic solvent such as formic acid.
En fremgangsmåte for å separere (6S) formen er beskrevet i PCT/EP88/00341 (W088/08844), hvor en oppløsning av kalsium-eller magnesiumsaltet av den stereoisomere blanding behandles med et amin, hvor kationsaltet som oksalatutfel-lende og det ønskete produkt erholdes ved fraksjonert krystallisering og rekonversjon til kalsium- eller magnesiumsaltet . A method for separating the (6S) form is described in PCT/EP88/00341 (W088/08844), where a solution of the calcium or magnesium salt of the stereoisomeric mixture is treated with an amine, where the cation salt as oxalate precipitate and the desired product is obtained by fractional crystallization and reconversion to the calcium or magnesium salt.
Utbyttene som er vist er relativt små og metoden involverer flere saltdannende og krystalliserende trinn. The yields shown are relatively small and the method involves several salt-forming and crystallizing steps.
I en ytterligere resolusjonsprosess blir den racemiske blanding, som kalsiumsalt (EP 367,902) tilsatt med et antall organiske og uorganiske salter, spesielt med natriumjodid, for således å erholde et produkt som viser en god ren-hetsgrad, men nok engang oppnås relativt dårlige utbytter, noe som må regnes som meningsløst fra et industrielt synspunkt. In a further resolution process, the racemic mixture, such as calcium salt (EP 367,902) is added with a number of organic and inorganic salts, especially with sodium iodide, in order to obtain a product that shows a good degree of purity, but once again relatively poor yields are obtained, which must be considered meaningless from an industrial point of view.
Enda en ytterligere metode angående separasjonen av isomerene av FTHF er beskrevet av Choi et al., Analytical Biochem. bind 168, s. 398-404 (1968), ifølge hvilket en kolonne av silikagel bundet med bovinserumalbumin (BSA) blir brukt. Yet another method regarding the separation of the isomers of FTHF is described by Choi et al., Analytical Biochem. vol. 168, pp. 398-404 (1968), according to which a column of silica gel bound with bovine serum albumin (BSA) is used.
Angående elueringen blir fosfatbufre ved en pH som ligger mellom 6,4 og 8,4, fortrinnsvis 7,4, brukt. Regarding the elution, phosphate buffers at a pH between 6.4 and 8.4, preferably 7.4, are used.
Det første eluat erholdt i henhold til Choi et al. inneholder (6S) isomeren i meget små mengder som kun kan brukes for analytiske formål. The first eluate obtained according to Choi et al. contains the (6S) isomer in very small amounts that can only be used for analytical purposes.
Choi et al. metoden anvendt ved industrielle betingelser ville gi en relativt høy oppholdstid for oppløsningen i kolonnen som et resultat på grunn av en dårlig resolusjon: den industrielle produktivitet ville følgelig vise seg utilstrekkelig fordi det vil være nødvendig å bruke kolonner som var ekstremt lange for å få en akseptabel resolusjon eller alternativt utsette oppløsningen i samme kolonne for flere sykler, hvilket ville innbefatte økete muligheter for nedbrytning av blandingen og et meget høyt forbruk av reagensene. Choi et al. the method applied under industrial conditions would give a relatively high residence time for the solution in the column as a result due to a poor resolution: the industrial productivity would consequently prove insufficient because it would be necessary to use columns that were extremely long to obtain an acceptable resolution or alternatively postpone the resolution in the same column for several cycles, which would include increased possibilities for degradation of the mixture and a very high consumption of the reagents.
Overraskende er det blitt funnet at i henhold til foreliggende oppfinnelse, ved å følge anvisningene til Choi et al., men ved å bruke med hensyn til elueringen av (6R,S) blandingene en bufret oppløsning i et meget snevert pH område (mellom 5,0 og 5,5), blir resolusjonen av (6S) isomeren erholdt med industrielle utbytter, billigt og ved en enkel prosedyre. Surprisingly, it has been found that according to the present invention, following the instructions of Choi et al., but using with regard to the elution of the (6R,S) mixtures a buffered solution in a very narrow pH range (between 5, 0 and 5.5), the resolution of the (6S) isomer is obtained with industrial yields, cheaply and by a simple procedure.
Faktisk, hvis man bruker to industrielle kolonner inneholdende silikagel bundet med et albumin for å adskille (6RS) blandingen i henhold til fremgangsmåten til Choi et al. og henholdsvis til foreliggende oppfinnelse blir et utbytte av henholdsvis (6S) isomeren lavere enn 5% og ikke lavere enn 2 0% erholdt. Indeed, if one uses two industrial columns containing silica gel bound with an albumin to separate the (6RS) mixture according to the method of Choi et al. and respectively to the present invention, a yield of the (6S) isomer respectively lower than 5% and not lower than 20% is obtained.
Ved siden av de ovennevnte metoder for separasjon av (6S) isomerene fra (6R,S) blandingen har forsøk blitt gjort for direkte å syntetisere (6S) isomerene. In addition to the above methods for the separation of the (6S) isomers from the (6R,S) mixture, attempts have been made to directly synthesize the (6S) isomers.
I henhold til EP-A-356,984 patentet har syntesen av (6S) isomeren blitt oppnådd ved en fremgangsmåte basert på den stereospesifikke hydrogenering av 5-6 dobbeltbindingen av 7,8-dihydrofolinsyre. According to the EP-A-356,984 patent, the synthesis of the (6S) isomer has been achieved by a method based on the stereospecific hydrogenation of the 5-6 double bond of 7,8-dihydrofolic acid.
Når nevnte hydrogenering utføres med dihydrofolatreduktase i nærvær av NADP blir en god konversjon erholdt: nevnte metode er imidlertid ekstremt komplisert og slett ikke anvendelig fra et industrielt synspunkt. When said hydrogenation is carried out with dihydrofolate reductase in the presence of NADP, a good conversion is obtained: said method is, however, extremely complicated and not at all applicable from an industrial point of view.
Andre typer av stereospesifikke hydrogeneringer som fremskaffer bruken av passende katalysatorer er blitt forsøkt, men alltid med dårligere resultater (TETRAHEDRON 42, 117, 1986). Other types of stereospecific hydrogenations that provide for the use of suitable catalysts have been attempted, but always with poorer results (TETRAHEDRON 42, 117, 1986).
Endelig eksisterer ingen billig metode som er anvendelig i industriell skala for å erholde (6S) isomerene av MTHF og FTHF. Med hensyn til den praktiske terapeutiske anvendelsen blir (6S) MTHF og (6S) FTHF ikke anvendt som sådanne, men som salter, vanligvis kalsiumsalter. Finally, no inexpensive method applicable on an industrial scale exists to obtain the (6S) isomers of MTHF and FTHF. With regard to the practical therapeutic application, (6S) MTHF and (6S) FTHF are not used as such, but as salts, usually calcium salts.
En slik omdannelse utføres i henhold til velkjente metoder, f.eks. i henhold til metodene beskrevet i US-A-2,688,018 patentet og av Weast i "Handbook fot-Chemistry and Physics" Such a conversion is carried out according to well-known methods, e.g. according to the methods described in the US-A-2,688,018 patent and by Weast in "Handbook fot-Chemistry and Physics"
(57. utg., s. B-100). (57th ed., p. B-100).
En blanding av (6RS) MTHF eller (6RS) FTHF blir adskilt i de respektive (6S) isomerer ved en metode i henhold til hvilken i en kromatografisk kolonne en vandig oppløsning av et albumin blir påsatt, derpå utføres en vask med en første bufret oppløsning, en vandig oppløsning av den diastereoisomere blanding av derivatet med formel (I) blir påsatt, derpå eluering med en andre bufret oppløsning for å erholde (6S) diastereoisomeren, og som er særpreget ved at konsentrasjonen av albuminet i oppløsningen derav ligger mellom 0,1 vekt% og 10 vekt%, og at oppløsningen av derivatene har en konsentrasjon mellom 0,1 vekt% og 10 vekt%, hvor pH av nevnte bufrete oppløsninger ligger mellom 4,8 og 5,8. A mixture of (6RS) MTHF or (6RS) FTHF is separated into the respective (6S) isomers by a method according to which in a chromatographic column an aqueous solution of an albumin is applied, followed by a wash with a first buffered solution , an aqueous solution of the diastereoisomeric mixture of the derivative of formula (I) is added, followed by elution with a second buffered solution to obtain the (6S) diastereoisomer, and which is characterized by the concentration of the albumin in the solution thereof being between 0.1 % by weight and 10% by weight, and that the solution of the derivatives has a concentration between 0.1% by weight and 10% by weight, where the pH of said buffered solutions is between 4.8 and 5.8.
Fortrinnsvis vaskes kolonnen med en bufret oppløsning ved pH 7 og deretter igjen ved pH 5, før den diastereoisomere blanding av derivatet av formel (I) tilsettes. Preferably, the column is washed with a buffered solution at pH 7 and then again at pH 5, before the diastereoisomeric mixture of the derivative of formula (I) is added.
Mere foretrukket er de vandige oppløsninger av albuminet og den diastereoisomere blanding bufret ved en pH som ligger mellom 4,8 og 5,8. More preferably, the aqueous solutions of the albumin and the diastereoisomeric mixture are buffered at a pH between 4.8 and 5.8.
Enda ytterligere foretrukket har silikagelen en granulometri som ligger mellom 25 og 60 um, og enda bedre mellom 35 og 45 um. Even more preferably, the silica gel has a granulometry that lies between 25 and 60 µm, and even better between 35 and 45 µm.
Med fordel er nevnte bufrete oppløsninger fosfatbufre med en konsentrasjon som ligger mellom 0,05 M og 0,15 M, hvor konsentrasjonen av nevnte albumin i den vandige oppløsning derav er omkring 1 vekt%, og den til den diastereoisomere blanding i sin oppløsning er omkring 5 vekt%, mens albuminet er valgt fra gruppen omfattende svine-, bovin-, ovin-, kanin-, kylling- og eggealbumin. Advantageously, said buffered solutions are phosphate buffers with a concentration between 0.05 M and 0.15 M, where the concentration of said albumin in the aqueous solution thereof is about 1% by weight, and that of the diastereoisomeric mixture in its solution is about 5% by weight, while the albumin is selected from the group comprising pig, bovine, ovine, rabbit, chicken and egg albumin.
Innlysende kan de bufrete oppløsninger som brukes i de forskjellige faser av fremgangsmåten ifølge foreliggende oppfinnelse være like eller forskjellige fra hverandre. Som bufrete oppløsninger kan ftalat, fosfat, acetat, etc. anvendes, fortrinnsvis fosfat. Obviously, the buffered solutions used in the different phases of the method according to the present invention can be the same or different from each other. Phthalate, phosphate, acetate, etc. can be used as buffered solutions, preferably phosphate.
I henhold til en foretrukken fremgangsmåte blir en 0,05 M fosfatbuffer brukt for å eluere den kromatografiske kolonne hvor en oppløsning av (6R,S)-metyl-tetrahydrofolinsyre, kalsiumsalt, på forhånd er blitt påsatt (en 0,15M fosfatbuffer i tilfelle med (6R,S)-formyl-tetrahydrofolinsyre, kalsiumsalt). According to a preferred method, a 0.05 M phosphate buffer is used to elute the chromatographic column to which a solution of (6R,S)-methyl-tetrahydrofolinic acid, calcium salt, has previously been applied (a 0.15 M phosphate buffer in the case of (6R,S)-formyl-tetrahydrofolinic acid, calcium salt).
Endelig i henhold til foreliggende oppfinnelse har det vært mulig å føre produktiviteten av separasjonsmetoden til industrielle nivå, idet det ble oppnådd ekstremt forkortete elueringstider. Finally, according to the present invention, it has been possible to bring the productivity of the separation method to industrial level, as extremely shortened elution times were achieved.
Nå vil foreliggende oppfinnelse bli beskrevet i detalj under henvisning til foretrukne utførelsesformer. Imidlertid bør det bli forstått at foreliggende oppfinnelse, på ingen måte er begrenset til disse spesielle utførelsesformer. The present invention will now be described in detail with reference to preferred embodiments. However, it should be understood that the present invention is in no way limited to these particular embodiments.
Utbyttene angitt i de følgende eksempler er blitt uttrykt som ponderalverdier, hvorved dette tilsvarer et dobbelt optisk utbytte. The yields indicated in the following examples have been expressed as ponderal values, whereby this corresponds to a double optical yield.
EKSEMPEL 1 EXAMPLE 1
I en industriell kolonne med en diameter på 0,90 m og en høyde på 6,0 m, er det tilsatt silikagel (partikkeldiameter 35/45 pm) suspendert i en 0,15 M bufret vandig oppløsning ved pH 5 inneholdende merkaptoetanol (0,1 vekt%) til nesten fullstendig fylling (høyde 5,5 m lik et volum på omkring 3600 g silikagel). In an industrial column with a diameter of 0.90 m and a height of 6.0 m, silica gel (particle diameter 35/45 pm) suspended in a 0.15 M buffered aqueous solution at pH 5 containing mercaptoethanol (0, 1 weight%) to almost complete filling (height 5.5 m equal to a volume of about 3600 g of silica gel).
Fremstillingen av det chirale substrat utføres på følgende måte: En oppløsning av eggserumalbumin (1% i 0,15 M fosfatbuffer, pH 5,0) settes på kolonnen under overvåkning av absorpsjonen i UV ved 230 nm på den eluerte væske. Mengden av det absorberte albumin på silikagelen er lik 200/400 kg. Eluering utføres med 10000 1 av 0,15 M fosfatbuffer ved pH 5,0 derpå med 10000 1 0,15 M fosfatbuffer ved pH 7 og endelig med 10000 1 av 0,15 M fosfatbuffer ved pH 5,0. En 5%'ig oppløsning av kalsium 5-formyl(6R,S)tetrahy-drofolat tilsvarende 5 kg med en strømningshastighet på 200/400 1 pr. time ble derpå tilsatt under eluering med en 0,15 M fosfatbuffer ved pH 5,0. Fraksjoner av 500 1 blir samlet opp. The preparation of the chiral substrate is carried out as follows: A solution of egg serum albumin (1% in 0.15 M phosphate buffer, pH 5.0) is placed on the column while monitoring the absorption in UV at 230 nm of the eluted liquid. The amount of albumin absorbed on the silica gel is equal to 200/400 kg. Elution is performed with 10,000 1 of 0.15 M phosphate buffer at pH 5.0, then with 10,000 1 of 0.15 M phosphate buffer at pH 7 and finally with 10,000 1 of 0.15 M phosphate buffer at pH 5.0. A 5% solution of calcium 5-formyl(6R,S)tetrahydrofolate corresponding to 5 kg with a flow rate of 200/400 1 per hour was then added while eluting with a 0.15 M phosphate buffer at pH 5.0. Fractions of 500 1 are collected.
(6S) fraksjonene elueres før (6R) fraksjonene. Elueringen av fraksjonene kan være linjekontrollert med et polarimeter utstyrt med strømningscelle og derpå nøyaktig dosert med HPLC. Fraksjonene som er tilstrekkelig rene isoleres i forhold til de ønskete formål. De rene fraksjoner samles opp og saltgjøres med kjente metoder. The (6S) fractions are eluted before the (6R) fractions. The elution of the fractions can be line-controlled with a polarimeter equipped with a flow cell and then accurately dosed with HPLC. The fractions that are sufficiently pure are isolated in relation to the desired purposes. The pure fractions are collected and salted using known methods.
Utbytte: 1,9 kg (28%) kalsium (6S) folinat Yield: 1.9 kg (28%) calcium (6S) folinate
Titer: HPLC > 98 % Titer: HPLC > 98%
Optisk Ren > 97 %. Optical Pure > 97%.
EKSEMPEL 2 EXAMPLE 2
I foreliggende eksempler har metodene til Choi et al. blitt reprodusert i industriell skala. In the present examples, the methods of Choi et al. been reproduced on an industrial scale.
Ved å anvende samme fremstillingsmetode av den stasjonære fase utføres metoden som i eksempel 1. By using the same production method of the stationary phase, the method is carried out as in example 1.
Elueringen utføres med 10000 1 av 0,15 M fosfatbuffer ved pH 7. En 5%'ig oppløsning av Ca (6R,S)-folinat tilsvarende ca. 5 kg tilsettes til kolonnen, strømningshastighet 200/400 l/time. Elueringen utføres med fosfatbuffer ved pH 7. Fraksjoner på 500 1 samles opp. The elution is carried out with 10,000 1 of 0.15 M phosphate buffer at pH 7. A 5% solution of Ca (6R,S)-folinate corresponding to approx. 5 kg is added to the column, flow rate 200/400 l/hour. The elution is carried out with phosphate buffer at pH 7. Fractions of 500 1 are collected.
Adskillelsen er utilfredsstillende; opprenskningssyklusen ble gjengatt fire ganger for å forsøke å erholde en partiell adskillelse. The separation is unsatisfactory; the purification cycle was repeated four times to try to obtain a partial separation.
Resten blir kastet idet det mangler adskillelse. The rest is thrown away as there is a lack of separation.
EKSEMPEL 3 EXAMPLE 3
Den samme type kolonne og den samme type metode for fremstilling av den stasjonære fase blir anvendt som i eksempel 1, men svineserumalbumin (PSA) blir brukt heri. The same type of column and the same type of method for preparing the stationary phase are used as in Example 1, but porcine serum albumin (PSA) is used herein.
Utbytte: 1,5 kg (30%) av Ca(6S) folinat Yield: 1.5 kg (30%) of Ca(6S) folinate
HPLC titer: > 98% HPLC titer: > 98%
Optisk Renhet: > 97% Optical Purity: > 97%
EKSEMPEL 4 EXAMPLE 4
Det er blitt anvendt samme type kolonne som i eksempel 1, men avgasset vann er blitt brukt heri med hensyn til fremstillingen av den stasjonære fase og med hensyn til de neste faser med absorpsjon og eluering. The same type of column as in example 1 has been used, but degassed water has been used here with regard to the preparation of the stationary phase and with regard to the next phases of absorption and elution.
10000 1 av 0,05 M fosfatbuffer ved pH 5,2 blir brukt for elueringen, en 5%'ig oppløsning av (6S)-metyltetrahydrofo-linsyre tilsvarende 10 kg med en strømningshastighet på 200/4 00 l/time blir tilsatt med dette, hvor eluering igjen blir utført med 0,05 M fosfatbuffer ved pH 5,2 og 500 l's fraksjoner blir så samlet opp. 10000 1 of 0.05 M phosphate buffer at pH 5.2 is used for the elution, a 5% solution of (6S)-methyltetrahydrofolinic acid corresponding to 10 kg at a flow rate of 200/400 l/hour is added with this , where elution is again carried out with 0.05 M phosphate buffer at pH 5.2 and 500 l's fractions are then collected.
(6S) fraksjonene elueres før (6R) fraksjonene. Fraksjonene som er tilstrekkelig rene blir isolert i henhold til de ønskete formål, samlet opp og behandlet med kjente metoder. Utbytte: 3,9 g (39%) kalsiummetyltetrahydrofolat (6S) The (6S) fractions are eluted before the (6R) fractions. The fractions that are sufficiently pure are isolated according to the desired purposes, collected and processed by known methods. Yield: 3.9 g (39%) calcium methyltetrahydrofolate (6S)
HPLC titer: > 97% HPLC titer: > 97%
Optisk Renhet: > 97% Optical Purity: > 97%
Claims (9)
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| NO932047A NO301166B1 (en) | 1993-06-04 | 1993-06-04 | Process for the preparation of folic acid derivatives |
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| NO932047A NO301166B1 (en) | 1993-06-04 | 1993-06-04 | Process for the preparation of folic acid derivatives |
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| NO301166B1 true NO301166B1 (en) | 1997-09-22 |
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