DK167978B1

DK167978B1 - GLYCERO-3 (2) -PHOSPHO-L-SERINE DERIVATIVES AND SALTS THEREOF, PROCEDURE FOR PREPARING SUCH COMPOUNDS AND PHARMACEUTICAL PREPARATIONS CONTAINING THESE

Info

Publication number: DK167978B1
Application number: DK106287A
Authority: DK
Inventors: Hans Brachwitz; Peter Langen; Christine Lehmann; Eckart Matthes; Juergen Schildt; Iduna Fichtner; Albin Hermetter; Friedrich Paltauf
Original assignee: Hafslund Nycomed Pharma
Priority date: 1985-07-03
Filing date: 1987-03-02
Publication date: 1994-01-10
Also published as: DK106287A; EP0229128A1; HU198076B; FI82473C; DK106287D0; FI82473B; JPH0751588B2; FI870732A; HUT43861A; NO169171B; NO870834D0; FI870732A0; NO870834L; DE3671630D1; JPS63500658A; WO1987000173A1; EP0229128B1; NO169171C

Description

i DK 167978 B1in DK 167978 B1

Den foreliggende opfindelse angår en gruppe af hidtil ukendte glycero-3(2)-phospho-L-serin-derivater , der er ejendommelige ved, at de har den almene formelThe present invention relates to a group of novel glycero-3 (2) -phospho-L-serine derivatives, characterized in that they have the general formula

ChL-AChla

I 2 CH-B (I)I 2 CH-B (I)

CH2-CCH2-C

5 hvori A betyder C^-C^g-alkoxy, halogen eller en gruppe med den almene formel —0—(CH2)n—CF3 (II) hvori n er 0, 1, 2 eller 3, og hvori den ene af grupperne B og C har en af de for gruppen A angivne betydninger, mens den anden gruppe 10 er en phosphatidy1-L-seringruppe med formlenWherein A is C C-CC g alkoxy, halogen or a group of the general formula -O- (CH2) n-CF3 (II) wherein n is 0, 1, 2 or 3 and wherein one of the groups is B and C have one of the meanings set forth for group A, while the other group 10 is a phosphatidyl1-L series group of the formula

0 ^COOHCOOH

-o-p-o-ch2-ch (III) OH ^"NH2 under den forudsætning, at mindst én af grupperne A, B eller C betyder C^-C^^-alkoxy. Opfindelsen angår endvidere de farmaceutisk acceptable salte af forbindelser med den ovenfor viste formel I med baser, en frem-13 gangsmåde til fremstilling af de omhandlede forbindel ser samt farmaceutiske præparater indeholdende de omhandlede forbindelser.The invention further relates to the pharmaceutically acceptable salts of compounds having the above-described compounds. Formula I with bases, a process for preparing the compounds of the invention, and pharmaceutical compositions containing the compounds of the invention.

DK 167978 B1 2DK 167978 B1 2

Glycerophospho-L-serin-derivaterne med den almene formel I har i hvert enkelt tilfælde et chiralitetscentrum i serin-delen af molekylet, og i afhængighed af betydningen af grupperne A, B og C kan derivaterne indehol-5 de endnu et chiralitetscentrum i glycerin-delen af mole kylet. Forbindelserne ifølge opfindelsen med den almene formel I omfatter dermed alle de mulige chirale og dia-stereomere former af forbindelserne.The glycerophospho-L-serine derivatives of the general formula I have in each case a center of chirality in the serine portion of the molecule, and depending on the significance of groups A, B and C, the derivatives may contain another center of chirality in the glycerine. part of the pier cooled. The compounds of the invention of the general formula I thus comprise all the possible chiral and diastereomeric forms of the compounds.

Udtrykket "alkoxy" refererer til ligekædede eller en 10 eller flere gange forgrende alkylethergrupper, som for trinsvis indeholder 14 - 20 carbonatomer, især 16 -18 carbonatomer. Som eksempler på foretrukne alkoxy-grupper kan anføres tetra-, penta-, hexa-, hepta-, octa-og nonadecyloxy, eicosyloxy samt deres forgrenede ana-15 loge.The term "alkoxy" refers to straight-chain or a 10 or more branched alkyl ether groups containing, for example, 14 to 20 carbon atoms, especially 16 to 18 carbon atoms. As examples of preferred alkoxy groups may be mentioned tetra-, penta-, hexa-, hepta-, octa- and nonadecyloxy, eicosyloxy and their branched analogs.

Udtrykket "halogen" henfører til de fire halogener chlor, brom, iod og fluor, blandt hvilke chlor og fluor er særligt foretrukne.The term "halogen" refers to the four halogens chlorine, bromine, iodine and fluorine, of which chlorine and fluorine are particularly preferred.

I en foretrukken klasse af forbindelser med den almene 20 formel I betyder C den ovenfor viste phosphatidy1-L-se- ringruppe med formlen III, og den ene af grupperne A og B er C^-C^g-alkoxy, mens den anden er halogen.In a preferred class of compounds of general formula I, C represents the phosphatidyl1-L-secreting group of formula III shown above, and one of groups A and B is C1-C6 alkoxy, while the other is halogen.

En anden foretrukken klasse består af forbindelser med den almene formel I, hvori C betyder den ovenfor viste 25 phosphatidy1-L-serin-gruppe med formlen III, og hvori den ene af grupperne A og B er C^-C^g-alkoxy, mens den anden er en endestillet fluor-substitueret alkoxygruppe med formlen II.Another preferred class consists of compounds of general formula I wherein C represents the phosphatidyl 1-L-serine group of formula III shown above and wherein one of groups A and B is C while the other is a terminal fluoro-substituted alkoxy group of formula II.

Man foretrækker også sådanne forbindelser med den alme-30 ne formel I, hvori A betyder C^-C^g-alkoxy, B betyder C^-C^Q-alkoxy, og C betyder den ovenfor viste phosphatidy 1-L-serin-gruppe med formlen III.Preferred are also such compounds of the general formula I wherein A represents C 1 -C 2 alkoxy, B represents C 1 -C 2 alkoxy, and C means the phosphatidyl 1-L-serine shown above. group of formula III.

DK 167978 Bl 3DK 167978 Pg 3

Endelig foretrækker man enhver klasse af forbindelser med den almene formel I, hvori B betegner phosphatidyl-L-serin-gruppen med formlen III, og hvori den ene af grupperne A og C betyder C^-C^g-alkoxy, mens den anden 5 betyder halogen.Finally, any class of compounds of the general formula I is preferred, wherein B represents the phosphatidyl L-serine group of formula III and wherein one of the groups A and C represents C1-C6 alkoxy while the other 5 means halogen.

Særligt foretrukne forbindelser ifølge opfindelsen er: l-0-hexadecyl“2-chlor-2-desoxy-glycero-3-phospho-L-serin l-0-hexadecyl-2-fluor-2-desoxy-glycero-3-phospho-L-serin l-chlor-l-desoxy-3-0-hexadecyl-glycero-2-phospho~L-serin 10 l-chlor-l-desoxy-2-0-hexadecyl-glycero-3-phospho-L-serin l-0-hexadecyl-2-desoxy-glycero-3-phospho-L-serin l,2-di-0-hexadecyl-glycero-3-phospho-L-serin 1-0-(2,2,2-trifluorethyl)-2-0-hexadecyl-glyeero-3-phos-pho-L-serin 15 l-0-hexadecyl-2-0-(2,2,2-tri fluorethyl)-glycero-3-phos- pho-L-serin l-chlor-l-desoxy-2-0-octadecyl-glycero-3-phospho-L-serin l-0-octadecyl-2-(2,2,2-trifluorethyl)-glycero-3-phospho-L-serin og 20 l-0-octadecyl-2-chlor-2-desoxy-glycero-3-phospho-L-serin.Particularly preferred compounds of the invention are: 1-O-hexadecyl 2-chloro-2-deoxy-glycero-3-phospho-L-serine 1-O-hexadecyl-2-fluoro-2-deoxy-glycero-3-phospho L-Serine 1-Chloro-1-deoxy-3-O-hexadecyl-glycero-2-phospho-L-serine 10-Chloro-1-deoxy-2-O-hexadecyl-glycero-3-phospho-L-serine 1-O-hexadecyl-2-deoxy-glycero-3-phospho-L-serine 1,2-di-O-hexadecyl-glycero-3-phospho-L-serine 1-O- (2,2,2-trifluoroethyl) ) -2-O-hexadecyl-glyeero-3-phos-pho-L-serine 1-O-hexadecyl-2-O- (2,2,2-trifluoroethyl) -glycero-3-phospho-L -serine 1-chloro-1-deoxy-2-o-octadecyl-glycero-3-phospho-L-serine 1-O-octadecyl-2- (2,2,2-trifluoroethyl) -glycero-3-phospho-L -serine and 20-O-octadecyl-2-chloro-2-deoxy-glycero-3-phospho-L-serine.

De omhandlede glycero-3(2)-phospho-L-serin-derivater med den almene formel I og deres farmaceutiske acceptable salte fremstilles ved en fremgangsmåde, der er ejendommelig ved, at man: 25 (a) omsætter et glycero-3(2)-phosphorsyre-derivat med den almene formel C «2—A (IV)The present glycero-3 (2) -phospho-L-serine derivatives of the general formula I and their pharmaceutically acceptable salts are prepared by a process which is characterized by reacting a glycero-3 (2) ) -phosphoric acid derivative of the general formula C 2 - A (IV)

CH —DCH —D

CH2-ECH2-E

DK 167978 B1 4 hvori A har den ovenfor anførte betydning, og hvori den ene af grupperne D og E har en af de ovenfor for A angivne betydninger, mens den anden er en gruppe med den almene formel Λ _o— P= 0 (V)DK 167978 B1 4 wherein A has the meaning given above and wherein one of the groups D and E has one of the meanings given above for A, while the other is a group of the general formula Λ _o— P = 0 (V)

^Y^ Y

5 hvori' X og Y enten er ens og betegner hydroxy el ler halogen, eller hvor Y betegner lavere alkoxy eller, ai^yloxy under den forudsætning, at mindst én af grupperne A, D eller E betegner C^-C^Q-alkoxy eller et salt deraf med et beskyttet L-serin-deri-10 vat med den almene formel ^/C00Z1 HO-CH2-CH (VI) xnh-z2 hvori betyder en carboxy-beskyttende gruppe og betyder en amino-beskyttende gruppe, fortrinsvis i nærværelse af et kondensationsmiddel, hvorefter man i vilkårlig rækkefølge eller samtidigt fra-15 spalter beskyttelsesgrupperne Z^ og Z^ og eventuelt hydrolyserer den dannede glycerophosphorsyreester eller det dannede giycerophosphorsyrehalogenid, eller (b) omsætter en glycero-3(2)-phosphoresyreester med 20 den almene formel DK 167978 B1 55 wherein X and Y are either the same and represent hydroxy or halogen, or wherein Y represents lower alkoxy or, ali, yloxy, provided that at least one of the groups A, D or E represents C 1 -C 2 Q alkoxy or a salt thereof having a protected L-serine derivative of the general formula ^ / C00Z1 HO-CH2-CH (VI) xnh-z2 wherein a carboxy protecting group means and an amino protecting group, preferably in the presence of a condensing agent, after which the protecting groups Z 1 and Z 2 are randomly or simultaneously decomposed and optionally hydrolyzed the glycerophosphoric acid ester or glycerophosphoric acid halide formed, or (b) reacting a glycero-3 (2) phosphoric acid ester with the general formula DK 167978 B1 5

CH - ACH - A

I 2 CH — L (VII) ch2~m hvori A har den ovenfor angivne betydning, og hvori den ene af grupperne L og M har en af de ovenfor for A angivne betydninger, mens den anden er en gruppe med den almene formel / OH yr — 0 —P = 0 — 0—P=0 /¾ X0R, X0-(CH2)n-N-!~R3 * • (Tina) eller (Mb) 5 hvor er C^-C^-alkyl, der eventuelt er substitue ret med hydroxy eller halogen, R2, R^ og R^, der er ens eller forskellige, hver for sig betegner hydrogen eller methyl, og n er et helt tal fra 1 til 6, forudsat at mindst én af grupperne A, L eller 10 M betyder C^-C^g-alkoxy, med L-serin i nærværelse af phospholipase D og isolerer den dannede forbindelse med formlen I eller et salt heraf, hvorefter man om ønsket (c) overfører en under (a) eller (b) opnået forbindelse 15 med den almene formel I eller et farmaceutisk ikke- acceptabelt salt deraf i et farmaceutisk acceptabelt DK 167978 B1 6 salt deraf.In 2 CH - L (VII) ch 2 ~ m wherein A has the meaning given above and wherein one of the groups L and M has one of the meanings given above for A, the other is a group of the general formula / OH yr - 0 - P = 0 - 0 - P = 0 / ¾ X0R, X0- (CH2) nN-! ~ R3 * • (Tina) or (Mb) 5 where is C1 -C4 alkyl optionally substituted with hydroxy or halogen, R 2, R 2 and R 2, which are the same or different, each represent hydrogen or methyl, and n is an integer from 1 to 6, provided that at least one of the groups A, L or M is C C-CC g alkoxy, with L-serine in the presence of phospholipase D and isolating the compound of formula I or a salt thereof, and then, if desired (c), transferring a sub (a) or (b) ) obtained compound 15 of the general formula I or a pharmaceutically unacceptable salt thereof in a pharmaceutically acceptable salt thereof.

Ifølge fremgangsmådevarianten (a) kan forbindelserne med den almene formel I således fremstilles ud fra glyce-ro-3(2)-phosphorsyrederivater med formlen IV og tilsva-5 rende beskyttede L-serin-forbindelser med formlen VIThus, according to the process variant (a), the compounds of the general formula I can be prepared from glycero-3 (2) phosphoric acid derivatives of the formula IV and corresponding protected L-serine compounds of the formula VI

ved i sig selv kendte metoder, der er nærliggende for fagmanden. Eksempelvis er det kendt at fremstille phos-phatidyIseriner med mættede og umættede acylgrupper ved kondensation af phosphatidsyrer med seriner, hvis 1G amino- og carboxylgrupper er beskyttede, eller ved omsæt ning af diacylglycerol-iodhydriner med beskyttede 0-phos-phoseriner, hvorved beskyttelsesgrupperne spaltes fra de som mellemprodukter dannede beskyttede phosphati-dylseriner, enten samtidigt eller efter hinanden, ved 15 passende metoder. I stedet for phosphatidsyrer kan man anvende phosphatidsyrechlorider i reaktionen. Alkyl-og alkylen-substituerede glycerophosphoseriner lader sig syntetisere på analog måde (A.J. Slotboom og P.P.by itself known methods which are readily apparent to those skilled in the art. For example, it is known to prepare phosphatidylserines with saturated and unsaturated acyl groups by condensation of phosphatidic acids with serines whose 1G amino and carboxyl groups are protected, or by reacting the diacylglycerol iodohydrins with protected 0-phosphoserines, thereby protecting the protected phosphatidylserines formed as intermediates, either simultaneously or in succession, by suitable methods. Instead of phosphatidic acids, phosphatidic acid chlorides can be used in the reaction. Alkyl and alkylene-substituted glycerophosphoserines can be synthesized in an analogous manner (A.J. Slotboom and P.P.

Bonsen, Chem. Phys. Lipids _5(1970), 301 - 398; M. Kates 20 i: E.D. Korn (udgiver) Methods in Membrane Biology,Bonsen, Chem. Phys. Lipids _5 (1970), 301 - 398; M. Kates 20 i: E.D. Grain (Publisher) Methods in Membrane Biology,

Vol. 8, Plenum Press, New York, 1977, s. 119 ff.; H.Vol. 8, Plenum Press, New York, 1977, pp. 119 ff .; H.

Eibl, Chem. Phys. Lipids 26^ (1980), 403 - 429; A. Her-metter, F. Paltauf og H. Hauser, Chem.Phys.Lipids M] (1982), 35 - 45).Eibl, Chem. Phys. Lipids 26 (1980), 403 - 429; A. Her-metter, F. Paltauf and H. Hauser, Chem.Phys.Lipids M] (1982), 35 - 45).

25 Til fremstilling af de hidtil ukendte glycero-3(2)-phos- pho-L-serin-derivater med den almene formel I anvender man fortrinsvis tilsvarende substituerede glycero-3(2 )-phosphorsyrer med den ovenfor viste formel IV, hvori den ene af grupperne D og E er en gruppe med formlen 30 V, hvori X og Y begge er hydroxy.For the preparation of the novel glycero-3 (2) -phospho-L-serine derivatives of the general formula I, preferably substituted glycero-3 (2) -phosphoric acids of the above formula IV are used, wherein the one of groups D and E is a group of formula 30 V in which X and Y are both hydroxy.

I stedet for de frie syrer kan man også anvende de tilsvarende phosphorsyredihalogenider (X og Y er halogen i formlen V), fortrinsvis phosphorsyredichloriderne, som DK 167978 B1 7 man opnår ved at omsætte tilsvarende substituerede glyceroler med phosphoroxyhalogenider. Ued omsætningen af et sådant phosphorsyredihalogenid med formlen IU (X og Y er halogen i formel U) med en beskyttet serin af ty-5 pen vist ved formlen VI opstår som mellemprodukt et på serin-gruppen beskyttet glycero-phospho-L-serin-chlorid af en forbindelse med den almene formel I, som før eller efter isolering og før, under eller efter fjernelse af de beskyttende grupper kan omdannes til en forbin-10 delse med den almene formel I eller et salt deraf.Instead of the free acids, the corresponding phosphoric acid dihalides (X and Y are halogen of formula V) may also be used, preferably the phosphoric acid dichlorides obtained by reacting similarly substituted glycerols with phosphorus oxyhalides. Without reacting such a phosphoric acid dihalide of formula IU (X and Y are halogen in formula U) with a protected serine of the type shown by formula VI, an intermediate glycero-phospho-L-serine chloride protected by the serine group of a compound of the general formula I which, before or after isolation and before, during or after removal of the protecting groups, can be converted into a compound of the general formula I or a salt thereof.

En anden variant af fremgangsmåden ifølge opfindelsen består i, at man omsætter den beskyttede serin med formlen VI med glycerophosporsyreester-halogenider med formlen IV, hvori en af grupperne D eller E betegner en 15 sådan gruppe med formlen V, hvor X er halogen, fortrins vis chlor, og Y er lavere alkoxy eller aryloxy. De som mellemprodukter opnåede serin-gruppe-beskyttede alkyl-eller arylestere af forbindelser med den almene formel I omdannes til de tilsvarende syrer med den almene for-20 mel I eller salte deraf ved sædvanlige kemiske metoder, herunder hydrolyse og fraspaltning af de beskyttende grupper.Another variant of the process of the invention consists in reacting the protected serine of formula VI with glycerophosphoric acid ester halides of formula IV, wherein one of groups D or E represents such a group of formula V wherein X is halogen, preferably chlorine, and Y is lower alkoxy or aryloxy. The serine group-protected alkyl or aryl esters of compounds of general formula I obtained as intermediates are converted to the corresponding acids of general formula I or salts thereof by conventional chemical methods including hydrolysis and decomposition of the protecting groups.

GlycerophosphorsyrealkyKeller aryl)ester-halogeniderne med formlen IV er let tilgængelige gennem omsætning 25 af tilsvarende substituerede glyceroler med phosphorsy- realkyl (eller ary 1)ester-dihalogenider.The glycerophosphoric acid alkyls or the aryl) ester halides of formula IV are readily available through the reaction of similarly substituted glycerols with phosphoric acid alkyl (or ary 1) ester dihalides.

Som beskyttede L-serin-derivater til brug ved fremgangsmåden ifølge opfindelsen egner sig sådanne forbindelser med formlen VI, hvori betegner en inden for peptid-30 kemien brugbar, let fraspaltelig carboxy-beskyttende gruppe, som eksempelvis kan fraspaltes ved katalytisk hydrogenolyse, hydrazinolyse, behandling med HC1 eller natriumthiophenolat eller ved hydrolyse, såsom benzyl, DK 167978 B1 8 tert.-butyl, phthalimidomethyloxy, isopropyl eller benz-hydryl, og hvori Z^ betegner en inden for peptidkemien sædvanlig amino-beskyttende gruppe, der eksempelvis kan fjernes ved katalytisk hydrogenolyse, hydrazino-5 lyse eller behandling med HC1 eller myresyre. Sådanne amino-beskyttende grupper er eksempelvis acylgrupper, såsom formyl, acetyl eller tri fluoracetyl, alkoxycarbo-nylgrupper, såsom ethoxycarbony1, tert.-butoxycarbony1, β,β,β-trichlorethoxycarbonyl eller β-iodethoxycarbonyl, 10 aralkoxycarbony1, såsom benzyloxycarbony1 eller p-methoxy- benzyloxycarbonyl, aryloxycarbonyl, såsom phenoxycarbo-nyl, silylgrupper, såsom trimethylsilyl og andre grupper, såsom trityl, tetrahydropyranyl, vinyloxycarbony1, o-nitrophenylsulfonyl, diphenylphosphinyl, p-toluensul-15 fonyl, benzyl og lignende.As protected L-serine derivatives for use in the process of the invention, such compounds of formula VI, wherein a useful peptide chemistry represents a readily cleavable carboxy protecting group which can be cleaved, for example, by catalytic hydrogenolysis, hydrazinolysis, are suitable. with HCl or sodium thiophenolate or by hydrolysis such as benzyl, tert.-butyl, phthalimidomethyloxy, isopropyl or benzhydryl, and wherein Z 2 represents an amino-protecting group usual within the peptide chemistry which can be removed, for example, by catalytic hydrogen. , hydrazinolysis or treatment with HCl or formic acid. Such amino protecting groups are, for example, acyl groups such as formyl, acetyl or trifluoroacetyl, alkoxycarbonyl groups such as ethoxycarbonyl, tert-butoxycarbonyl, β, β, β-trichloroethoxycarbonyl or β-iodoethoxycarbonyl, aralkoxycarbonyl, or benzyloxycarbonyl, benzyloxycarbonyl, aryloxycarbonyl such as phenoxycarbonyl, silyl groups such as trimethylsilyl and other groups such as trityl, tetrahydropyranyl, vinyloxycarbonyl, o-nitrophenylsulfonyl, diphenylphosphinyl, p-toluenesulfonyl, benzyl and the like.

De beskyttende grupper og Z^ kan vælges vilkårligt, men det er dog i reglen en fordel, at man til beskyttelse af L-serin vælger sådanne kombinationer af beskyttende grupper, som kan fraspaltes i et enkelt reakfcåons-20 trin, idet man eksempelvis som beskyttet serin-derivat med formel VI anvender N-tert.-butyloxy-L-serin-benzhy-drylester eller lignende.The protecting groups and Z 1 may be selected at random, but it is, however, generally advantageous to select such combinations of protective groups which can be cleaved off in a single reaction step, for example as protected serine derivative of formula VI uses N-tert-butyloxy-L-serine benzyl dry ester or the like.

Omsætningen af en forbindelse med formlen IV med en forbindelse med formlen VI, hvorved der dannes et mellempro-25 . dukt i form af et derivat af en forbindelse med den almene formel I, som stadig er beskyttet på serin-gruppen, ifølge metode (a), gennemføres sædvanligvis på en sådan måde, at man omsætter et glycero-3(2)-phosphorsyre-deri-vat med formlen IV, fortrinsvis et omhyggeligt tørret 30 salt af en glycero-3(2)-phosphorsyre med formlen IV, eksempelvis pyridiniumsaltet, med den beskyttede serin med formlen IV. Man anvender et molforhold på mellem 1:1 og 1:5, og man gennemfører omsætningen i nærværelse af en stærk base, såsom pyridin, triethylamin, "Hiining- DK 167978 B1 9 base" eller lignende, og eventuelt også i nærværelse af et inert, ikke-polært organisk opløsningsmiddel, såsom chloroform, ethylacetat, diethylether, diisopro-pylether, benzen, chlorbenzen, tetrahydrofuran eller 5 lignende.The reaction of a compound of formula IV with a compound of formula VI, thereby forming an intermediate sample. Cloths in the form of a derivative of a compound of general formula I still protected on the serine group according to method (a) are usually carried out in such a way as to react a glycero-3 (2) phosphoric acid. derivative of formula IV, preferably a carefully dried salt of a glycero-3 (2) phosphoric acid of formula IV, for example the pyridinium salt, of the protected serine of formula IV. A molar ratio of between 1: 1 and 1: 5 is used and the reaction is carried out in the presence of a strong base such as pyridine, triethylamine, "Hiining-DK 167978 B1 9 base" or the like, and possibly also in the presence of an inert , non-polar organic solvent such as chloroform, ethyl acetate, diethyl ether, diisopropyl ether, benzene, chlorobenzene, tetrahydrofuran or the like.

Omsætningen gennemføres fortrinsvis i nærværelse af et egnet kondensationsmiddel, eksempelvis 2,4,6-triiso-propylbenzensulfonylchlorid, ved en temperatur x området omkring stuetemperatur.The reaction is preferably carried out in the presence of a suitable condensing agent, for example 2,4,6-triisopropylbenzenesulfonyl chloride, at a temperature x the range of room temperature.

10 Fraspaltningen af de beskyttende grupper fra det ved den ovennævnte omsætning opnåede beskyttede mellemprodukt kan, i afhængighed af de valgte beskyttelsesgrupper, foregå ved metoder, der er velkendte for fagmanden. Eksempelvis kan mellemprodukter i form af benzhydryles-15 tere, der er beskyttet med alkoxycarbonylgrupper, over føres i forbindelser med formlen I under fraspaltning af begge beskyttelsesgrupper ved behandling med HC1, fortrinsvis ved at lede HC1 igennem opløsninger af mellemprodukterne i organiske opløsningsmidler. Eventuelle 20 trityl-beskyttelsesgrupper kan fjernes ved hydrogeno- lyse, mens andre beskyttende grupper kan fjernes ved hydrolyse, hydrazinolyse eller lignende.The cleavage of the protecting groups from the protected intermediate obtained by the above reaction may, depending on the selected protecting groups, take place by methods well known to those skilled in the art. For example, intermediates in the form of benzhydryl esters protected by alkoxycarbonyl groups can be transferred into compounds of formula I under the cleavage of both protecting groups by treatment with HCl, preferably by passing HCl through solutions of the intermediates in organic solvents. Any 20 trityl protecting groups can be removed by hydrogenolysis, while other protective groups can be removed by hydrolysis, hydrazinolysis or the like.

Ifølge fremgangsmådevarianten (b) kan man udvinde forbindelserne med den almene formel I fra glycero-3(2)-25 phosphorsyreestere med den almene formel VII gennem en enzymatisk katalyseret forestring ved hjælp af phospholipase D ved i sig selv kendte metoder.According to process variant (b), the compounds of general formula I can be recovered from glycero-3 (2) -25 phosphoric acid esters of general formula VII through an enzymatically catalyzed esterification by phospholipase D by methods known per se.

Man har allerede fremstillet enkelte phospholipider ved hjælp af phospholipase D (H. Eibl et al., Methods 30 in Enzymology, TZ, 1981, 632 - 639), idet man som substra ter ved reaktionen til dels anvendte glycerophosphor-syrealkylestere og hermed analoge forbindelser, som DK 167978 B1 10 ikke var naturligt forekommende. Ved den enzymatiske syntese af phosphatidylseriner ud fra naturlige phospha-tidylcholiner (P. Comfurius et al., Biochim.Biophys.Some phospholipids have already been prepared by means of phospholipase D (H. Eibl et al., Methods 30 in Enzymology, TZ, 1981, 632-639), using as substrates in the reaction partly glycerophosphoric acid alkyl esters and analogous compounds thereof. which DK 167978 B1 10 was not naturally occurring. In the enzymatic synthesis of phosphatidylserines from natural phosphatidylcholines (P. Comfurius et al., Biochim.Biophys.

Acta 488, 1977, 36 - 42) har man hidtil kun kunnet opnå 5 beskedne udbytter af phosphatidylserinerne, og man har især kunne iagttage en hydrolyse under dannelse af phos-phatidsyrer. Man har hidtil ikke anvendt andre substrater end de naturligt forekommende substrater til fremstilling af phosphatidylseriner ved forestring ved hjælp 10 af phospholipase D.Acta 488, 1977, 36 - 42), so far only 5 modest yields of the phosphatidylserines have been obtained, and in particular hydrolysis has been observed to form phosphatidic acids. So far no substrates other than the naturally occurring substrates have been used to prepare phosphatidylserines during esterification by phospholipase D.

I det væsentlige foregår fremstillingen af de omhandlede glycero-3(2)-phospho-L-serin-derivater på den måde, at man lader en forbindelse med den almene formel VII i vandig opløsning eller suspension reagere med L-serin 15 under tilsætning af organiske opløsningsmidler, eksempelEssentially, the preparation of the present glycero-3 (2) phospho-L-serine derivatives is made by reacting a compound of the general formula VII in aqueous solution or suspension with L-serine 15 with the addition of organic solvents, for example

vis ether og/eller chloroform, og en puffer, eksempelvis en natriumacetat- eller tris-puffer, ved en pH-værdi på mellem 4,8 og 8 i nærværelse af et calciumsalt (ved en molaritet, der fortrinsvis er mellem 0,01 og 0,1 20 mol/liter) under tilstedeværelse af phospholipase Dvis ether and / or chloroform, and a buffer, for example a sodium acetate or tris buffer, at a pH of between 4.8 and 8 in the presence of a calcium salt (at a molarity preferably between 0.01 and 0.1 20 mol / liter) in the presence of phospholipase D

ved temperaturer på mellem 10 og 50 °C. En særlig fordel ved den omhandlede fremgangsmåde ligger i, at man ved omsætningen kan anvende ikke-beskyttet serin.at temperatures between 10 and 50 ° C. A particular advantage of the present process lies in the fact that non-protected serine can be used in the reaction.

Efter gennemført omsætning (der eksempelvis kan følges 25 ved tyndtlagschromatografi) inaktiveres enzymet, for trinsvis ved tilsætning af en 0,1 M ethylendiamintetra-eddikesyreopløsning (EDTA), hvorefter det dannede glycero-3(2)-phospho-L-serin-derivat med den almene formel I isoleres og renses på sædvanlig måde, f.eks. ved hjælp 30 af chromatografiske metoder, såsom tyndtlagschromatogra- fi, kolonnechromatografi eller højtryksvæskechromatografi.After completion of the reaction (which can be followed, for example, by thin layer chromatography), the enzyme is inactivated, step by step, by the addition of a 0.1 M ethylenediaminetetraacetic acid solution (EDTA) and the resulting glycero-3 (2) -phospho-L-serine derivative the general formula I is isolated and purified in the usual manner, e.g. by means of chromatographic methods such as thin layer chromatography, column chromatography or high pressure liquid chromatography.

Som allerede anført omfatter den almene formel I alle de mulige chirale og diastereomere former af forbindel- DK 167978 B1 11 seme Ifølge opfindelsen. Såvel ifølge fremgangsmådeva-rianten (a) som ifølge fremgangsmådevarianten (b) kan man opnå enten chirale eller diastereomere slutprodukter i afhængighed af de steriske forhold i udgangsmaterialer-5 ne med formlen IV, henholdsvis formlen VII. Hvis man i en af de to beskrevne fremgangsmådevarianter anvender chirale udgangsmaterialer eller sådanne udgangsmaterialer, som ikke indeholder et chiralitetscentrum, opnår man chirale former af forbindelserne med den almene 10 formel I. Anvender man derimod racemiske udgangsmateria ler med formlen IV eller formlen VII, fører den omhandlede omsætning til blandinger af diastereomere forbindelser med den almene formel I, uanset om man går ud fra et beskyttet L-serin-derivat eller fra L-serin i sig selv.As already stated, the general formula I comprises all the possible chiral and diastereomeric forms of compound according to the invention. Both the process variant (a) and the process variant (b) provide either chiral or diastereomeric end products depending on the steric ratios of the starting materials of formula IV and formula VII, respectively. If one of the two process variants described uses chiral starting materials or such starting materials which do not contain a center of chirality, then chiral forms of the compounds of general formula I are obtained, if racemic starting materials of formula IV or formula VII are used The present invention relates to mixtures of diastereomeric compounds of general formula I, whether from a protected L-serine derivative or from L-serine itself.

15 De ved fremgangsmådevarianterne (a) eller (b) opnåede forbindelser med den almene formel I eller disses farmaceutisk ikke-acceptable salte kan ifølge procestrin (c) overføres i tilsvarende farmaceutisk acceptable salte på sædvanlig måde ved hjælp af uorganiske eller 20 organiske baser. Denne saltdannelse kan eksempelvis gennemføres ved, at man opløser den pågældende forbindelse med formlen I i et egnet organisk opløsningsmiddel, eksempelvis en lavere alifatisk alkohol, tilsætter en ækvivalent mængde af den ønskede base og sørger for 25 en omhyggelig blanding. Efter afsluttet saltdannelse afdamper man opløsningsmidlet i vakuum.The compounds obtained by process variants (a) or (b) of the general formula I or their pharmaceutically unacceptable salts can, according to process step (c), be transferred into corresponding pharmaceutically acceptable salts in the usual manner by means of inorganic or 20 organic bases. This salt formation can be carried out, for example, by dissolving the compound of formula I in a suitable organic solvent, for example a lower aliphatic alcohol, adding an equivalent amount of the desired base and providing a careful mixture. After completion of salt formation, the solvent is evaporated in vacuo.

Farmaceutisk acceptable salte er eksempelvis metalsalte, især alkalimetal- og jordalkalimetalsalte, såsom natrium-, kalium-, magnesium- eller calciumsalte. Som eksemp-30 ler på andre farmaceutisk acceptable salte kan nævnes ammoniumsalte, som er afledt af ammoniak eller organiske aminer, eksempelvis mono-, di- eller tri-lavere -(alkyl, cycloalkyl eller hydroxyalkyl)-aminer, lavere alkylen-diaminer eller heterocycliske baser, f.eks. methylamin, DK 167978 B1 12 diethylamin, triethylamin, dicyclohexylamin, triethanol-amin, ethylendiamin, pyridin, piperidin, piperazin, morpholin og lignende.Pharmaceutically acceptable salts are, for example, metal salts, especially alkali metal and alkaline earth metal salts such as sodium, potassium, magnesium or calcium salts. Examples of other pharmaceutically acceptable salts include ammonium salts derived from ammonia or organic amines, e.g., mono-, di- or tri-lower - (alkyl, cycloalkyl or hydroxyalkyl) amines, lower alkylene diamines or heterocyclic bases, e.g. methylamine, DK 167978 B1 12 diethylamine, triethylamine, dicyclohexylamine, triethanolamine, ethylenediamine, pyridine, piperidine, piperazine, morpholine and the like.

Farmaceutisk ikke-acceptable salte af forbindelser med 5 den almene formel I kan på sædvanlig måde omdannes til farmaceutisk acceptable salte, idet den farmaceutisk ikke-acceptable kation udskiftes med en farmaceutisk anvendelig kation. Som et alternativ hertil kan man neutralisere et farmaceutisk ikke-acceptabelt salt og 10 derefter omsætte den således opnåede frie syre med en base, hvorved der opnås et farmaceutisk acceptabelt salt.Pharmaceutically unacceptable salts of compounds of general formula I can be converted in the usual manner to pharmaceutically acceptable salts, replacing the pharmaceutically unacceptable cation with a pharmaceutically useful cation. As an alternative, a pharmaceutically unacceptable salt can be neutralized and then the free acid thus obtained is reacted with a base to obtain a pharmaceutically acceptable salt.

De glycero-3(2)-phosphorsyre-derivater med den almene formel IV, som anvendes som udgangsmaterialer ved frem-13 gangsmådevarianten (a), er enten i sig selv kendte for bindelser eller forbindelser, der kan fremstilles ved i sig selv kendte metoder (se f.eks. H. Brachwitz et al., Chem. Phys. Lipids 3_1> 1982, 33 - 52, og A. Hermet-ter et al., Chem. Phys. Lipids 3JD, 1982, 35 - 45). Der 20 kan især henvises til de nærmere angivelser i udførelses- eksemplerne i de ovennævnte artikler.The glycero-3 (2) phosphoric acid derivatives of the general formula IV used as starting materials in the process variant (a) are either known per se or as compounds which can be prepared by methods known per se. (see, e.g., H. Brachwitz et al., Chem. Phys. Lipids 3_1> 1982, 33-52, and A. Hermetter et al., Chem. Phys. Lipids 3JD, 1982, 35-45). In particular, reference may be made to the details of the exemplary embodiments of the above articles.

De glycero-3(2)-phosphorsyreestere med den almene formel VII, som anvendes som udgangsmaterialer ved fremgangsmådevarianten (b), er ligeledes enten i sig selv kendte 25 forbindelser eller forbindelser, som kan opnås ved i sig selv kendte metoder (se f.eks. DDR-patentskrifterne nr. 222 594 og nr. 222 595).The glycero-3 (2) phosphoric acid esters of the general formula VII used as starting materials of the process variant (b) are also either compounds known per se or compounds which can be obtained by methods known per se (see f. for example, DDR Patent Nos. 222,594 and 222,595).

En phospholipase D, som egner sig til den enzymatiske omsætning, kan i analogi med allerede kendte metoder 30 opnås på enkelt måde ud fra hvidkål, som man homogeni serer, hvorefter man filtrerer homogenisatet og centrifugerer den vandige fase i 45 minutter ved 25000 g.A phospholipase D suitable for the enzymatic reaction can be obtained, by analogy with already known methods 30, from white cabbage which is homogenized, after which the homogenate is filtered and centrifuged for 45 minutes at 25000 g.

DK 167978 B1 13DK 167978 B1 13

Den klare overvæske omsættes med 2 volumener acetone.The clear supernatant is reacted with 2 volumes of acetone.

Væsken dekanteres fra det dannede bundfald under anvendelse af en glasfritte, og resten centrifugeres i 20 minutter ved 13000 g og en temperatur på 5 °C. Det med 5 acetone fugtede, enziymholdige præparat tørres over phos- phorpentoxid i vakuum.The liquid is decanted from the formed precipitate using a glass frit and the residue is centrifuged for 20 minutes at 13000 g and a temperature of 5 ° C. The 5-acetone-wetted, enzyme-containing preparation is dried over phosphorus pentoxide in vacuo.

Forbindelserne med den almene formel I og saltene deraf er biologisk højaktive, og de har i særdeleshed en udpræget antitumorvirkning. Disse værdifulde farmakologiske 10 egenskaber kan eftervises såvel in vitro som in vivo under anvendelse af standardmetoder, idet man f.eks. bestemmer hæmningen af væksten af Erhlich-Ascites-tu-morceller in vitro under indvirkning af glycero-3(2 )-phospho-L-serin-derivater med den almene formel I.The compounds of general formula I and their salts are biologically highly active and in particular have a pronounced anti-tumor effect. These valuable pharmacological properties can be demonstrated both in vitro and in vivo using standard methods, e.g. determine the inhibition of the growth of Erhlich-Ascites tumor cells in vitro under the action of glycero-3 (2) -phospho-L-serine derivatives of the general formula I.

15 I denne test (tabel I) bevirker de omhandlede forbin delser, som eksempelvis er l-0-hexadecyl-2-chlor-2-desoxy-glycero-3-phospho-L-serin (forbindelse nr. 1) l-0-hexadecyl-2-fluor-2-desoxy-glycero-3-phospho-L-serin 20 (forbindelse nr. 2) l-chlor-l-desoxy-3-0-hexadecyl-glycero-2-phospho-L-serin (forbindelse nr. 3) og 1-0-(2,2,2-trifluorethyl)-2-0-hexadecyl-glycero-3-phos-pho-L-serin (forbindelse nr. 4), 25 allerede i meget ringe koncentrationer en signifikant hæmning af cellevæksten af Erhlich-Ascites-tumorceller.In this test (Table I), the subject compounds, which are, for example, 1-O-hexadecyl-2-chloro-2-deoxy-glycero-3-phospho-L-serine (compound # 1) cause 1- hexadecyl-2-fluoro-2-deoxy-glycero-3-phospho-L-serine (compound # 2) 1-chloro-1-deoxy-3-O-hexadecyl-glycero-2-phospho-L-serine ( compound 3) and 1-O- (2,2,2-trifluoroethyl) -2-O-hexadecyl-glycero-3-phos-pho-L-serine (compound # 4), already at very low concentrations a significant inhibition of the cell growth of Erhlich-Ascites tumor cells.

DK 167978 B1 14DK 167978 B1 14

TABEL ITABLE I

Hæmning af væksten (%) af Erhlich-Ascites-tumorceller in vitro med forbindelser med den almene formel I i afhængighed af koncentrationen 5 Forbindelse nr. Koncentration (^uM) 100 20 10 5 2 1 % hæmning 1 95 80 55 30 10 16 2 95 82 51 18 13 10 10 3 93 81 68 38 17 15 4 100 74 36 10 V 100 62 23 4 0 0 V = sammenligningsforbindelse: l-0-octadecyl-2-G-methyl-glycero-3-phosphocholin.Inhibition of the Growth (%) of Erhlich-Ascites Tumor Cells in vitro with Compounds of General Formula I Depending on the Concentration 5 Compound No. Concentration (µM) 100 20 10 5 2 1% Inhibition 1 95 80 55 30 10 16 2 95 82 51 18 13 10 10 3 93 81 68 38 17 15 4 100 74 36 10 V 100 62 23 4 0 0 V = Comparative Compound: 1- O -octadecyl-2-G-methyl-glycero-3-phosphocholine.

15 Virkningen in vitro af de omhandlede forbindelser er kvalitativt sammenlignelig med den cytostatisk virksomme forbindelse l-0-octadecyl-2-0-methyl-glycero-3-phos-phocholin, som allerede har fundet klinisk anvendelse inden for cancerterapien (P.G. Munder et al. i: "Aug-20 menting Agents in Cancer Therapy", s. 441 - 458, Raven Press, New York, 1981; W.E. Berdel et al. Cancer 50, 1982, 2011 - 2015). I forhold til sammenligningsforbindelsen har forbindelserne ifølge opfindelsen den fordel, at der indtræder en signifikant antitumorvirkning allere-25 de ved væsentligt lavere koncentrationer, således at man for at opnå den samme cytostatiske virkning kan nøjes med at indgive betydeligt mindre dosisenheder.The in vitro action of the compounds of this invention is qualitatively comparable to the cytostatically active compound 1-O-octadecyl-2-O-methyl-glycero-3-phos-phocholine, which has already found clinical use in cancer therapy (PG Munder et al . in: "Aug-20 Mentoring Agents in Cancer Therapy", pp. 441 - 458, Raven Press, New York, 1981; WE Berdel et al. Cancer 50, 1982, 2011 - 2015). Compared to the comparative compound, the compounds of the invention have the advantage of having a significant antitumor effect already at substantially lower concentrations, so that in order to achieve the same cytostatic effect, it is sufficient to administer significantly smaller dosage units.

På grund af disse egenskaber giver de omhandlede forbindelser forventninger om en fordelagtig anvendelse inden 30 for den humane medicin til behandling og forebyggelse af tumorsygdomme.Because of these properties, the compounds of the present invention provide expectations of an advantageous use within the human medicine for the treatment and prevention of tumor diseases.

DK 167978 B1 15DK 167978 B1 15

Alt i alt er den udprægede antitumorvirkning af de omhandlede glycero-3(2)-phospho-L-serin-derivater overraskende, eftersom man hidtil har antaget (se f.eks. D.R.All in all, the pronounced antitumor effect of the glycero-3 (2) -phospho-L-serine derivatives in question is surprising since it has been assumed so far (see, for example, D.R.

Hoffman et al., Research Commun. in Chem. Pathology 5 and Pharmacology 44, 1984, 239 - 306), at antitumorvirk- ningen af alkylphospholipid-analoge er begrænset til forbindelser, som indeholder en phosphocholin-gruppe.Hoffman et al., Research Commun. in Chem. Pathology 5 and Pharmacology 44, 1984, 239-306) that the antitumor effect of alkylphospholipid analogs is limited to compounds containing a phosphocholine group.

De omhandlede forbindelser med formlen I finder anvendelse som lægemidler, og den foreliggende opfindelse 10 angår derfor også et farmaceutisk præparat, som er ejen dommeligt ved, at det indeholder en forbindelse med formlen I eller et farmaceutisk acceptabelt salt deraf i kombination med sædvanlige galeniske hjælpestoffer og/eller bærermaterialer. Hjælpestofferne og bærermate-15 rialerne kan være både organiske og uorganiske, og ti.l enteral eller parenteral indgivelse egner sig f.eks. farmaceutisk harmløse opløsningsmidler, gelatine, gummi arabicum, mælkesukker, stivelse, magnesiumstearat, talkum, planteolier, polyalkylenglycoler, vaseline eller 20 lignende.The present compounds of formula I find use as pharmaceuticals, and the present invention therefore also relates to a pharmaceutical composition which is particularly judgmental in that it contains a compound of formula I or a pharmaceutically acceptable salt thereof in combination with usual galenic excipients and / or carrier materials. The adjuvants and carrier materials can be both organic and inorganic, and for enteral or parenteral administration, e.g. pharmaceutically harmless solvents, gelatin, gum arabic, milk sugar, starch, magnesium stearate, talc, plant oils, polyalkylene glycols, vaseline or the like.

De farmaceutiske præparater kan foreligge i fast form, f.eks. som tabletter, drageer, suppositorier, kapsler eller lignende, eller i flydende form, f.eks. som opløsninger, suspensioner eller emulsioner. De kan eventuelt 25 være underkastet sterilisering, og de kan indeholde hjælpestoffer, såsom konserverings-, stabiliserings-eller emulgeringsmidler, salte til forandring af det osmotiske tryk og lignende.The pharmaceutical compositions may be in solid form, e.g. as tablets, dragons, suppositories, capsules or the like, or in liquid form, e.g. as solutions, suspensions or emulsions. They may optionally be subjected to sterilization and may contain auxiliaries such as preservatives, stabilizers or emulsifiers, salts for changing the osmotic pressure and the like.

De farmaceutiske præparater kan især indeholde de omhand-30 lede forbindelser i kombination med andre terapeutisk værdifulde stoffer. Sammen med sådanne stoffer kan de omhandlede forbindelser være formuleret til kombinations-præparater ved hjælp af de ovenfor angivne hjælpemidler DK 167978 B1 16 og/eller bærerstoffer.In particular, the pharmaceutical compositions may contain the compounds of this invention in combination with other therapeutically valuable substances. Together with such substances, the compounds of the present invention may be formulated for combination preparations by means of the auxiliary agents DK 167978 B1 16 and / or carriers indicated above.

Opfindelsen illustreres nærmere ved de følgende eksempler. EKSEMPEL 1 l-0-Hexadecyl-2-chlor-2-desoxy-glycero-3-phospho-L-serin 5 l-0-Hexedecyl-2-chlor-2-desoxy-glycero-3-phosphorsyre: 335 mg (1 mmol) l-0-hexadecyl-2-chlor-2-desoxy-glycerol opløses i 10 ml tør tetrahydrofuran, og der tilsættes 0,6 ml pyridin, hvorefter den således opnåede opløsning tildryppes en opløsning af 0,35 ml (3,755 mmol) phosphor-10 oxychlorid i 3,5 ml vandfri tetrahydrofuran ved 0 °C under omrøring. Der omrøres i yderligere 3 timer ved 0 QC. Der tilsættes 16 ml af en 10¾ natriumbicarbonat-suspension, og der omrøres i 15 minutter, hvorefter blandingen indstilles til en pH-værdi på 7 ved hjælp 15 af fortyndet saltsyre. Derpå ekstraheres blandingen flere gange med en blanding af ether og chloroform. Ekstrakterne inddampes i vakuum, hvorved man opnår 390 mg (92¾ af det teoretiske udbytte) råprodukt, som er tilstrækkeligt rent til at kunne anvendes i de følgende 20 reaktioner.The invention is further illustrated by the following examples. Example 1 1- O-Hexadecyl-2-chloro-2-deoxy-glycero-3-phospho-L-serine 5-O-Hexedecyl-2-chloro-2-deoxy-glycero-3-phosphoric acid: 335 mg (1 Dissolve 1-O-hexadecyl-2-chloro-2-deoxy-glycerol in 10 ml of dry tetrahydrofuran and add 0.6 ml of pyridine and then drop the solution thus obtained to a solution of 0.35 ml (3.755 mmol) phosphorus oxychloride in 3.5 ml of anhydrous tetrahydrofuran at 0 ° C with stirring. Stir for an additional 3 hours at 0 ° C. Add 16 ml of a 10¾ sodium bicarbonate suspension and stir for 15 minutes, then adjust the mixture to a pH of 7 using 15 of dilute hydrochloric acid. Then the mixture is extracted several times with a mixture of ether and chloroform. The extracts are evaporated in vacuo to give 390 mg (92¾ of theoretical yield) of crude product which is sufficiently pure to be used in the following 20 reactions.

Rf 0,15 (silicagel 60, Alufolie Merckj 01101^/011^01)/25¾ NH3 = 50:25:6, v/v/v).Rf 0.15 (silica gel 60, Alufolie Merckj 01101 ^ / 011 ^ 01) / 25¾ NH3 = 50: 25: 6, v / v / v).

l-0-Hexadecyl-2-chlor-2-desoxy-glycero-3-phospho-N-tert.-butoxycarbonyl-L-serinbenzhydrylester: 25 225 mg (0,54 mmol) l-0-hexadecyl-2-chlor-2-desoxy-glyce- ro-3-phosphorsyre, 0,3 g (0,81 mmol) N-tert.-butoxy-carbonyl-L-serin-benzhydrylester og 0,657 g (2,35 mmol) 2,4,6-triisopropylbenzensulfonylchlorid omrøres med DK 167978 Bl 17 13 ml vandfri pyridin ved stuetemperatur i 36 timer.1-O-Hexadecyl-2-chloro-2-deoxy-glycero-3-phospho-N-tert-butoxycarbonyl-L-serine benzhydryl ester: 225 mg (0.54 mmol) 1- O -hexadecyl-2-chloro 2-deoxy-glycero-3-phosphoric acid, 0.3 g (0.81 mmol) N-tert.-butoxy-carbonyl-L-serine benzhydryl ester and 0.657 g (2.35 mmol) 2.4.6 -Triisopropylbenzenesulfonyl chloride is stirred with DK 167978 B1 17 ml of anhydrous pyridine at room temperature for 36 hours.

Derefter tilsættes nogle dråber vand, og der inddampes i vakuum, hvorpå inddampningsresten efterdesti1leres flere gange med toluen. Resten optages i ether, og blan-5 dingen filtreres og inddampes. Det herved opnåede rå produkt adsorberes på silicagel (35 g, KG 60, Merck 40 - 63 ,/Um) og elueres med 50 ml chloroform efterfulgt af 50 ml af en blanding af chloroform og methanol (9:1).Then, a few drops of water are added and evaporated in vacuo, after which the residue is distilled several times with toluene. The residue is taken up in ether and the mixture is filtered and evaporated. The crude product thus obtained is adsorbed on silica gel (35 g, KG 60, Merck 40-63, µm) and eluted with 50 ml of chloroform followed by 50 ml of a mixture of chloroform and methanol (9: 1).

Der opsamles fraktioner på hver 15 ml. Fraktionerne 10 6-12 sammenlægges og inddampes. På denne måde opnår man 161 mg (38?ό af det teoretiske udbytte) rent produkt.Fractions are collected every 15 ml. Fractions 10 6-12 are combined and evaporated. In this way, 161 mg (38? Of theoretical yield) of pure product is obtained.

Rf: 0,72 (silicagel 60, Alufolie Merck; CHCl^/CH^0H/25?i • NH3 = 65:35:5, v/v/v).Rf: 0.72 (silica gel 60, Alufolie Merck; CHCl3 / CH2OH / 25? NH3 = 65: 35: 5, v / v / v).

l-0-Hexadecyl-2-chlor-2-desoxy-glycero-3-phospho-L-serin: 15 161 mg (0,21 mmol) l-0-hexadecyl-2-chlor-2-desoxy-glyce- ro-3-phospho-N-tert.-butoxycarbonyl-L-serin-benzhydryl-ester opløses i 30 ml tør chloroform, og under omrøring ved 0 °C ledes tør HCl-gas igennem opløsningen i 15 minutter. Derefter ledes tørt nitrogen igennem opløsnin-20 gen i 1 time. Efter denne behandling vaskes opløsnin gen med fortyndet vandig ammoniak og derefter med vand, hvorpå den inddampes.1-O-Hexadecyl-2-chloro-2-deoxy-glycero-3-phospho-L-serine: 161 mg (0.21 mmol) of 1-O-hexadecyl-2-chloro-2-deoxy-glycero Dissolve -3-phospho-N-tert-butoxycarbonyl-L-serine benzhydryl ester in 30 ml of dry chloroform, and with stirring at 0 ° C, dry HCl gas is passed through the solution for 15 minutes. Then, dry nitrogen is passed through the solution for 1 hour. After this treatment, the solution is washed with dilute aqueous ammonia and then with water and then evaporated.

Det herved opnåede råprodukt renses over silicagel (10 g KG 60, Merck 40 - 63 ^um; elueringsmiddel: CHCl^/CH^OH, 25 2:1 v/v med stigende methanol-gradient). Efter forening af de i tyndtlagschromatogrammet ensartede fraktioner og afdampning af opløsningsmidlet opnår man 38 mg rent produkt (36¾ af det teoretiske udbytte).The crude product thus obtained is purified over silica gel (10 g KG 60, Merck 40 - 63 µm; eluent: CHCl 2 / CH 2 OH, 2: 1 v / v with increasing methanol gradient). After combining the uniform fractions in the thin layer chromatogram and evaporation of the solvent, 38 mg of pure product (36¾ of theoretical yield) is obtained.

Rf: 0,13 (silicagel 60, Alufolie Merck; CHC1^/CH^OH/h^O = 30 50:25:4, v/v/v).Rf: 0.13 (silica gel 60, Alufolie Merck; CHCl3 / CH2 OH / h2 O = 30 50: 25: 4, v / v / v).

C22H45CIPN07 (50Z>02) Beregnet: C 52,63 H 9,04 N 2,79C22H45CIPN07 (50Z> 02) Calculated: C 52.63 H 9.04 N 2.79

Fundet : C 51,76 H 8,82 N 2,60.Found: C, 51.76; H, 8.82; N, 2.60.

DK 167978 B1 18DK 167978 B1 18

Analogt med den i eksempel 1 beskrevne arbejdsgang fremstilles de i eksemplerne 2-9 angivne forbindelser: EKSEMPEL 2 l-0-Hexadecyl-2-fluor-2-desoxy-qlycero-3-phospho-L-serin 3 l-0-Hexadecyl-2-fluor-2-desoxy-glycero-3-phosphorsyre:Analogous to the procedure described in Example 1, the compounds of Examples 2-9 are prepared: EXAMPLE 2 1- 0-Hexadecyl-2-fluoro-2-deoxy-glycero-3-phospho-L-serine 3-1-O-Hexadecyl 2-fluoro-2-deoxy-glycero-3-phosphoric acid:

Ud fra 334 mg (1,05 mmol) l-0-hexadecyl-2-fluor-2-desoxy-glycerol. Udbytte: 320 mg (76% af det teoretiske udbytte).From 334 mg (1.05 mmol) of 1-O-hexadecyl-2-fluoro-2-deoxy-glycerol. Yield: 320 mg (76% of theoretical yield).

Rf: 0,15 (silicagel 60, Alufolie Merck; CHClj/CH^OH/255i NH3 = 50:25:6, v/v/v).Rf: 0.15 (silica gel 60, Alufolie Merck; CHCl3 / CH2 OH / 255i NH3 = 50: 25: 6, v / v / v).

10 l-0-Hexadecyl-2-fluor-2-desoxy-glycerO“3-phospho-N-tert.- butoxycarbonyl-L-serin-benzhydrylester:10-O-Hexadecyl-2-fluoro-2-deoxy-glycero-3-phospho-N-tert-butoxycarbonyl-L-serine benzhydryl ester:

Ud fra 184 mg (0,46 mmol) l-0-hexadecyl-2-fluor-2-des-oxy-glycero-3-phosphorsyre. Udbytte: 190 mg (54% af det teoretiske udbytte) rent produkt.From 184 mg (0.46 mmol) of 1-O-hexadecyl-2-fluoro-2-desoxy-glycero-3-phosphoric acid. Yield: 190 mg (54% of theory) of pure product.

15 Rf: 0,75 (silicagel 60, Alufolie Merck; CHCl^/CH^OH/25% NH3 = 65:35:5, v/v/v).Rf: 0.75 (silica gel 60, Alufolie Merck; CHCl3 / CH2 OH / 25% NH3 = 65: 35: 5, v / v / v).

l-0-Hexadecyl-2-fluor-2-desoxy-glycero-3-phospho-L-serin:l-0-hexadecyl-2-fluoro-2-deoxy-glycero-3-phospho-L-serine:

Ud fra 190 mg (0,25 mmol) l-0-hexadecyl-2-fluor-2-desoxy-glycero-3-phospho-N-tert.-butoxycarbonyl-L-serin-benz-20 hydrylester. Udbytte: 88 mg (72% af det teoretiske ud bytte) rent produkt.From 190 mg (0.25 mmol) of 1-O-hexadecyl-2-fluoro-2-deoxy-glycero-3-phospho-N-tert.-butoxycarbonyl-L-serine-benz-hydryl ester. Yield: 88 mg (72% of theoretical yield) pure product.

Rf: 0,12 (silicagel 60, Alufolie Merck; CHClj/Ch^OH/t^O = 50:25:4, v/v/v).Rf: 0.12 (silica gel 60, Alufolie Merck; CHCl 2 / Ch 2 OH / t 2 O = 50: 25: 4, v / v / v).

l-Chlor-l-desoxy-3-0-hexadecyl-qlycero-2-phospho-L-serin EKSEMPEL 3 DK 167978 B1 19 l-Chlor-l-desoxy-3-0-hexadecyl-glycero-2-phosphorsyre:1-Chloro-1-deoxy-3-O-hexadecyl-glycero-2-phospho-L-serine EXAMPLE 3 DK-167978 B1 19 1-Chloro-1-deoxy-3-O-hexadecyl-glycero-2-phosphoric acid:

Ud fra 350 mg (1,05 mmol) l-chlor-l-desoxy-3-O-hexadecyl-5 glycerol. Udbytte: 322 mg (74% af det teoretiske udbytte).From 350 mg (1.05 mmol) of 1-chloro-1-deoxy-3-O-hexadecyl-5 glycerol. Yield: 322 mg (74% of theory).

Rf: 0,18 (silicagel 60, Alufolie Merck; CHCl3/CH30H/25% NH3 = 50:25:6, v/v/v).Rf: 0.18 (silica gel 60, Alufolie Merck; CHCl 3 / CH 3 OH / 25% NH 3 = 50: 25: 6, v / v / v).

l-Chlor-l-desoxy-3-0-hexadecyl-glycero-2-phospho-N-tert.- butoxycarbonyl-L-serin-benzhydrylester: 10 Ud fra 322 mg (0,77 mmol) l-chlor-l-desoxy-3-O-hexadecyl-glycero-2-phosphorsyre. Udbytte: 318 mg (53% af det teoretiske udbytte) rent produkt.1-Chloro-1-deoxy-3-O-hexadecyl-glycero-2-phospho-N-tert.-butoxycarbonyl-L-serine-benzhydryl ester: From 322 mg (0.77 mmol) of 1-chloro-1- deoxy-3-O-hexadecyl-glycero-2-phosphoric acid. Yield: 318 mg (53% of theory) of pure product.

Rf: 0,80 (silicagel 60, Alufolie Merck; CHClj/CHjOH/25% NH3 = 65:35:5, v/v/v).Rf: 0.80 (silica gel 60, Alufolie Merck; CHCl 2 / CH 2 OH / 25% NH 3 = 65: 35: 5, v / v / v).

15 · l-Chlor-l-desoxy-3-0-hexadecyl-glycero-2-phospho-L-serin:· 1-Chloro-1-deoxy-3-O-hexadecyl-glycero-2-phospho-L-serine:

Ud fra 308 mg (0,40 mmol) l-chlor-l-desoxy-3-O-hexadecyl-glycero-2-phospho-N-tert.-butoxycarbonyl-L-serin-benz-hydrylester. Udbytte: 87 mg (43% af det teoretiske udbytte) rent produkt.From 308 mg (0.40 mmol) of 1-chloro-1-deoxy-3-O-hexadecyl-glycero-2-phospho-N-tert.-butoxycarbonyl-L-serine-benz hydrolyster. Yield: 87 mg (43% of theory) of pure product.

20 Rf: 0,13 (silicagel 60, Alufolie Merck; CHC^/Ch^QH/t^O = 50:25:4, v/v/v).Rf: 0.13 (silica gel 60, Alufolie Merck; CHCl3 / Ch2QH / t2 O = 50: 25: 4, v / v / v).

l-Chlor-l-desoxy-3-0-octadecyl-qlycero-2-phospho-L-serin EKSEMPEL 4 DK 167978 B1 20 l-Chlor-l-desoxy-3-0-octadecyl-glycero-2-phosphorsyre:1-Chloro-1-deoxy-3-o-octadecyl-glycero-2-phospho-L-serine EXAMPLE 4 DK-167978 B1 1-Chloro-1-deoxy-3-o-octadecyl-glycero-2-phosphoric acid:

Ud fra 417 mg (1,15 mmol) l-chlor-l-desoxy-3-O-octade-5 cyl-glycerol. Udbytte: 450 mg (89¾ af det teoretiske udbytte).From 417 mg (1.15 mmol) of 1-chloro-1-deoxy-3-O-octadecyl-glycerol. Yield: 450 mg (89¾ of theoretical yield).

Rf: 0,18 (silicagel 60, Alufolie Merck; CHC1 ^/CH^0H/25?0 NH3 = 50:25:6, v/v/v).Rf: 0.18 (silica gel 60, Alufolie Merck; CHCl3 / CH2 OH / 25 NH3 = 50: 25: 6, v / v / v).

l-Chlor-l-desoxy-3-0-octadecyl-glycero-2-phospho-N-tert.-10 butoxycarbonyl-L-serinbenzhydrylester:1-Chloro-1-deoxy-3-o-octadecyl-glycero-2-phospho-N-tert-butoxycarbonyl-L-serine benzhydryl ester:

Ud fra 320 mg (0,72 mmol) l-chlor-l-desoxy-3-O-octade-cy1-glycero-2-phosphorsyre. Udbytte: 258 mg (45¾ af det teoretiske udbytte) rent produkt.From 320 mg (0.72 mmol) of 1-chloro-1-deoxy-3-O-octadecyl-glycero-2-phosphoric acid. Yield: 258 mg (45¾ of theoretical yield) pure product.

Rf: 0,80 (silicagel 60, Alufolie Merck; CHCl^/CH^OH/2550 15 · NH3 = 65:35:5, v/v/v).Rf: 0.80 (silica gel 60, Alufolie Merck; CHCl3 / CH2 OH / 2550 · NH3 = 65: 35: 5, v / v / v).

l-Chlor-l-desoxy-3-0-octadecyl-glycero-2-phospho-L-serin:l-Chloro-l-deoxy-3-0-octadecyl-glycero-2-phospho-L-serine:

Ud fra 250 mg (0,32 mmol) l-chlor-l-desoxy-3-O-octade-cyl-glycero-2-phospho-N-tert.-butoxycarbonyl-L-serin-benzhydrylester. Udbytte: 61 mg (36¾ af det teoretiske 20 udbytte) rent produkt.From 250 mg (0.32 mmol) of 1-chloro-1-deoxy-3-O-octadecyl-glycero-2-phospho-N-tert-butoxycarbonyl-L-serine benzhydryl ester. Yield: 61 mg (36¾ of theoretical 20 yield) pure product.

Rf: 0,13 (silicagel 60, Alufolie Merck; CHCl-j/CH-jOH/^O = 50:25:4, v/v/v).Rf: 0.13 (silica gel 60, Alufolie Merck; CHCl3 / CH2OH = 50: 25: 4, v / v / v).

l-O-Octadecvl-2-fluor-2-desoxy-qlycero-3-phospho-L-serin EKSEMPEL 5 DK 167978 B1 21 l-0-0ctadecyl-2-fluor-2-desoxy-glycero-3-phosphorsyre:1- O-Octadecyl-2-fluoro-2-deoxy-glycero-3-phospho-L-serine EXAMPLE 5 DK-16-O-Octadecyl-2-fluoro-2-deoxy-glycero-3-phosphoric acid:

Ud fra 346 mg (1 mmol) l-0-octadecyl-2-fluor-2-desoxy-5 glycerol. Udbytte: 324 mg (76% af det teoretiske udbytte).From 346 mg (1 mmol) of 1-O-octadecyl-2-fluoro-2-deoxy-glycerol. Yield: 324 mg (76% of theory).

Rf: 0,15 (silicagel 60, Alufolie Merck,· CHCl^/CH^OH/25% NH3 = 50:25:6, v/v/v).Rf: 0.15 (silica gel 60, Alufolie Merck, · CHCl3 / CH2 OH / 25% NH3 = 50: 25: 6, v / v / v).

l-0-0ctadecyl-2-fluor-2-desoxy-glycero-3-phospho-N-tert.-butoxycarbonyl-L-serin-benzhydrylester: 10 Ud fra 307 mg (0,72 mmol) l-0-octadecyl-2-fluor-2-des- oxy-glycero-3-phosphorsyre: Udbytte: 281 mg (50% af det teoretiske udbytte) rent produkt.1-O-octadecyl-2-fluoro-2-deoxy-glycero-3-phospho-N-tert.-butoxycarbonyl-L-serine benzhydryl ester: From 307 mg (0.72 mmol) of 1- O-octadecyl 2-Fluoro-2-desoxy-glycero-3-phosphoric acid: Yield: 281 mg (50% of theory) of pure product.

Rf: 0,75 (silicagel 60, Alufolie Merck; CHCl3/CH30H/25% NH3 = 65:35:5, v/v/v).Rf: 0.75 (silica gel 60, Alufolie Merck; CHCl 3 / CH 3 OH / 25% NH 3 = 65: 35: 5, v / v / v).

15 l-0-0ctadecyl-2-fluor-2-desoxy-glycero-3-phospho-L-serin:1-O-octadecyl-2-fluoro-2-deoxy-glycero-3-phospho-L-serine:

Ud fra 281 mg (0,36 mmol) 1-0-octadecy1-2-fluor-2-desoxy-glycerol-3-phospho-N-tert.-butoxycarbonyl-L-serin-benzhy-drylester. Udbytte: 54 mg (29% af det teoretiske udbytte) rent produkt.From 281 mg (0.36 mmol) of 1-O-octadecyl-2-fluoro-2-deoxy-glycerol-3-phospho-N-tert-butoxycarbonyl-L-serine-benzhydryl ester. Yield: 54 mg (29% of theory) of pure product.

20 Rf: 0,13 (silicagel 60, Alufolie Merck; = 50:25:4, v/v/w).Rf: 0.13 (silica gel 60, Alufolie Merck; = 50: 25: 4, v / v / w).

l-0-0ctadecyl-2-chlor-2-desoxy--qlycero-3-phospho-L-serin EKSEMPEL 6 DK 167978 B1 22 l-0-0ctadecyl-2-chlor-2-desoxy-glycero-3-phosphorsyre:1-O-octadecyl-2-chloro-2-deoxy-glycero-3-phospho-L-serine EXAMPLE 6 DK-16-O-octadecyl-2-chloro-2-deoxy-glycero-3-phosphoric acid:

Ud fra 544 mg (1,5 mmol) l-0-octadecyl-2-chlor-2-desoxy-5 glycerol. Udbytte: 597 mg (90¾ af det teoretiske udbytte).From 544 mg (1.5 mmol) of 1-O-octadecyl-2-chloro-2-deoxy-5 glycerol. Yield: 597 mg (90¾ of theoretical yield).

Rf: 0,16 (silicagel 60, Alufolie Merck; CHCl^/CH^0H/25?o NH3 = 50:25:6, v/v/v).Rf: 0.16 (silica gel 60, Alufolie Merck; CHCl3 / CH2 OH / 25? NH3 = 50: 25: 6, v / v / v).

l-0-0ctadecyl-2-chlor-2-desoxy-glycero-3-phospho-N-tert.~ butoxycarbonyl-L-serin-benzhydrylester: 10 Ud fra 443 mg (1 mmol) l-0-octadecyl-2-chlor-2-desoxy- glycero-3-phosphorsyre. Udbytte: 382 mg (48¾ af det teoretiske udbytte) rent produkt.1-O-Octadecyl-2-chloro-2-deoxy-glycero-3-phospho-N-tert-butoxycarbonyl-L-serine-benzhydryl ester: From 443 mg (1 mmol) of 1-O-octadecyl-2 chloro-2-deoxy-glycero-3-phosphoric acid. Yield: 382 mg (48¾ of theoretical yield) of pure product.

Rf: 0,80 (silicagel 60, Alufolie Merck; CHCl^/CH^OH/25% NH3 = 65:35:5, v/v/v).Rf: 0.80 (silica gel 60, Alufolie Merck; CHCl3 / CH2 OH / 25% NH3 = 65: 35: 5, v / v / v).

15 l-0-0ctadecyl-2-chlor-2-desoxy-glycero-3-phospho-L-serin:1-O-octadecyl-2-chloro-2-deoxy-glycero-3-phospho-L-serine:

Ud fra 382 mg (0,48 mmol) l-0-octadecyl-2-chlor-2-desoxy-glycero-3-phospho-IM-tert.-butoxycarbonyl-L-serin-benz-hydrylester. Udbytte: 102 mg (40¾ af det teoretiske udbytte) rent produkt.From 382 mg (0.48 mmol) of 1-O-octadecyl-2-chloro-2-deoxy-glycero-3-phospho-IM-tert.-butoxycarbonyl-L-serine-benz hydrolyster. Yield: 102 mg (40¾ of theoretical yield) pure product.

20 Rf: 0,13 (silicagel 60, Alufolie Merck; CHClj/CHjOH/l-^O = 50:25:4, v/v/v).Rf: 0.13 (silica gel 60, Alufolie Merck; CHCl₂ / CH₂OH / L₂O = 50: 25: 4, v / v / v).

EKSEMPEL 7 l-Q-Tetradecyl-2-chlor-2-desoxy-qlycero-3-phospho-L-serin DK 167978 B1 23 l-0-Tetradecyl-2-chlor-2-desoxy-glycero-3-phosphorsyre:EXAMPLE 7 1- Q-Tetradecyl-2-chloro-2-deoxy-glycero-3-phospho-L-serine 1-O-Tetradecyl-2-chloro-2-deoxy-glycero-3-phosphoric acid:

Ud fra 368 mg (1,2 mmol) l-0-tetradecyl-3-chlor-2-desoxy-5 glycerol. Udbytte: 445 mg (96% af det teoretiske udbytte).From 368 mg (1.2 mmol) of 1-O-tetradecyl-3-chloro-2-deoxy-5 glycerol. Yield: 445 mg (96% of theoretical yield).

Rf: 0,14 (silicagel 60, Alufolie Merck; CHCl3/CH30H/25% NH3 = 50:25:6, v/v/v).Rf: 0.14 (silica gel 60, Alufolie Merck; CHCl 3 / CH 3 OH / 25% NH 3 = 50: 25: 6, v / v / v).

l-0-Tetradecyl-2-chlor-2-desoxy-glycero-3-phospho-N-tert.- butoxycarbonyl-L-serin-benzhydrylester: 10· Ud fra 425 mg (1,1 mmol) 1-0-tetradecyl-2-chlor-2-desoxy- glycero-3-phosphorsyre. Udbytte: 423 mg (52% af det teoretiske udbytte) rent produkt.1- O-Tetradecyl-2-chloro-2-deoxy-glycero-3-phospho-N-tert.-butoxycarbonyl-L-serine benzhydryl ester: 10 · From 425 mg (1.1 mmol) of 1-O-tetradecyl 2-chloro-2-deoxy-glycero-3-phosphoric acid. Yield: 423 mg (52% of theory) of pure product.

Rf: 0,72 (silicagel 60, Alufolie Merck; CHCl3/CH30H/25% NH3 = 65:35:5, v/v/v).Rf: 0.72 (silica gel 60, Alufolie Merck; CHCl 3 / CH 3 OH / 25% NH 3 = 65: 35: 5, v / v / v).

15 l-0-Tetradecyl-2-chlor-2-desoxy-glycero-3-phospho-L-serin:1-O-Tetradecyl-2-chloro-2-deoxy-glycero-3-phospho-L-serine:

Ud fra 192 mg (0,26 mmol) l-0-tetradecyl-2-chlor-2-des-oxy-glycero-3-phospho-N-tert.-butoxycarbonyl-L-serin-benzhydrylester. Udbytte: 39,5 mg (32% af det teoretiske udbytte) rent produkt.From 192 mg (0.26 mmol) of 1-O-tetradecyl-2-chloro-2-desoxy-glycero-3-phospho-N-tert.-butoxycarbonyl-L-serine benzhydryl ester. Yield: 39.5 mg (32% of theory) of pure product.

20 ' Rf: 0,13 (silicagel 60, Alufolie Merck; CHC13/CH30H/H20 = 50:25:4, v/v/v).20 Rf: 0.13 (silica gel 60, Alufolie Merck; CHCl3 / CH3 OH / H2 O = 50: 25: 4, v / v / v).

l-0-Pentyl-2-chlor-2-desoxy-qlycero-3-phospho-L-serin DK 167978 B1 24 EKSEMPEL 8 l-0-Pentyl-2-chlor-2-desoxy-glycero-3-phosphorsyre:1-O-Pentyl-2-chloro-2-deoxy-glycero-3-phospho-L-serine EXAMPLE 8 1- O-Pentyl-2-chloro-2-deoxy-glycero-3-phosphoric acid:

Ud fra 271 mg (1,5 mmol) 1-0-pentyl-2-chlor-2-desoxy-5 glycerol. Udbytte: 250 mg (64¾ af det teoretiske udbytte).From 271 mg (1.5 mmol) of 1-O-pentyl-2-chloro-2-deoxy-5-glycerol. Yield: 250 mg (64¾ of theoretical yield).

Rf: 0,10 (silicagel 60, Alufolie Merck; CHC1^/CHjOH/25% NH3 = 50:25:6, v/v/v).Rf: 0.10 (silica gel 60, Alufolie Merck; CHCl3 / CH2OH / 25% NH3 = 50: 25: 6, v / v / v).

l-0-Pentyl-2-chlor-2-desoxy-glycero-3-phospho-N-tert.- butoxycarbonyl-L-serin-benzhydrylester: 10 Ud fra 208 mg (0,8 mmol) 1-0-pentyl-2-chlor-2-desoxy- glycero-3-phosphorsyre. Udbytte: 407 mg (83¾ af det teoretiske udbytte) rent produkt.1- O-Pentyl-2-chloro-2-deoxy-glycero-3-phospho-N-tert.-butoxycarbonyl-L-serine-benzhydryl ester: From 208 mg (0.8 mmol) of 1-O-pentyl 2-chloro-2-deoxy-glycero-3-phosphoric acid. Yield: 407 mg (83¾ of theoretical yield) pure product.

Rf: 0,70 (silicagel 60, Alufolie Merck; CHCl3/CH30H/25?i NHj = 65:35:5, v/v/v).Rf: 0.70 (silica gel 60, Alufolie Merck; CHCl3 / CH3OH / 25? NH4 = 65: 35: 5, v / v / v).

15 1-0-Penty1-2-chlor-2-desoxy-giycero-3-phospho-L-serin:1-O-Penty1-2-chloro-2-deoxy-glycero-3-phospho-L-serine:

Ud fra 406 mg (0,66 mmol) l-0-pentyl-2-chlor-2-desoxy-glycero-3-phospho-N-tert.-butoxy-L-serin-benzhydrylester. Udbytte: 64 mg (28¾ af det teoretiske udbytte) rent produkt.From 406 mg (0.66 mmol) of 1-O-pentyl-2-chloro-2-deoxy-glycero-3-phospho-N-tert-butoxy-L-serine benzhydryl ester. Yield: 64 mg (28¾ of theoretical yield) of pure product.

20 Rf: 0,11 (silicagel 60, Alufolie Merck; CHCl^/CH^OH/H^O = 50:25:4, v/v/v).Rf: 0.11 (silica gel 60, Alufolie Merck; CHCl 3 / CH 2 OH / H 2 O = 50: 25: 4, v / v / v).

EKSEMPEL 9 l-0-Hexadecyl-2-desoxy-qlycero-3-phospho-L-serin DK 167978 B1 25Example 9 1- O-Hexadecyl-2-deoxy-glycero-3-phospho-L-serine DK 167978 B1

En blanding af 1 g L-serin, 1,9 ml 0,1 M acetatpuffer (pH 5,6) indeholdende 0,1 M CaC^, 40 mg 1-0-hexadecy1-5 2-desoxy-glycero-phosphorsyreethylester, 2 ml af en blanding af ether og chloroform (9:1, v/v) og 100 mg af et phospholipase-D-præparat, der er udvundet fra ca. 500 g hvidkål, omrøres kraftigt i 40 timer ved 40 °C.A mixture of 1 g of L-serine, 1.9 ml of 0.1 M acetate buffer (pH 5.6) containing 0.1 M CaCl 2, 40 mg of 1-O-hexadecyl-5-2-deoxy-glycero-phosphoric acid ethyl ester, 2 ml of a mixture of ether and chloroform (9: 1, v / v) and 100 mg of a phospholipase D preparation recovered from ca. 500 g of cabbage, stir vigorously for 40 hours at 40 ° C.

Efter afkøling til stuetemperatur tilsættes 4,35 ml 10 0,1 M ethylendiamintetraeddikesyre (EDTA). De organiske opløsningsmidler fjernes ved gennemblæsning med nitrogen. Derefter omrøres blandingen med et 4,3 gange så stort volumen af en blanding af chloroform og methanol (5:8, v/v) i 30 minutter, og den ikke-omsatte serin, som derved 15 skiller sig ud, fjernes ved frasugning.After cooling to room temperature, 4.35 ml of 10 0.1 M ethylenediaminetetraacetic acid (EDTA) is added. The organic solvents are removed by purging with nitrogen. Then the mixture is stirred with a 4.3 times the volume of a mixture of chloroform and methanol (5: 8, v / v) for 30 minutes, and the unreacted serine, which thus separates, is removed by suction.

Filtratet omrøres i 10 minutter med 1 volumen vand og 3,7 volumener chloroform, hvorefter den organiske fase frasepareres og inddampes. Den herved opnåede rest separeres kolonnechromatografisk på 20 g carboxymethylcel-20 lulose (Servacel CM 52), idet man først eluerer med 75 ml chloroform (fraktion 1) og derefter med 500 ml af fire forskellige blandinger af chloroform og methanol (9:1, 8:2, 7:3 og 1:1, v/v), som giver fraktionerne 2-5. Slutproduktet udvindes i ren form fra fraktion 25 5.The filtrate is stirred for 10 minutes with 1 volume of water and 3.7 volumes of chloroform, after which the organic phase is separated and evaporated. The resulting residue is separated by column chromatography on 20 g of carboxymethyl cellulose (Servacel CM 52), first eluting with 75 ml of chloroform (fraction 1) and then with 500 ml of four different mixtures of chloroform and methanol (9: 1, 8). : 2, 7: 3 and 1: 1, v / v) giving the fractions 2-5. The final product is recovered in pure form from fraction 25 5.

Udbytte: 15 mg rent produkt (33?□ af det teoretiske udbytte).Yield: 15 mg of pure product (33? □ of theoretical yield).

Rf: 0,13 (Merck, silicagel 60, færdig plade,· CHC13/CH30H/H20 = 50:25:4, v/v/v).Rf: 0.13 (Merck, silica gel 60, finished plate, · CHCl3 / CH3 OH / H2 O = 50: 25: 4, v / v / v).

DK 167978 B1 26DK 167978 B1 26

Analogt med den i eksempel 9 beskrevne arbejdsgang fremstilles de i eksemplerne 10 - 19 angivne forbindelser.By analogy with the procedure described in Example 9, the compounds of Examples 10-19 are prepared.

EKSEMPEL 10 1-0-Hexadecy1-2-0-(2,2,2-trifluorethyl)-glycero-3-phospho-5 L-serin_EXAMPLE 10 1-O-Hexadecyl-2-O- (2,2,2-trifluoroethyl) -glycero-3-phospho-5-L-serine

Ud fra 40 mg 1-0-hexadecy1-2-0-(2,2,2-trifluorethyl)-glycero-3-phosphocholin. Udbytte: 12 mg (30¾ af teorien) rent produkt.From 40 mg of 1-O-hexadecyl-2-O- (2,2,2-trifluoroethyl) -glycero-3-phosphocholine. Yield: 12 mg (30¾ of theory) pure product.

Rf: 0,13 (Merck silicagel 60, færdig plade; 10 CHC13/CH30H/H20 = 50:25:4, v/v/v).Rf: 0.13 (Merck silica gel 60, finished plate; CHCl3 / CH3 OH / H2 O = 50: 25: 4, v / v / v).

EKSEMPEL 11 l-0-Hexadecyl-2-0-(2,2,2-trifluorethyl)-glycero-3-phos-pho-L-serin_Example 11 1- O-Hexadecyl-2-O- (2,2,2-trifluoroethyl) -glycero-3-phos-pho-L-serine

Ud fra 40 mg l-O-hexadecyl-2-O-(2,2,2-trifluorethy 1) -15 glycero-3-phosphorsyre-2-bromethylester. Udbytte: 13,5 mg (35¾ af teorien) rent produkt.From 40 mg of 1-O-hexadecyl-2-O- (2,2,2-trifluoroethyl) -glycero-3-phosphoric acid-2-bromomethyl ester. Yield: 13.5 mg (35¾ of theory) pure product.

Rf: 0,13 (Merck silicagel 60, færdig plade; CHC13/CH3OH/H20 = 50:25:4, v/v/v).Rf: 0.13 (Merck silica gel 60, finished plate; CHCl3 / CH3OH / H2 O = 50: 25: 4, v / v / v).

EKSEMPEL 12 20 · l-Chlor-l-desoxy-2-0-hexadecyl-qlycero-3-phospho-L-serinEXAMPLE 12 1-Chloro-1-deoxy-2-O-hexadecyl-glycero-3-phospho-L-serine

Ud fra 40 mg l-chlor-l-desoxy-2-0-hexadecyl-glycero-3-phosphocholin. Udbytte: 15 mg (37¾ af teorien) rent produkt.From 40 mg of 1-chloro-1-deoxy-2-O-hexadecyl-glycero-3-phosphocholine. Yield: 15 mg (37¾ of theory) pure product.

DK 167978 B1 27DK 167978 B1 27

Rf: 0,14 (Merck silicagel 60, færdig plade; CHC13/CH30H/H20 = 50:25:4, v/v/v).Rf: 0.14 (Merck silica gel 60, finished plate; CHCl3 / CH3 OH / H2 O = 50: 25: 4, v / v / v).

• EKSEMPEL 13 1-0-(2,2,2-T ri fluorethyl)-2-0-hexadecyl-glycero-3-phos-5 pho-L-serin_EXAMPLE 13 1-O- (2,2,2-Trifluoroethyl) -2-O-hexadecyl-glycero-3-phos-5-pho-L-serine

Ud fra 40 mg 1-0-(2,2,2-trifluorethy1)-2-0-hexadecy1-glycero-3-phosphocholin. Udbytte: 18 mg (44% af teorien) rent produkt.From 40 mg of 1-O- (2,2,2-trifluoroethyl) -2-O-hexadecyl-glycero-3-phosphocholine. Yield: 18 mg (44% of theory) pure product.

Rf: 0,13 (Merck silicagel 60, færdig plade; 10 CHC13/CH3OH/H20 = 50:25:4, v/v/v).Rf: 0.13 (Merck silica gel 60, finished plate; CHCl3 / CH3OH / H2 O = 50: 25: 4, v / v / v).

EKSEMPEL 14 l-0-Eicosanyl-2-chlor-2-desoxy-qlycero-3-phospho-L-serinExample 14 1- O-Eicosanyl-2-chloro-2-deoxy-glycero-3-phospho-L-serine

Ud fra 40 mg l-0-eicosanyl-2-chlor-2-desoxy-glycero-3-phosphorsyre-n-butylester. Udbytte: 14,5 mg (34% af 15 teorien) rent produkt.From 40 mg of 1-O-eicosanyl-2-chloro-2-deoxy-glycero-3-phosphoric acid n-butyl ester. Yield: 14.5 mg (34% of theory) pure product.

Rf: 0,15 (Merck silicagel 60, færdig plade; CHC13/CH30H/H20 = 50:25:4, v/v/v).Rf: 0.15 (Merck silica gel 60, finished plate; CHCl3 / CH3 OH / H2 O = 50: 25: 4, v / v / v).

EKSEMPEL 15 l-0-Triacontyl-2-chlor-2-desoxy-glycero-3-phospho-L-20 serin_____Example 15 1- O-Triacontyl-2-chloro-2-deoxy-glycero-3-phospho-L-20 serine

Ud fra 60 mg 1-0-triacontyl-2-chlor-2-desoxy-glycero- 3-phosphorsyreethylester. Udbytte: 14 mg (33% af teorien) rent produkt.From 60 mg of 1-O-triacontyl-2-chloro-2-deoxy-glycero-3-phosphoric acid ethyl ester. Yield: 14 mg (33% of theory) pure product.

DK 167978 B1 28DK 167978 B1 28

Rf: 0,20 (Merck silicagel 60, færdig plade; CHC13/CH30H/H20 = 50:25:4, v/v/v).Rf: 0.20 (Merck silica gel 60, finished plate; CHCl3 / CH3 OH / H2 O = 50: 25: 4, v / v / v).

EKSEMPEL 16 l-0-0ctadecyl-2-0-(2,2,2-trifluorethyl)-glycero-3-phos-5 pho-L-senn___EXAMPLE 16 1- O-Octadecyl-2-O- (2,2,2-trifluoroethyl) -glycero-3-phos-5-pho-L-sen

Ud fra 40 mg l-0-octadecyl-2-0-(2,2,2-trifluorethyl)-glycero-3-phosphorsyre-2-bromethylester. Udbytte: 13,5 mg (32¾ af teorien) rent produkt.From 40 mg of 1-O-octadecyl-2-O- (2,2,2-trifluoroethyl) -glycero-3-phosphoric acid-2-bromomethyl ester. Yield: 13.5 mg (32¾ of theory) pure product.

Rf: 0,14 (Merck silicagel 60, færdig plade; 10 CHC13/CH30H/H20 = 50:25:4, v/v/v).Rf: 0.14 (Merck silica gel 60, finished plate; CHCl3 / CH3 OH / H2 O = 50: 25: 4, v / v / v).

EKSEMPEL 17 l,2-Di-Q-hexadecyl-qlycero-3-phospho-L-serinEXAMPLE 17 1,2-Di-Q-hexadecyl-glycero-3-phospho-L-serine

Ud fra 40 mg 1,2-di-0-hexadecyl-glycero-3-phosphocholin. Udbytte: 13 mg (32¾ af teorien) rent produkt.From 40 mg of 1,2-di-O-hexadecyl-glycero-3-phosphocholine. Yield: 13 mg (32¾ of theory) pure product.

15 Rf: 0,22 (Merck silicagel 60, færdig plade; CHC13/CH30H/H20 = 50:25:4, v/v/v).Rf: 0.22 (Merck silica gel 60, finished plate; CHCl3 / CH3OH / H2O = 50: 25: 4, v / v / v).

EKSEMPEL 18 1-0-Eicosany1-2-0-(2,2,2-trifluorethyl)-glycero-3-phos-pho-L-serin_ 20 Ud fra 40 mg l-0-eicosanyl-2-0-(2,2,2-trifluorethyl)- glycero-3-phosphocholin. Udbytte: 16 mg (40¾ af teorien) rent produkt.EXAMPLE 18 1-O-Eicosanyl-2-O- (2,2,2-trifluoroethyl) -glycero-3-phos-pho-L-serine 20 From 40 mg of 1-O-eicosanyl-2-0- (2 (2,2-trifluoroethyl) -glycero-3-phosphocholine. Yield: 16 mg (40¾ of theory) pure product.

Rf: 0,14 (Merck silicagel 60, færdig plade; CHC13/CH3OH/H20 = 50:25:4, v/v/v).Rf: 0.14 (Merck silica gel 60, finished plate; CHCl3 / CH3OH / H2 O = 50: 25: 4, v / v / v).

EKSEMPEL 19 29 DK 167978 B1 l-0-(2-Methoxy-octadecyl)-2-chlor-2-desoxy-glycero-3-phospho-L-senn______Example 19 29 DK-167978 B1-1- (2-Methoxy-octadecyl) -2-chloro-2-deoxy-glycero-3-phospho-L-sen ______

Ud fra 50 mg l-0-(2-methoxy-octadecyl)-2-chlor-2-desoxy-5 glycero-3-phosphorsyreethylester. Udbytte: 16 mg (28% af teorien) rent produkt.From 50 mg of 1- (2- methoxy-octadecyl) -2-chloro-2-deoxy-5-glycero-3-phosphoric acid ethyl ester. Yield: 16 mg (28% of theory) pure product.

Claims

Glycero-3 (2) -phospho-L-serine derivatives, characterized in that they have the general formula CH, -AIL CH-B (I) CH 2 -C wherein A represents C , halogen or a group of the general formula - 0 - (CH 2) n - CF 2 (II) 5. which n is 0, 1, 2 or 3 and wherein one of the groups B and C has one of the group A, while the other is a phosphatidyl-L-serine group of the formula 0 '.C00H-O-P-O-CH 2 -CH (III) OH DK 167978 B1 31, provided that at least one of the groups A , B and C represent C 1 -C 2 Q alkoxy, and salts thereof.

A compound according to claim 1, characterized in that at least one of the groups A, B and C represents C1 - C1Q-1610 5 alkoxy.

A compound according to claim 1, characterized in that one of the groups A, B and C represents chlorine or fluorine.

A compound according to claim 1, characterized in that C represents the phosphatidyl-L-serine group of formula III and one of the groups A and B represents C 1 -C 2 alkoxy, while the other represents halogen.

A compound according to claim 1, characterized in that B represents the phosphatidyl1-L-serine group of formula III and one of groups A and C represents C 1 -C 2 alkoxy, while the other represents halogen. .

A compound according to claim 1, characterized in that A represents C ^-C ^ g alkoxy, B represents C ^-C ^ g alkoxy, and C represents the phosphatidyl-L-serine group of formula III.

A compound according to claim 1, characterized in that C represents the phosphatidyl-L-serine group of formula III and one of groups A and B represents C 1 -C 2 alkoxy, while the other represents an terminated 25 fluorinated alkoxy group of formula II.

A compound according to any one of claims 1-7, characterized in that it is in the form of diastereomers. A process for preparing glycero-3 (2) -phos-pho-L-serine derivatives of general formula I according to claim 1, characterized in that one (a) is reacted with a glycero-3 (2). -phosphoric acid derivative of the general formula CHO-AI 2 CH -D (iv) of CH2-E 5 wherein A has the meaning given above and wherein one of groups D and E has one of the meanings given to group A while the another is a group of the general formula - 0 - p = 0 (V) wherein X and Y are either identical and represent hydroxy or halogen, or wherein Y represents lower alkoxy or aryloxy, or a salt thereof, provided that at least one of the groups A, D and E represents C 1 -C 2 alkoxy, with a protected L-serine derivative of the general formula 1 / C00Z1 H0 - CH2 - CH (vi) xnh-z2 wherein is a carboxy protecting group and Z 1 is an amino protecting group, preferably in the presence of a condensing agent and then in any order or contemporaneous γ decomposes the protecting groups Z 2 and I 2 and hydrolyzes any glycerophosphoric acid esters or halides formed, or (b) converts a glycero-3 (2) phosphoric acid ester of the general formula CH M 10 wherein A has the meaning given above and wherein one of groups L and M has one of the meanings given for group A, while the other is a group of the general formula OH / 0- - 0 - p = 0 - 0 -P = 0 / "-OR. 0- (CH2) nN1 R3 (Mia) or (Mb) wherein Rn is C1 -C1 alkyl optionally substituted with hydroxy or halogen and wherein and R4 are the same or various are hydrogen or methyl and n is an integer from 1-6, provided that at least one of groups A, L and M represents C 1 -C 2 alkoxy, with L-serine in the near-20 ' phospholipase D and isolates the compound of general formula I or a salt thereof, and if desired, a compound of formula (I) or (b) obtained according to (a) or (b) unacceptable salt thereof in a pharmaceutically acceptable salt thereof. Pharmaceutical composition, characterized in that it contains a compound of the general formula I according to claim 1 or a pharmaceutically acceptable salt thereof in combination with conventional galenic excipients and / or carriers.