EP0531452A1 - Synthese de derives de diphosphate et de triphosphate de glycerine - Google Patents

Synthese de derives de diphosphate et de triphosphate de glycerine

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Publication number
EP0531452A1
EP0531452A1 EP91912531A EP91912531A EP0531452A1 EP 0531452 A1 EP0531452 A1 EP 0531452A1 EP 91912531 A EP91912531 A EP 91912531A EP 91912531 A EP91912531 A EP 91912531A EP 0531452 A1 EP0531452 A1 EP 0531452A1
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EP
European Patent Office
Prior art keywords
nucleoside
diphosphate
glycerol
group
derivative
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
EP91912531A
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German (de)
English (en)
Other versions
EP0531452A4 (en
Inventor
Henk Ven Den Bosch
Bert Van Wijk
Raj Kumar
Karl Y. Hostetler
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Nexstar Pharmaceuticals Inc
Original Assignee
Vical Inc
Nexstar Pharmaceuticals Inc
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Publication of EP0531452A1 publication Critical patent/EP0531452A1/fr
Publication of EP0531452A4 publication Critical patent/EP0531452A4/en
Ceased legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/06—Pyrimidine radicals
    • C07H19/10—Pyrimidine radicals with the saccharide radical esterified by phosphoric or polyphosphoric acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/06—Phosphorus compounds without P—C bonds
    • C07F9/08—Esters of oxyacids of phosphorus
    • C07F9/09—Esters of phosphoric acids
    • C07F9/10—Phosphatides, e.g. lecithin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/28—Phosphorus compounds with one or more P—C bonds
    • C07F9/38—Phosphonic acids [RP(=O)(OH)2]; Thiophosphonic acids ; [RP(=X1)(X2H)2(X1, X2 are each independently O, S or Se)]
    • C07F9/40—Esters thereof
    • C07F9/4071—Esters thereof the ester moiety containing a substituent or a structure which is considered as characteristic
    • C07F9/409—Compounds containing the structure P(=X)-X-acyl, P(=X) -X-heteroatom, P(=X)-X-CN (X = O, S, Se)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
    • C07F9/6527—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having nitrogen and oxygen atoms as the only ring hetero atoms
    • C07F9/6533—Six-membered rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/16—Purine radicals
    • C07H19/20—Purine radicals with the saccharide radical esterified by phosphoric or polyphosphoric acids
    • C07H19/207—Purine radicals with the saccharide radical esterified by phosphoric or polyphosphoric acids the phosphoric or polyphosphoric acids being esterified by a further hydroxylic compound, e.g. flavine adenine dinucleotide or nicotinamide-adenine dinucleotide

Definitions

  • the present invention relates to an improved chemical synthesis for the preparation of biologically important compounds. More particularly, the present invention concerns an improved method for the synthesis of glycerol di- and triphosphate derivatives, preferably nucleoside di- and triphosphate esters of glycerol lipids, such as nucleoside diphosphate mono- and diglycerides.
  • the glycerol monophosphate amidate intermediates of the new synthesis are novel compounds.
  • nucleoside di- and triphosphate esters of glycerol and glycerol derivatives are known in the art. Among them, nucleoside diphosphate diglycerides are of particular importance due to their role in biochemical processes. The synthesis and the biological importance of a naturally- occurring liponucleotide, cytidine diphosphate diglyceride (CDP-DG) in lipid biosynthesis have been well documented since the early 1960's. In eukaryotes, CDP-DG is a precursor of phosphatidylglycerol, cardiolipin and phosphatidylinositol
  • the efficacy of other clinically used anti-neoplastic pyrimidine nucleosides is limited by kinase activity. Since the release of ara-CMP from ara-CDP-DL-dipalmitin during phosphatidyl ⁇ inositol synthesis is independent of kinase activity, adminis ⁇ tration of ara-C and analogous compounds in the form of phospholipid prodrugs is expected to enhance antitumor activity, and lower toxicity.
  • the nucleotides ara-C, ara-A and TTJ are known chemotherapeutic agents for treatment of various types of cancer.
  • CDP-DG The chemical synthesis of CDP-DG in low yields was first described by Paulus, H. and Kennedy, E.P., J. Biol. Chem. 235, 1303 (1960) , and later by Agranoff and Suomi, Biochem. Prep. .10, 47-51 (1963) .
  • CMP-morpholidate condensed cytidine- ⁇ '-monophosphate-morpholidate
  • DL-diacylglycerol phosphate DL-diacylglycerol phosphate
  • racemic 1-0- hexadecyl-2-0-palmitoylglycero-3-phosphate morpholidate with ara-CMP was allowed to proceed for seven days, and the yield of the desired racemic l-0-hexadecyl-2-0-palmitoylglycero-3- phosphate was reported to be 30%.
  • nucleoside diphosphate diglycerides is modified such that instead of reacting a nucleoside-5'-monophosphate morpholidate with a phosphatidic acid derivative, first the phosphatidic acid derivative is converted into a corresponding amidate, for example morpholidate, which is then reacted with the free acid or salt form of the desired nucleoside-5'-monophosphate, the yields are substantially increased, and the reaction time is significantly shorter.
  • a idates other phosphatidic acid derivatives in which one of the phosphate hydroxyls is replaced by a leaving group, may also be employed with similar results.
  • nucleoside diphosphate diglycerides were synthesized by the improved methods of the present invention, the reaction time was reduced from several days to 3 to 10 hours, and the yield was increased to about 60 to 80%. Furthermore, the purification of the nucleoside diphosphate diglycerides is highly facilitated. When synthesizing the target compounds by the new route, phosphatidic acid is almost completely absent in the reaction mixture, which greatly simplifies and speeds up purification of the desired product. Crude reaction mixtures can easily be purified in a single HPLC procedure, resulting in faster elution, and higher yields of pure compound.
  • the improved results are not limited to nucleoside diphosphate diglyceride synthesis; the synthesis route according to the present invention is generally applicable to the preparation of monoglyceride diphosphate, diglyceride diphosphate and and corresponding triphosphate derivatives of various compounds, such as nucleosides, phosphonoformates, and nucleoside phosphonoformates and analogues thereof.
  • the invention therefore provides an improved process for coupling a monoglyceride or diglyceride monophosphate species to a compound having a terminal phosphate group by means of a pyrophosphate linkage.
  • the present invention relates to an improved method for the synthesis of mono- or diglyceride di- or triphosphate derivatives wherein a phospholipid having the formula
  • R 1 and R 2 are independently hydroxyl or branched or unbranched aliphatic groups having from 1 to 24 carbon atoms and 0 to 6 sites of unsaturation;
  • L is a leaving group, is reacted with a compound having a terminal monophosphate or diphosphate group, in the presence of a basic catalyst, under anhydrous conditions, whereby a glyceride di- or triphosphate derivative is formed; provided that said phospholipid derivative is not a 1-O-alkyl-
  • the leaving group, L is preferably an amine, which can be a morpholino or imidazole group; the process can be carried out at a temperature between about 4°C and 80°C, preferably at room temperature; the preferred solvent for the coupling reaction is pyridine, and anhydrous pyridine is particularly preferred.
  • the present invention concerns a process for the preparation of a glyceride di- or triphosphate derivative of formula (II) wherein
  • A is oxygen, sulfur, or methylene k is 0 or 1;
  • Nu is a nucleoside, or a nucleoside analogue; and salts thereof, comprising: reacting a phospholipid derivative of formula (I) as hereinabove defined, with a mono- or diphosphate having the formula
  • A, Nu, and k are as hereinabove defined, in the presence of a basic catalyst, under anhydrous conditions, whereby a phospholipid nucleoside derivative is formed; providing that when A is oxygen, and k is 0, said phospholipid derivative is not a l-0-alkyl-2-0-acylglycero-3-phosphate morpholidate when said second compound is a nucleoside or nucleoside analogue comprising an adenine, cytosine, 5- fluorouracil, 5-azacytosine, 6-mercaptopurine, or 7- deazaadenine group attached to a pentose which is a ribose or arabinose.
  • a molar ratio between the glyceride monophosphate species and nucleoside reactants is between about 2:1 and about 1:2, preferably between 2:1 and 1:2, and most preferably about 1:1.
  • the preferred basic catalyst is pyridine and the reaction is preferably performed in anhydrous pyridine as a solvent.
  • the reaction time preferably does not exceed 10 hours.
  • the reaction temperature preferably is between about 4"C and about 80°C, most preferably room temperature.
  • the invention includes a further step of purifying the obtained nucleoside diphosphate diglyceride, performed, for example, by high pressure liquid chromatography, or on a DEAE Sephadex ® column.
  • the process can be used in the preparation of naturally occurring complex lipid, for example, any glyceride derivatives of the naturally occurring ribose and 2*- deoxyribose derivatives of adenine, guanine, cytosine and thymine, including the diphosphate diglycerides of cytosine (CDP diglyceride) .
  • the process can be used in the preparation of glyceride derivatives of nucleoside analogues wherein either a purine or pyrimidine base or a sugar moiety is an analogue of a naturally occurring base or sugar.
  • the process is particularly useful in the preparation of lipid derivatives of arabinose containing nucleosides, for example l-(2 , -deoxy-2'- fluoro-l- ⁇ -arabinosyl)-5-iodocytosine (FIAC) ; l-(2'-deoxy-2'- fluoro-l- ⁇ -D-arabinofuranosyl)-5-iodouracil (FIAU) , l-(2'- deoxy-2'-fluoro-l- ⁇ -D-arabinofuranosyl)-5-methyluracil (FMAU) ; 1-(2'-deoxy-2•-fluoro-1- ⁇ -D-arabino-furanosyl)-5-ethylurac
  • the invention further provides an improved process for the preparation of a glyceride phosphate phosphonoacid derivative having the formula
  • D is a -(CH 2 ) m -C(0)0- group; m is 0 or 1; k is 0 or 1;
  • Nu is a nucleoside or a nucleoside analogue; and n is 0 or 1 and salts thereof, comprising: reacting a glyceride monophosphate derivative of formula (I) as hereinabove defined with a phosphonoacid having the formula
  • At least one of R 1 and R 2 has the structure
  • compounds which are diglyceride mono- or diphosphates of nucleosides or nucleoside analogues, or diglycerides of phosphonoacid ⁇ , phosphononucleosides, or phosphononucleoside analogues comprise at least one of R 1 and R 2 having the formula CH 3 - (CH 2 ) a -C(0)0- wherein a is an integer from 10 to 16.
  • the glyceride diphosphate or triphosphate derivatives can be obtained in the form of their salts, for example metal salts.
  • the leaving group in the starting phospholipid derivative preferably is an amino group, most preferably a cyclic amino group, such as a morpholino group or an imidazole group.
  • the present invention relates to the new phospholipid derivatives of the formula (I)
  • L is an amino group. Morpholine is a preferred amino group.
  • Preferred glyceride monophosphate derivatives are l,2-dilauroyl-sn-glycero-3-phosphoro-morpholidate;
  • the present invention relates to a process for the preparation of the new intermediates of formula (I) , wherein the substituents are as defined above, by reacting a phospholipid of formula (VI)
  • Figure 1 illustrates the biosynthesis of phosphatidylinositol (PI) , phosphatidylglycerol (PG) and cardiolipin in mammals via the CDP-DG pathway. All three conversions give rise to the release of cytidine-5•- monophosphate (CMP) .
  • PI phosphatidylinositol
  • PG phosphatidylglycerol
  • CMP cytidine-5•- monophosphate
  • Figure 2 illustrates a preferred embodiment of the chemical synthesis of nucleoside diphosphate diglycerides according to the present invention.
  • the symbols X, Y and n are as defined in the legend.
  • Figure 3 is a comparison of the yields and reaction times of two different syntheses of AZT-5'-diphosphate-(1,2- dimyristoyl)glycerol (AZT-DP-DMG) . Dashed line, Method A: present invention; solid line. Method B: conventional procedure (Agranoff and Suomi, supra) . The figure clearly shows the advantage of Method A. The different yields were obtained quantitatively, based on P £ and UV intensities with HPTLC. The final yields were determined after HPLC purification.
  • Figure 4 shows the HPLC profiles of purifications of AZT- 5'-diphosphate-(1,2-dimyristoyl)glycerol (AZT-DP-DMG) from crude reaction mixtures obtained by Methods A and B, respectively.
  • Solvent n-hexane/2-propanol/25%NH 3 /H 2 0 (43:57:3:7 v/v). Detection at 206 nm; flow: 14 ml/min.
  • A Method A
  • B Method B.
  • AZT-DP-DMG eluted at 12 min.
  • PA phosphatidic acid
  • Figure 5 shows the HPTLC pictures of the crude reaction mixtures obtained by the synthesis of AZT-DP-DMG according to Methods A and B, respectively.
  • Plate A stained with phosphorus reagent; Plate B: ultraviolet detection at 254 nm.
  • Lane 1 Method A after 5 hours.
  • Lane 2 Method A after 10 hours.
  • Lane 3 Method B after 10 hours.
  • Lane 4 Method B after 5 days.
  • the AZT-DP-DMG product is indicated by arrows. Note the large amount of remaining PA in Method B (lanes 3 and 4 in plate A, below the product) .
  • Figure 6 illustrates the time course of the reaction of 3'- deoxythymidine-monophosphate (3dTMP) with the morpholidate of 1,2-dimyristoyl phosphatidic acid (DMPA morpholidate) as analyzed by determining the phosphorus (Pi) content of the different spots after HPTLC by U.V. absorption.
  • nucleoside as used throughout the specification and claims includes naturally occurring nucleosides and their analogues.
  • the naturally occurring nucleoside are those nucleoside species comprising a pyrimidine or purine base e.g., adenine, guanine, cytosine, uracil, inosine, or thymine, linked to a ribose (ribonucleoside) or 2•-deoxyribose (deoxyribonucleoside) 5-carbon cyclic sugar group.
  • Ribonucleosides and deoxynucleosides are phosphorylated at the 5' site and enzymatically assembled into RNA and DNA respectively in vivo.
  • Nucleoside analogues may comprise a naturally occurring purine or pyrimidine base attached to an analogue of the naturally occurring ribose group, an analogue of a purine or pyrimidine base attached to a ribose or 2'-deoxyribose group which is present in naturally occurring nucleosides, or alternatively, both the base and the ribose moieties of the nucleoside analogues may be different from the moieties found in nature.
  • a nucleoside analogue may also comprise either a naturally occurring base or a base analogue attached to a nonribose sugar moiety.
  • Analogs of both the purine or pyrimidine base and the ribose group can differ from a corresponding naturally occurring moiety by having new substituent groups attached thereto, by having naturally occurring substituent groups deleted therefrom, or by having atoms normally present replaced by others.
  • Naturally occurring nucleosides have a purine or pyrimidine base attached to ribose or a ribose residue through the nitrogen in the 9 position of the purines and through the nitrogen in the 1 position of the pyrimidines. These nitrogens are linked by a ⁇ -N-glycosyl linkage to the 1* carbon of the pentose residue.
  • Nucleoside analogues may comprise a purine or pyrimidine base attached to the pentose moiety in a non-naturally occurring linkage such as, for example, through the nitrogen at the 3 position rather than the 1 position of pyrimidine.
  • Nucleoside analogues are believed to have cytotoxic or antiviral effects because they inhibit DNA or RNA synthesis in the proliferation of tumor cells or in the process of viral replication. Specific classes of nucleoside analogues found to have these effects are as follows:
  • Dideoxynucleosides wherein the hydroxyl groups at both the 2' and 3'-position of ribose are replaced by hydrogen for example, 2' ,3 *-dideoxycytidine (ddc) ; 2' ,3'-dideoxyinosine (ddl) ; 2' ,3 •-dideoxyadenosine (ddA) ; 3'-deoxythymidine (3dT) ; and 2' ,3'-dideoxyguanosine (ddG) ; .
  • Dideoxynucleosides are particularly useful in treating retroviral infections such as AIDS, hairy cell leukemia, topical spastic paraparesis and hepatitis B, where viral replication requires the transcription of viral RNA into DNA by viral reverse transcriptase.
  • Acyclic nucleosides wherein the acyclic pentose residue is a fragment of a cyclic pentose, such as an hydroxylated 2-propoxymethyl residue or an hydroxylated ethoxymethyl residue.
  • Particular nucleoside residues having these structures include 2-amino-l,9-dihydro-9-[ (2-hydroxy- ethoxy)methyl]-6H-purine-6-one (acyclovir) or ganciclovir (DHPG) , pencyclovir and famcyclovir.
  • the phosphate groups are generally connected to the 5' carbon of the pentoses in the nucleoside monophosphate reactants in the methods of the present invention, it is important to recognize that in analogues having pentose residues that are not complete pentoses, the phosphate groups are connected to the carbon that would have been the 5' carbon if the pentose were complete. In these pentose fragments, the 2' and/or 3' carbons may be missing; nevertheless, they are considered to be nucleoside derivatives within the meaning of present invention, and the carbon atom to which the phosphate groups are connected will be referred to herein as the 5' carbon for purposes of consistency of usage.
  • 3'-azido-2' .3'-dideoxypyrimidine nucleosides wherein the 3'-hydroxyl of the nucleoside pentose is replaced by N 3 , , for example AZT, AZT-P-AZT, AZT-P-dda, AZT-P-ddi, AzddClU, AzddMeC, AzddMeC N4-0H, AzddMeC N4Me, AZT-P-CyE-dda, AzddEtU(CS-85) , AzddU(CS-87) , AzddC(CS-91) , AzdddFC, AzddBrU, and AzddlU.
  • N 3 for example AZT, AZT-P-AZT, AZT-P-dda, AZT-P-ddi, AzddClU, AzddMeC, AzddMeC N4-0H, Az
  • Arabinose-containing nucleosides wherein the naturally- occurring pentose moiety of the nucleoside, ribose, is replaced by its 2'-epimer, arabinose, which may be in furanose form, for example: 1- (2 ' -deoxy-2 * -f luoro-1- ⁇ -arabinosyl) -5-iodocytosine (FIAC) ; 1- (2 ' -deoxy-2 • -f luoro-1- ⁇ -D-arabinofuranosyl) -5-iodouraci (FIAU) ; l-(2'-deoxy-2'-fluoro-l- ⁇ -D-arabinofuranosyl)-5 methyluracil (FMAU) ; l-(2'-deoxy-2'-fluoro-l- ⁇ -D-arabino furanosyl)-5-ethyluracil (FEAU) ; 9- ⁇ -D-arabinofura
  • 3 ' -halopyrimidine dideoxynucleosides wherein the 3 ' hydroxyl of the nucleoside pentose is replaced by a halogen, usually fluorine, for example 3 ' -f luoro-5-methyl-deoxycytidine (FddMeCyt) , 3 ' -chloro-5-methyl-deoxycytidine (ClddMeCyt) , 3- FddClU, 3-FddU, 3-FddT, 3-FddBrU, and 3-FddEtU.
  • a halogen usually fluorine
  • FddMeCyt 3 ' -f luoro-5-methyl-deoxycytidine
  • ClddMeCyt 3 ' -chloro-5-methyl-deoxycytidine
  • D4T D4C, D4MeC, and D4A.
  • nucleoside analogues may comprise more than one analogous feature, for example, 5-F-ddC; 2 ' , 3 ' -dideoxy-3 ' - fluorothymidine (FddThd) ; 3 ' -f luoro-5-methyl-deoxycytidine (FddMeCyt) ; 3' -chloro-5-methyl-deoxycytidine (ClddMeCyt) ; 3'- amino-5-methyl-deoxycytidine (AddMeCyt) ; ddDAPR(diaminopurine) ; ddMeA(N6 methyl) ; and the class comprising sugar- substituted dideoxypurine nucleosides, for example, 3-N 3 ddDAPR, 3-N 3 ddG, 3-FddDAPR, 3-FddG, 3-FddaraA, and 3-FddA.
  • FUDR 2'-deoxy-5-fluorouridine (Floxuridine ® , Roche Laboratories, Nutley, NJ 07110).
  • Preferred nucleoside analogues for use in preparing lipid derivatives according to the invention are those used in the treatment of AIDS, including 3'-azido, 3 • -deoxythymidine (azidothymidine or AZT) ; 3'-deoxythymidine (3dT) ; 2',3'- dideoxycytidine (ddC) ; 2' ,3'-dideoxyadenosine (ddA) ; and 2' ,3•-dideoxyguanosine (ddG) .
  • AZT, 3dT, ddC, and ddG are most preferred analogues at present.
  • the didehydropyrimidines as well as carbovir, a carbocyclic 2' ,3'-didehydroguanosine, are also preferred.
  • the 3'-azido derivatives of deoxyguanosine (AZG) and the pyrimidine, deoxyuridine, and the 3'-fluoro derivatives of deoxythymidine and deoxyguanosine are preferred as well.
  • the 2' ,6*-diaminopurines the 2•,3*-deoxyriboside and its 3'-fluoro and 3'-azido derivatives are preferred.
  • acyclic sugar derivatives 9-(4,-hydroxy-1' ,2'- butadienyl)adenine (adenallene) and its cytosine equivalent are preferred.
  • Preferred acyclic derivatives having a purine or diaminopurine base are 9-(2-phosphonylmethoxyethyl)adenine and phosphonomethoxyethyl deoxydiaminopurine (PMEDADP) .
  • Stereoisomers of these nucleosides may be advantageous because of their resistance to acid- catalyzed hydrolysis of the glycosidic bond, which prolongs their antiviral activity. In such cases, they are preferred.
  • Diglyceride diphosphate derivatives ofnucleoside analogues having an antiviral effect have been found to be more effective than the nucleoside analogue alone in the treatment of herpes, cytomegalovirus and hepatitis B infections.
  • FIAC 1-(2'-deoxy-2'-fluoro-l- / 3-D- arabinofuranosyl)-5-iodocytosine
  • FIAU 1(2'-deoxy-2'-fluoro- 1- ⁇ -D
  • nucleoside as used in connection with the present invention.
  • nucleoside as used in connection with the present invention.
  • the terms "glycerol monophosphate derivative”, “glycerol diphosphate derivative” and “glycerol triphosphate derivative” and their grammatical variants, as used throughout the specification and claims refer to glycerol derivatives in which one of the glycerol hydroxyls of the structure is replaced by a moiety comprising one, two or three phosphate groups.
  • “Glyceride” include lipid moieties wherein one or both of the glyceryl hydroxyls of the glycerol phosphate derivatives are replaced by an aliphatic group, as defined below.
  • phosphatidic acid is most often used to describe phospholipids in which two hydroxyl groups of the glycerol moiety are esterified by C_. z aliphatic groups and the third one by a phosphate group.
  • this term includes naturally occurring phosphatidic acids, synthetic phosphatidic acid species, and synthetic analogs of phosphatidic acid, including racemic, sn-glycerol-1-phosphate and sn-glycerol-3-phosphate.
  • Naturally occurring phosphatidic acid can be readily obtained by cleavage of plant or animal phosphoglycerides, such as phosphatidylcholine, with phospholipase D [Kates, M. and
  • phosphatidic acid is not a single molecular species, rather is a mixture of various diacylglycerol phosphates.
  • phosphatidic acid is also used to include lyso species, having only one glyceryl hydroxyl replaced by an aliphatic group. It also include those species having one or both glyceryl hydroxyls replaced by aliphatic groups in ether, rather than ester linkage.
  • Phosphatidic acids and their synthetic analogs may, for example, be synthesized as described by Lapidot et al.,
  • aliphatic group is used in the broadest sense to describe non-aromatic groups and is not limited to aliphatic groups containing only hydrogen and carbon.
  • Aliphatic groups including one or more heteroatoms, such as oxygen or sulfur are also within this definition.
  • ester thioester, ether or thioether groups attached to an aliphatic hydrocarbon moiety.
  • a preferred group of phosphatidic acids can be encompassed by the following formula (A)
  • R 1 and R 2 may be the same or different, and are aliphatic hydrocarbon groups having from 1 to 24 carbon atoms, and 0 to 6 sites of unsaturation.
  • the aliphatic hydrocarbon groups represented by R 1 and R 2 preferably have the structure
  • aliphatic groups in acyl ester linkage as shown in formula (A) comprise naturally occurring saturated fatty acids, such as lauric, myristic, palmitic, stearic, arachidic and lignoceric acids, and naturally occurring unsaturated fatty acids, such as palmitoleic, oleic, linoleic, linolenic and arachidonic acids.
  • the aliphatic groups R 1 and R 2 can be branched chains of the same carbon atom number, and comprise primary or secondary alkanol or alkoxy groups, cyclopropane groups, and internal ether linkages.
  • the term "leaving group” is used to refer to any group that is readily removed from the phosphate moiety of the phospholipid derivative (e.g. phosphatidic acid) it is attached to, under the conditions of the condensation reaction with a corresponding compound containing a terminal phosphate group, for example a nucleoside-5'-monophosphate (either in free acid or in salt form) . Since in the synthesis of the present invention amidates are prefereably used, the leaving group preferably is an amino group. However, other leaving groups, such as diphenylphosphate [Heinz et al., Eur. J. Biochem. 184. 445 (1989)], or diphenyl pyrophosphate are also suitable.
  • amino group is used in a broad sense and includes primary, secondary and tertiary amines, for example, aliphatic amines, such as diisopropylamine, triethylamine, tributyla ine (mono-, di- or amines, or aromatic amines, such as diphenylamine, benzidine or toluidines, or heterocyclic amines, such as pyridine, picolines, pyrrole, pyrazole, quinoline, carbazole or quinaldine, in which the nitrogen atom of the amino group is part of a heterocyclic ring.
  • aliphatic amines such as diisopropylamine, triethylamine, tributyla ine (mono-, di- or amines, or aromatic amines, such as diphenylamine, benzidine or toluidines, or heterocyclic amines, such as pyridine, picolines, pyrrole, pyrazole, quino
  • the preferred phosphatidic acid amidate is phosphatidic acid morpholidate, wherein the "amino group" is a morpholino group.
  • suitable amidates include, but are not limited to, imid- azolidate, anisidate, piperidate and l,l'-carbonyl- diimidazole.
  • the phospholipid amidates of the present invention (Formula I) are new compounds, and can be prepared by reacting a corresponding phospholipid, in free acid or salt form, with a suitable amine. The preparation of phosphatidic acid morpholidate is illustrated in the Examples hereinafter.
  • the basic catalyst used in the process of the present invention serves to convert the hydroxyl of the phosphate group to O " , and may, for example, be pyridine or 4'- dimethylaminopyridine. 2. Description of Preferred Embodiments
  • nucleoside diphosphate or triphosphate diglycerides are prepared by reacting corresponding phosphatidic acid morpholidates with nucleoside-5'-monophosphates or -5'- diphosphates in anhydrous pyridine.
  • the phosphatidic acid morpholidates may be prepared and further reacted in a salt form, for example in the form of 4'-morpholine-N,N'- dicyclohexylcarboxamidinium salt, as shown hereinbelow, in the Example.
  • the target nucleoside di- or triphosphate diglycerides can be obtained in the form of their salts, for example, as metal salts, by means of treatment with a base, preferably an inorganic base, as known to those in the art.
  • Phosphatidic acid morpholidates may be prepared from "free" phosphatidic acids and morpholine, preferably in a solvent mixture of chloroform and tert-butanol.
  • the resultant phosphatidic acid morpholidate is lyophilized. Thereafter, the lyophilized morpholidate and the corresponding nucleoside- 5'-monophosphate are dissolved in anhydrous pyridine, and the reaction is allowed to proceed at room temperature.
  • the molar ratio of phosphatidic acid morpholidate and nucleoside-5'- monophosphate typically is between about 2:1 and 1:2, preferably between about 2:1 and 1:1.
  • the progress of the reaction can be monitored by thin layer chromatography (TLC) .
  • the speed of the reaction varies depending on the actual reactants. In some instances optimum conversions is reached in less than an hour. Generally, the reaction is complete within about 5 to 10 hours. The yields typically are between about 60% and about 80%.
  • the obtained nucleoside di- and triphosphate diglycerides are essentially free of phosphatidic acid, which highly simplifies and speeds up their purification. Crude reaction mixtures can easily be purified in a single HPLC procedure, resulting in larger amounts of pure product and faster elution.
  • phosphatidic acid morpholidates may also be synthesized directly from the disodium salt of phosphatidic acid, without prior conversion to the free acid form. This is done under the same reaction conditions as hereinabove described, except that small amounts of methanol/water (1:1 v/v) are usually added to obtain a clear solution. The yields obtained by this variant of the process do not differ significantly from the yields obtained when using free phosphatidic acid as a starting compound. Also, the morpholidate prepared this way reacts equally well with the corresponding nucleoside-5'-mono- or diphosphate.
  • nucleoside diphosphate diglycerides that can be prepared in accordance with the method of the present invention is encompassed by the following formula (II)
  • R x and R 2 independently are hydroxyl or aliphatic groups having from 1 to 24 carbon atoms, and 0 to 6 sites of unsaturation;
  • A is oxygen, sulfur, or methylene, k is 0 or 1; n is 0 or 1; and
  • Nu is a nucleoside or nucleoside analogue.
  • the method of the invention can be used to prepare glycerol, monoglyceride and diglyceride derivatives of naturally occurring nucleosides, for example, adenine diphosphate, and to prepare the naturally occurring intermediate of lipid metabolism, cytidine diphosphate diglyceride.
  • the methods are also useful in preparing diglyceride diphosphate derivatives of cytotoxic and antiviral nucleoside analogues. Particularly preferred are within this group: (3'-azido-3* -deoxy) thymidine-5 ' -diphosphate- ( 1, 2- dilauroyl)glycerol (AZT-DP-DLG) ;
  • diacylglycerol phosphate phosphonoacids are synthesized by preparing the morpholidate of the corresponding phosphatidic acid, and coupling to the corresponding phosphonoacid, which can be phosphonoformate or phosphonoacetate.
  • the new synthesis is adapted for the preparation of diacylglycerol phosphate phosphonoacids to which nucleosides including those having a cytotoxic or antiviralactivity are coupled, for example, by a carboxyl ester linkage.
  • Preferred phosphonoacid derivatives are 1,2-dilauroylglycero- 3-phosphate-(pyro)-phosphonoformate; or 1,2-dimyristoylglycero-3-phosphate-(pyro)-phosphonoformate.
  • the chemical reactions described above are generally disclosed in terms of their broadest application to the methods of the invention. Occasionally, the reactions may not be applicable as described to the synthesis of each compound suggested within the disclosed scope. The compounds for which this occurs will be readily recognized by those skilled in the art. In all such cases, either the reactions can be successfully performed by conventional modifications known to those skilled in the art, e.g., by appropriate protection of interfering groups, by changing to alternative conventional reagents, or by routine modification of reaction conditions. In all preparative methods, all starting materials are known or readily preparable from known starting materials.
  • Dilauroyl and dimyristoyl phosphatidic acids, disodium salts were obtained from Avanti Polar lipids (Pelham, AL, USA) .
  • the reaction was mostly completed within 45 to 75 minutes as judged by this method, and the reaction product was hydrolyzed and neutralized with 2 volumes of aqueous sodium hydroxide to a final pH of 7. Purification was as described above for the analysis of the reaction mixture. By this method, 10-20 mg of nucleoside-5'- monophosphate could be purified. Larger amounts were purified on a Sepharose Q fast flow column using the same elution conditions. Yields varied between 80 and 96% after repeated lyophilization from water.
  • Phosphatidic acids, di-sodium salts were acidified by application of an extraction procedure according to Bligh and Dyer, Can. J. Biochem. 37. 911-917 (1959) .
  • 1 mmol of lipid was dissolved in a homogenous mixture of 100 ml CHC1 3 , 200 ml MeOH, 100 ml 0.1 M HC1 and stirred at room temperature for one hour.
  • 100 ml H 2 0 and 100 ml CHC1 3 were added, the separated CHC1 3 layer was isolated and the aqueous phase was extracted twice with 200 ml CHC1 3 .
  • the combined CHC1 3 extracts were evaporated to dryness and lyophilized. Yield: 95-100% phosphatidate as the free acid.
  • the reaction was monitored by thin-layer chromatography using silica 60 F254 HPTLC plates and CHCl 3 /MeOH/25% NH 3 /H 2 0 (70:38:8:2 v/v) as developing system.
  • the reaction mixture was taken to dryness and suspended in 50 ml H 2 0 and transferred to a dropping funnel. The suspension was extracted three times with diethylether, evaporated to dryness and lyophilized.
  • Method B From phosphatidic acid,disodium salt: This reaction was performed essentially as described above. Sometimes, however, the reaction mixture had to be clarified by the addition of a minimum amount of methanol/water (1:1, v/v) . The aqueous phase was extracted with chloroform or diethylether, evaporated, lyophilized and used in the condensation reaction without further purification.
  • E. Synthesis of Nucleoside-Diphosphate-Diglvcerides Lyophilized mixtures of phosphatidic acid morpholidates and nucleoside-5' monophosphates were dissolved in pyridine and evaporated to dryness, only letting N 2 into the apparatus.
  • AZT-DP-DMG has also been synthesized on a 50 ⁇ molar scale in a 1:1 ratio of DMPA-morpholidate and AZT-5 '-monophosphate with similar yields.
  • the crude reaction products were purified without further processing.
  • the lyophilized reaction mixtures were dissolved in elution solvent or, alternatively, in a 1:1 (v./v.) mixture of chloroform and methanol, and purified by means of HPLC, using a silica ⁇ Porasil ® column (Waters Associates Inc. , Milford, MA. USA; 19mm (I.D.) x 30 cm (length)) and the solvent system hexane/2-propanol/25% NH 3 /H 2 0 (43:57:3:7 v/v), [Geurts van Kessel, et al., Biochim. Biophys. Acta 486. 524- 530 (1977)]. Detection was performed by UV absorption at 206 nm. By this method 50-100 mg of crude product could be purified in half an hour.
  • Fig. 3 a comparison of the yields and reaction times of the synthesis of AZT-DP-DMG is made between the two condensation procedures for the preparation of nucleoside- diphosphate-diglycerides.
  • Method A is the process according to the present invention
  • Method B is the procedure that has been used widely in the literature, namely the condensation of phosphatidic acid and a nucleoside-5'- monophosphoromorpholidate (Agranoff et al., supra) .
  • the reactions were performed on scales varying from 0.05 to 0.5 mmolar, followed qualitatively by HPTLC and the yields quantified by weighing the product after purification by HPLC, as described.
  • Figs. 4A and 4B show pictures of HPLC purifications of compound 1, which were synthesized by Method A and B respectively.
  • Fig. 5 shows HPTLC pictures of the reaction mixtures. Comparison of both HPTLC and HPLC profiles shows the almost complete absence of phosphatidic acid (Fig. 4A and Fig, 5A, lanes 1 and 2) , or its abundant presence (Fig. 4B and Fig 5A, lanes 3 and 4) in the respective reaction mixtures. Also the enrichment of the desired product in the reaction mixture of Method A when compared to that of Method B is clearly visualized both with respect to phosphate- containing and UV-positive compounds.
  • nucleoside-diphosphate-diglycerides with potential anti- retroviral activity by a new method, which is based on the condensation of a l,2-diacyl-sn-glycero-3-phosphoro- morpholidate and a nucleoside-5'-monophosphate.
  • the method seems to be applicable generally for the synthesis of these compounds, independent of the nature of the nucleoside.
  • the method has several advantages over the state of art procedure.
  • Method A Acyclovir-Diphosphate(l,2-Dimyristoyl)Glycerol
  • dimyristoylphosphatidic acid (Avanti Polar Lipids, Birmingham, AL) was converted to free acid as described in Example 1, Part C.
  • Dry dimyristoylphosphatidic acid was converted to the corresponding morpholidate as described in Example 1, Part D.
  • 1.48 g of the lyophilized morpholidate compound and 0.610 g of dry acyclovir monophosphate were combined in 50 ml of dry pyridine, and evaporated to dryness under vacuum on a rotary evaporator. Finally, 50 ml of dry pyridine was added and concentrated to approximately 20 ml.
  • acyclovir monophosphate To bring acyclovir monophosphate into solution required the addition of 10 ml of anhydrous dimethyl sulfoxide (DMSO) and heating the reaction vessel to 85°C for 2 hours and at 45°C for an additional 16 hours.
  • DMSO dimethyl sulfoxide
  • Purified acyclovir-5'-diphosphate-(l, 2 - dimyri ⁇ toyl)glycerol was isolated by HPLC as described in Example 1, eluting from the column at 18-20 minutes. The fractions were combined and lyophilized to yield a white powder.
  • acyclovir diphosphate diglycerides may be purified by DEAE sephadex column chromatography as noted in Example 4 below.
  • the compound was dissolved in chloroform/methanol (1:1 v/v) and spotted at the origin of a silica gel G plate and developed with chloroform/ methanol/concentrated ammonia (70:38:8 v/v) .
  • the product gave a U.V. and phosphorus positive spot with an Rf value of 0.23.
  • Method B Acyclovir-Diphosphate(l,2-dipalmitoyl)glycerol (ACV-DP-DPG) Dipalmitoyl phosphatidic acid morpholidate (DPPA morpholidate) was prepared as described in Example 1, using the sodium salt of phosphatidic acid directly for activation.
  • ACV-DP-DPG Acyclovir-Diphosphate(l,2-dipalmitoyl)glycerol
  • DPPA morpholidate Dipalmitoyl phosphatidic acid morpholidate
  • TBA tributylamine
  • TOA trioctylamine
  • the combined chloroform layers were evaporated to dryness and dissolved in 15 ml warm chloroform/methanol/25% ammonia/water (70:38:8:2, v/v) as developing system, were pooled, evaporated to dryness, and lyophilized.
  • the compound has a fatty acid to P ratio (Shapiro, B. , Biochem. J. 53:663 (1953)) of 1.05, confirming the absence of PA.
  • Example 2 The methods of Example 2 are particularly suitable for guanosine-containing nucleosides or nucleoside analogues that are relatively difficult to solubilize.
  • EXAMPLE 3
  • ACV-diphosphate (1-O-octadecyl, 2-acetyl)glycerol is purified as described above using a column of Q-Sepharose ® eluted with a linear gradient of chloroform/methanol/0.25M NH 4 HC0 3 (2:3:1, v/v).
  • the fractions containing pure ACV diphosphate (1-O-octadecyl, 2-acetyl)glycerol were combined and evaporated to dryness.
  • the product was taken up in a small volume of chloroform/methanol (1:1) and treated with methanolic KOH as described by Chang and Kennedy, J. Biol. Chem.
  • Dipalmitoylphosphatidic acid (950 mg, 1.47 mmol) was prepared from its disodium salt, essentially as described in Example 1, Part C. Free phosphatidic acid was dissolved in 30 ml chloroform, and the obtained solution was transferred to a two-neck round bottom flask, which contained 30 ml tert- butanol, morpholine (0.53 ml, 6 mmol), and distilled water (0.1 ml, 6 mmol). This mixture was gently refluxed and a solution of dicyclohexylcarbodiimide (1.20 g, 5.9 mmol) in 30 ml tert-butanol was added stepwise from a dropping funnel within 2 hours.
  • the solvent was evaporated under vacuum and the residue was added to 50 ml water.
  • This aqueous suspension was extracted five-times with 75-ml portions of chloroform.
  • the chloroform layers were collected and evaporated to dryness and then lyophilized from cyclohexane three times to yield a white foam. This compound was used without further purification in the subsequent synthesis steps.
  • FIAU-MP l-(2'-deoxy-2'-fluoro- ⁇ -D-arabinofuranosyl)- 5'-monophosphate
  • FIAU-MP precipitated as a white crystal.
  • the supernatant was discarded and the precipitate was washed with anhydrous ether (5x10 ml) .
  • the precipitate was redissolved in water (20 ml) and washed with chloroform (3x20 ml) .
  • the aqueous layers were combined and lyophilized to yield crude FIAU-MP (800 mg, 1.83 mmol, 85% yield) .
  • HPLC retention time of FIAU-MP was 15.3 min using a 250 x 4.6 mm, 5 micron Brownlee silica column eluted with hexane:2-propanol:ammonium hydroxide:water (43:57:3:7, v/v).
  • the compound had an Rf of 0.32 on silica 60A F254 TLC plate eluted with acetic acid:n-butanol:water (1:4:1, v/v).
  • UV ⁇ 254 nm (hexane:2-propanol:ammonium hydroxide:water,
  • anhydrous 1,2-dipalmitoyl- sn-glycero-3-phosphoromorpholidate 400 mg, 0.55 mmol
  • FIAU-MP 200 mg, 0.48 mmol
  • anhydrous pyridine 15 ml
  • the solution was evaporated to dryness in vacuum 5-times from anhydrous pyridine, and then 7 ml of anhydrous pyridine were added. This solution was stirred at room temperature overnight under argon. The progress of the reaction was monitored by TLC (chloroform:methanol:ammonium hydroxide:water, 70:38:8:2, v/v).
  • reaction mixture was then evaporated from toluene (4x10 ml) .
  • This residue was dissolved in 15 ml of chloroform:methanol:water (2:3:1, v/v), and acidified to pH 3 with 0.1N hydrochloric acid. Two layers formed, and the aqueous layer was washed with chloroform (2x10 ml) .
  • the combined organic layers were evaporated to dryness, and the residue was dissolved in chloroform:methanol:water (2:3:1, v/v) and applied to a DEAE Sephadex (acetate form) column (2.8 x 30 cm).
  • HPLC retention time of FIAU-DP-DPG diammonium salt was 12.65 min. using a 250x4.6 mm, 5 micron Brownlee silica column eluted with hexane:2-propanol:ammonium hydroxide: water (43:57:3:7, v/v) as the developing system.
  • the compound had an Rf of 0.23 on silica 60A F254 TLC plate eluted with chloroform:methanol:ammonium hydroxide:water (70:28:8:2, v/v) .
  • DMPA dimyristoylphosphatidic acid
  • the sodium salt of phosphonoformic acid (PFA) was converted to the acid form by passage through a Dowex AG50W-H+ column (Biorad, Richmond, CA) .
  • the acid form was lyophilized overnight and 120 mg was added to a reaction vessel which contained DMPA morpholidate (125 mg) dissolved in 5 ml of dry chloroform and 1 ml of dry pyridine.
  • the reaction was sealed under nitrogen and stirred overnight at room temperature.
  • the reaction was stopped by the addition of 10 ml of chloroform/methanol/water (1/2/0.8 by volume) and the chloroform layer was removed after further addition of 2.5 ml each of chloroform and water.
  • the organic (lower) phase was dried over sodium sulfate, evaporated, and purified on silica gel G thin layers developed with a solvent system of chloroform/methanol/20% aqueous methylamine (60/30/10 by volume).
  • the purified product had an Rf of 0.33.
  • Dimyristoyl phosphatidic acid morpholidate (DMPA morpholidate) and 3'-deoxythymidine monophosphate (3dTMP) were prepared essentially following the process described in Example 1. In this particular case, 650 ⁇ mol DMPA morpholidate was condensed with 350 ⁇ mol 3dTMP in 10 ml pyridine.
  • AZTDP-DG it has been shown that both 2:1 and 1:1 ratios of PA morpholidate and AZT-MP give rise to comparable yields. Analysis of the reaction course:
  • Figure 6 illustrates the time course of the reaction as analyzed by P ⁇ content of the different spots:
  • the reaction is essentially completed within 30 minutes as indie ated by the amount of 3dTDP-DG formed (A) (71%) and the sharp decrease in the amounts of 3dTMP (E) and DMPA morpholidate (D) .
  • the yield of 3dtDP-DMG does not improve and the amounts of by-products increase as a result of further reaction of DMPA morpholidate (descending curve D, rising curves B and C) .
  • the yields of the syntheses described are between about 50% and 80%, primarily depending on losses in purification.
  • the formation of by-product is controlled by terminating the reaction within a few hours.

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Abstract

Procédé servant à la préparation de dérivés de glycérophospholipides comprenant le couplage du groupe phosphate d'un dérivé de monophosphate de glycérine dans lequel un des hydroxyles de phosphate est remplacé par un groupe partant, avec le groupe phosphate de terminaison d'un composé de mono- ou de diphospate ou un de ses sels, en présence d'un catalyseur basique, dans des conditions anhydres.
EP19910912531 1990-05-29 1991-05-29 Synthesis of glycerol di- and triphosphate derivatives Ceased EP0531452A4 (en)

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