EP2424874A1 - Als substrate für polymerasen und antivirale mittel geeignete neue phosphatmodifizierte nukleoside - Google Patents

Als substrate für polymerasen und antivirale mittel geeignete neue phosphatmodifizierte nukleoside

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Publication number
EP2424874A1
EP2424874A1 EP10725631A EP10725631A EP2424874A1 EP 2424874 A1 EP2424874 A1 EP 2424874A1 EP 10725631 A EP10725631 A EP 10725631A EP 10725631 A EP10725631 A EP 10725631A EP 2424874 A1 EP2424874 A1 EP 2424874A1
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EP
European Patent Office
Prior art keywords
phosphoramidate
group
structural formula
hydrogen
represented
Prior art date
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Application number
EP10725631A
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English (en)
French (fr)
Inventor
Piet Herdewijn
Philippe Marlière
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Katholieke Universiteit Leuven
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Katholieke Universiteit Leuven
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Publication date
Priority claimed from GB0907436A external-priority patent/GB0907436D0/en
Priority claimed from US12/549,117 external-priority patent/US8242087B2/en
Priority claimed from GB0921664A external-priority patent/GB0921664D0/en
Application filed by Katholieke Universiteit Leuven filed Critical Katholieke Universiteit Leuven
Publication of EP2424874A1 publication Critical patent/EP2424874A1/de
Withdrawn legal-status Critical Current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
    • C07H19/16—Purine radicals
    • C07H19/20—Purine radicals with the saccharide radical esterified by phosphoric or polyphosphoric acids
    • 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
    • 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 novel phosphate-modified nucleosides, such as carboxylyc acid containing phosphoramidate nucleosides.
  • the present invention also relates to the phosphate-modified nucleosides as substrates for wild type and/or mutated DNA or RNA polymerases.
  • the present invention provides for the use of these novel phosphate-modified nucleosides for the production of oligonucleotides such as DNA or RNA and of polypeptides or proteins.
  • the invention also relates to the use of these phosphate- modified nucleosides for growing or selecting specific micro-organisms, such as bacteria.
  • the invention further provides for the use of these novel phosphate- modified nucleosides to treat or prevent viral infections and their use to manufacture a medicine to treat or prevent viral infections, particularly infections with viruses belonging to the HIV family.
  • the present invention furthermore relates to a method for the production of oligonucleotides, peptides or proteins by using said phosphate-modified nucleosides.
  • nucleotide analogues bearing a modified nucleobase moiety or unnatural sugar and that are substrates for polymerases. Modifications at the phosphate moiety are introduced to increase the stability of a nucleotide toward enzymatic degradation or to mask the phosphate negative charge and facilitate its penetration into a cell.
  • Common strategy in nucleotide prodrug design is protecting a phosphate moiety with a labile masking group. Removal of a masking group liberates a nucleoside monophosphate entity to be transformed into a nucleoside triphosphate (hereinafter referred as NTP), a substrate for intracellular enzymes.
  • NTP nucleoside triphosphate
  • nucleoside monophosphate Even after removal of the masking group, phosphorylation and activation of nucleoside monophosphate remains a problem due to substrate specificity of cellular kinases. Therefore, design of a nucleotide analogue that would allow bypassing the kinase activation pathway while behaving as a direct polymerase substrate would be a considerable challenge.
  • HIV RT HIV reverse transcriptase
  • the function of this enzyme is to use a viral RNA genome and a reverse transcriptase to synthesize a double stranded DNA for integration into a host genome. Because this step is critical for the propagation of the viral infection, HIV reverse transcriptase (RT) is an excellent target for anti-viral treatment.
  • RTIs RT inhibitors
  • Non-nucleoside reverse transcriptase inhibitors are a group of compounds that act through the allosteric inhibition by binding to a hydrophobic site, or a pocket in close proximity to the active site of HIV RT.
  • the other group of RTIs is represented by nucleoside reverse transcriptase inhibitors (NRTIs) that bind directly to the active site and interfere with the polymerization reaction and DNA synthesis.
  • NRTIs nucleoside reverse transcriptase inhibitors
  • Nucleoside reverse transcriptase inhibitors are designed to be recognized as substrates for RT and incorporated into a growing strand for further termination of chain elongation. Inhibition of reverse transcriptase activity and chain termination by NRTIs is achieved by introduction of structural modifications to the sugar moiety.
  • the elongation of the DNA strand by a polymerase requires a nucleophilic attack of the 3'- OH group to the ⁇ phosphorus atom of an incoming nucleotide. Therefore, nucleoside analogs that lack the 3'-OH group or have it substituted with other functional groups (for instance, N 3 , F, H) not capable of the nucleophilic attack and formation of phosphodiester bond would act as chain terminators.
  • nucleoside analogues Termination of DNA or RNA synthesis with nucleoside analogues is a common and one of the most efficient strategies in the treatment of viral infections, regardless of various side effects and cell toxicity.
  • the therapeutically active form of a nucleoside analogue is a nucleoside triphosphate.
  • nucleoside triphosphates At the physiological pH nucleoside triphosphates are negatively charged molecules and thus they can not penetrate cellular membranes.
  • RT inhibitors are usually administered as biologically inactive free nucleosides or as monophosphate pro-drugs where a phosphate group is masked with a lipophilic group.
  • kinase-mediated activation of anti-viral nucleosides There are three steps of kinase-mediated activation of anti-viral nucleosides. At first, transformation to a monophosphate derivative takes place through the action of a cytoplasmic nucleoside kinase (for instance, thymidine kinase and deoxycytidine kinase). Furthermore, a nucleoside 5'-monophosphate kinase catalyzes the conversion of a nucleoside monophosphate to a nucleoside diphosphate. Finally, a diphosphate derivative is phosphorylated by a nucleoside 5'-diphosphate kinase (NDK) to provide an anti-viral nucleoside analog in its activated (phosphorylated) form.
  • NDK nucleoside 5'-diphosphate kinase
  • TMPK thymidylate kinase
  • NRTIs which often relies on intracellular phosphorylation and activation
  • a prodrug or pronucleotide approach In the prodrug approach, the monophosphate moiety is "masked" with a labile functional group which also serves to facilitate passage of a "masked” nucleotide inside the cell. Once inside the cell, a masking group is removed either enzymatically or through chemical activation. Removal of the masking group affords a free nucleoside monophosphate intracellular ⁇ where it can be further phosphorylated by TMPK and NDK.
  • the prodrug approach facilitates delivery of an inhibitory nucleoside inside the cell and eliminates the need for initial phosphorylation by a nucleoside kinase, phosphorylation by TMPK and NDK are still required.
  • HSV herpes simplex virus
  • WO 00/47591 discloses phosphoramidates of 4-(6-amino-purin-9-yl)-2- cyclopentene-1 -methanol being useful in treating viral infections. These compounds however do not include a ribose or deoxyribose sugar moiety as required in any nucleoside.
  • WO 2007/020193 discloses antiviral nucleoside phosphoramidates wherein one carbon atom of the ribose or deoxyribose sugar moiety is substituted with a group selected from the group consisting of azido, ethynyl and chloroethenyl, and wherein the nitrogen atom of said phosphoramidate is substituted with hydrogen or C- 1 -3 alkyl. These compounds however do not include any pending aryl or carboxylic acid groups on the phosphoramidate moiety attached to the nucleoside or deoxynucleoside.
  • J. Org. Chem. (2005) 70:1 100-1 103 discloses (scheme 2) the use of resin- bound phosphitylating reagents to yield monophosphorylated unprotected nucleosides such as thymine, uridine and adenosine.
  • the document does not teach nucleoside or deoxynucleoside phosphoramidates or phosphates including any pending aryl or carboxylic acid groups.
  • J. Med. Chem. (1992) 35:2728-2735 discloses (compounds 12 and 13) the 5'- phenyl phosphates of 2',3'-didehydro-2',3'-dideoxyadenosine and 2',3'-didehydro- 2',3'-dideoxycytidine which in serum-containing medium can function as pro-drugs of the antiviral nucleosides which are released before their introduction into cells.
  • nucleoside or deoxynucleoside phosphoramidates or any nucleoside or deoxynucleoside phosphates including pending aryl groups linked to the phosphate group via a short alkylene chain, or pending carboxylic acid groups.
  • Antiviral Chemistry and Chemotherapy (1998) 9;1 -8 discloses (compound 1 1 ) 5'-[(hydroxyl)(phenyl)phosphinyl]thymidine which was tested for herpes simplex virus inhibition. The document however does not teach nucleoside or deoxynucleoside phosphoramidates or phosphates including any pending aryl or carboxylic acid groups.
  • French Patent No. 2,781 ,229 discloses on the one hand 2',3'-didehydro-2',3'- dideoxythymidine-5'-dibenzylphosphate (example 1 ) and 2',3'-didehydro-2',3'- dideoxythymidine-5'-diethylphosphate (example 3) made from dibenzylphosphite or diethylphosphite respectively, and on the other hand a family of 5'-H-phosphonates of 2',3'-didehydro-2',3'-dideoxynucleosides made from O-benzyl H-phosphonate such as 2',3'-didehydro-2',3'-dideoxythymidine-5'-benzyl H-phosphonate (example 4).
  • the document does not teach any nucleoside or deoxynucleoside phosphoramidates or any nucleoside or deoxynucle
  • JP 2004-043371 discloses the 5'-dibenzylphosphate of a ribofuranosyl- pyrazine carboxamide. The document however does not teach any nucleoside or deoxynucleoside phosphoramidates or any nucleoside or deoxynucleoside phosphates including pending carboxylic acid groups.
  • Bioorg. Med. Chem. (2006) 14:1924-1934 discloses 5'-aryl H-phosphonates and 5'-aryl ⁇ -hydroxy(aryl)methanephosphonates of 2',3'-dideoxyinosine or 3'-azido- 2',3'-dideoxythymidine wherein aryl may be phenyl, 4-methylphenyl, A- methoxyphenyl, 2,6-dimethylphenyl, 4-chlorophenyl, 4-nitrophenyl or pyridin-3-yl.
  • the document however does not teach any nucleoside or deoxynucleoside phosphoramidates or any nucleoside or deoxynucleoside phosphates including pending aryl or carboxylic acid groups.
  • WO 2008/104408 discloses nucleoside phosphates, phosporothioates and phosphoramidates including a pending imidazolyl or carboxylic group attached to their O or S atom or NH group.
  • WO 01/34622 discloses (page 8 and claim 13) nucleoside derivatives being both: substituted at carbon 2 with a R 3 or OR 3 group, and - phosphorylated with a group PXYR, wherein each of X and Y may be O, S, OR or NR 1 R 2 wherein R is a hydrophobic group having 1 to 18 carbon atoms, and wherein each of R 1 , R 2 and R 3 is as defined by R.
  • Nucleic Acids Research (1989) 17:8979-89 discloses N,N-dimethylguanosine 5'-(S-phenyl hydrogen phosphorothioate. This document however does not teach any nucleoside or deoxynucleoside phosphoramidates, or any nucleoside or deoxynucleoside phosphates including pending carboxylic acid groups or pending aryl groups linked to the phosphate group via a short alkylene chain.
  • Nucleosides & Nucleotides (1987) 6:913-34 discloses adenosine 5'-(hydrogen phenylphosphoramidate) and adenosine 5'-[hydrogen (phenylmethyl)- phosphoramidate]. This document however does not teach any nucleoside or deoxynucleoside phosphates including pending carboxylic acid groups or aryl groups, or any nucleoside or deoxynucleoside phosphoramidates including pending carboxylic acid groups.
  • Nucleic Acids Research (1974) discloses uridine 5'-[hydrogen (4- chlorophenyl)phosphoramidate], uridine 5'-[hydrogen (4-bromophenyl)- phosphoramidate] and uridine 5'-[hydrogen (4-iodophenyl)phosphoramidate].
  • This document does not teach any nucleoside or deoxynucleoside phosphates including pending carboxylic acid groups or aryl groups, or any nucleoside or deoxynucleoside phosphoramidates including pending carboxylic acid groups.
  • nucleotide analogue that would not depend on activation by nucleoside/nucleotide kinases whilst serving as a natural substrate mimic, would be of a great interest. Also, there is still a need for the development of novel phosphate-modified nucleosides that meet the requirements for successful polymerase recognition, including good chelating properties and spatial features to form stable enzyme-substrate complexes, and whereby the incorporation reaction is not stalled.
  • the present invention provides novel phosphate-modified nucleosides which can act as substrates of DNA- or RNA-polymerases and/or as antiviral agents.
  • the present invention provides novel phosphate-modified nucleosides that can be used as alternative (compared to natural NTPs or dNTPs) efficient substrates for DNA- or RNA-polymerases.
  • these phosphate- modified nucleosides are such that the pyrophosphate group of nucleosides/nucleotides is replaced by an easily leaving group, more particularly a leaving group in a nucleotidyl transfer mechanism.
  • the leaving group comprises at least a pending aryl (e.g.
  • this leaving group includes or is a carboxylic acid- containing group coupled to the nucleoside by a phosphoramide binding moiety, yet more particularly this carboxylic acid containing group comprises at least two carboxylic acid groups being each linked via 0, 1 , 2 or 3 CH 2 -groups to the N of the phosphoramide binding moiety.
  • this leaving group is a aryl-containing group coupled to the nucleoside by a phosphoramide, phosphate or phosphorothioate binding moiety
  • this aryl-containing group comprises one, two or three phenyl groups being each linked via 0, 1 , 2 or 3 methylene (CH 2 ) groups to said phosphoramide, phosphate or phosphorothioate binding moiety.
  • said phenyl group(s) is (are) substituted with one or two carboxylic acid groups.
  • the present invention encompasses modified nucleosides represented by the structural formula (A): W
  • - Nuc is a natural nucleoside or a nucleoside analogue, wherein said natural nucleoside or nucleoside analogue can be non-substituted or substituted as defined below;
  • R 3 is selected from the group consisting of hydrogen, Ci -6 alkyl, C 3 . 6 cycloalkyl, aryl-
  • any one of alkyl, cycloalkyl or arylalkyl may optionally be substituted with 1 , 2 or 3 substituents independently selected from the group consisting of halogen, OH, C 1-6 alkoxy, trifluoromethyl, trifluoromethoxy, nitro, cyano and amino;
  • - W is O or S
  • - Z is selected from the group consisting of O; S; NH and NCH 3 ;
  • - Ar is an aryl group as defined below, provided that when W is S and Z is O, n is not 1 or 2, and stereoisomers, pharmaceutically acceptable salts and pro-drugs thereof, or R 2 is represented by the structural formula (II)
  • R 4 is an aryl group or COOR 6 , wherein R 6 is hydrogen or C 1-6 alkyl or benzyl; and R is a group represented by the structural formula (III):
  • - m is 0, 1 , 2, or 3;
  • R 11 is selected from the group consisting of aryl, imidazolyl and COOR 6 wherein R 6 is hydrogen or C 1-6 alkyl or benzyl; or R 5 is a group represented by the structural formula (IV)
  • each of R 12 and R 13 is independently selected from the group consisting of aryl; hydrogen; (CH 2 ) q -imidazolyl; and (CH 2 ) q - COOR 6 , wherein R 6 is hydrogen or C 1-6 alkyl or benzyl, and wherein q is 0, 1 or 2, provided that if p is 0, R 12 and R 13 are not both hydrogen; and stereoisomers, pharmaceutically acceptable salts and pro-drugs thereof, provided that said modified nucleoside is not:
  • said natural nucleoside or nucleoside analogue (Nuc) is coupled via its 5' position (referring to the Standard numbering of atoms for cyclic sugar moieties) to the phosphorus atom P in the structural formula (A).
  • the modified nucleoside is one wherein R 2 is represented by the structural formula (II), wherein R 5 is represented by the structural formula (III), and wherein at least one of R 4 and R 11 is COOR 6 .
  • the modified nucleoside is one wherein Nuc, W and R 3 are as broadly defined hereinabove, wherein R 2 is represented by the structural formula (II), wherein R 5 is represented by the structural formula (III), and wherein R 4 and R 11 are both COOR 6 .
  • the modified nucleoside is one wherein Nuc, W and R 3 are as broadly defined hereinabove, wherein R 2 is represented by the structural formula (II), wherein R 5 is represented by the structural formula (IV), and wherein at least one of R 12 and R 13 is
  • the modified nucleoside is one wherein Nuc, W and R 3 are as broadly defined hereinabove, wherein R 2 is represented by the structural formula (II), wherein R 5 is represented by the structural formula (IV), and wherein R 12 and R 13 are both COOR 6 .
  • the modified nucleoside is one wherein Nuc, W and R 3 are as broadly defined hereinabove, wherein R 2 is represented by the structural formula (II), wherein R 5 is represented by the structural formula (IV), and wherein at least one of R 4 , R 12 and R 13 is COOR 6 .
  • the modified nucleoside is one wherein Nuc, W and R 3 are as broadly defined hereinabove, wherein R 2 is represented by the structural formula (II), wherein R 5 is represented by the structural formula (IV), and wherein at least two of R 4 , R 12 and R 13 are COOR 6 .
  • the modified nucleoside is one wherein Nuc, W and R 3 are as broadly defined hereinabove, wherein R 2 is represented by the structural formula (II), wherein R 5 is represented by the structural formula (IV), and wherein all of R 4 , R 12 and R 13 are COOR 6 .
  • the modified nucleoside is one wherein Nuc, W and R 3 are as broadly defined hereinabove, wherein R 2 is represented by the structural formula (II), wherein R 5 is represented by the structural formula (III), and wherein at least one of R 4 and R 11 is COOH.
  • the modified nucleoside is one wherein Nuc, W and R 3 are as broadly defined hereinabove, wherein R 2 is represented by the structural formula (II), wherein R 5 is represented by the structural formula (III), and wherein R 4 and R 11 are both COOH.
  • the modified nucleoside is one wherein Nuc, W and R 3 are as broadly defined hereinabove, wherein R 2 is represented by the structural formula (II), wherein R 5 is represented by the structural formula (IV), and wherein at least one of R 12 and R 13 is COOH.
  • the modified nucleoside is one wherein Nuc, W and R 3 are as broadly defined hereinabove, wherein R 2 is represented by the structural formula (II), wherein R 5 is represented by the structural formula (IV), and wherein R 12 and R 13 are both COOH.
  • the modified nucleoside is one wherein Nuc, W and R 3 are as broadly defined hereinabove, wherein R 2 is represented by the structural formula (II), wherein R 5 is represented by the structural formula (IV), and wherein at least one of R 4 , R 12 and R 13 is COOH.
  • the modified nucleoside is one wherein Nuc, W and R 3 are as broadly defined hereinabove, wherein R 2 is represented by the structural formula (II), wherein R 5 is represented by the structural formula (IV), and wherein at least two of R 4 , R 12 and R 13 are COOH.
  • the modified nucleoside is one wherein Nuc, W and R 3 are as broadly defined hereinabove, wherein R 2 is represented by the structural formula (II), wherein R 5 is represented by the structural formula (IV), and wherein all of R 4 , R 12 and R 13 are COOH.
  • the modified nucleoside is one wherein Nuc, W and R 3 are as broadly defined hereinabove, wherein R 2 is represented by the structural formula (II), wherein R 5 is represented by the structural formula (III), and wherein m differs from n.
  • the modified nucleoside is one wherein Nuc, W and R 3 are as broadly defined hereinabove, wherein R 2 is represented by the structural formula (II), and wherein at each occurrence of R 6 in the definitions of R 4 and R 5 , R 6 is hydrogen or C 1-6 alkyl.
  • a second more specific aspect of the present invention relates to modified nucleosides represented by the structural formula (I):
  • B is a pyrimidine or purine base, or an analogue thereof (such as defined below), optionally substituted with one or two substituents independently selected from the group consisting of halogen, hydroxyl, sulfhydryl, methyl, ethyl, isopropyl, amino, methylamino, ethylamino, trifluoromethyl and cyano;
  • R 1 is H or OH
  • R 3 is selected from the group consisting of hydrogen, C 1-6 alkyl, C 3 - 6 cycloalkyl, aryl-C 1 -6 alkyl and 2-cyanoethyl, wherein said C 1-6 alkyl, C 3 - 6 cycloalkyl or aryl-C 1-6 alkyl is optionally substituted with one or more, preferably 1 , 2 or 3, substituents independently selected from the group consisting of halogen, OH, C 1-6 alkoxy, trifluoromethyl, trifluoromethoxy, nitro, cyano and amino;
  • W is O or S
  • R 2 is represented by the structural formula (V):
  • - n 0,1 or 2;
  • - Z is selected from the group consisting of O; S; NH and NCH 3 ;
  • - Ar is an aryl group as defined below, provided that when W is S and Z is O, n is not 1 or 2, and stereoisomers, pharmaceutically acceptable salts and pro-drugs thereof, or R 2 is represented by the structural formula (II):
  • n O, 1 , 2, or 3;
  • R 4 is selected from the group consisting of aryl, imidazolyl and
  • R 6 is H or Ci -6 alkyl or benzyl
  • R 5 is a group represented by the structural formula (CH 2 ) »1 1 m -R '
  • - m is O, 1 , 2, or 3;
  • R 11 is selected from the group consisting of: aryl, imidazolyl and COOR 6 wherein R 6 is hydrogen or Ci -6 alkyl or benzyl; or R 5 is a group represented by the structural formula (IV):
  • - p is 0, 1 , 2, or 3;
  • each of R 12 and R 13 is independently selected from the group consisting of aryl; hydrogen; (CH 2 ) q -imidazolyl; and (CH 2 ) q -
  • W is preferably O (oxygen) but it can be replaced by S by chemical reactions known in the art.
  • the molecular weight of the group R 2 is not above 500.
  • the second aspect of the present invention relates to phosphate-modified nucleosides represented by the structural formula (I) wherein
  • R 1 , R 2 , R 3 and W have any of the values as described herein-above, and wherein B is adenine; guanine; cytosine; thymine; uracil, or a substituted uracil as described below.
  • the second aspect of the present invention relates to phosphate-modified nucleosides represented by the structural formula (I) wherein R 1 , R 3 and W have any of the values as described herein-above, wherein R 2 is represented by the structural formula (V), and wherein B is a pyrimidine base substituted with one or two substituents independently selected from the group consisting of halogen, hydroxyl, sulfhydryl, methyl, ethyl, amino and methylamino.
  • the second aspect of the present invention relates to phosphate-modified nucleosides represented by the structural formula (I) wherein R 1 , R 3 and W have any of the values as described herein-above, wherein R 2 is represented by the structural formula (V), and wherein B is a purine base substituted with one or two substituents independently selected from the group consisting of halogen, hydroxyl, sulfhydryl, methyl, ethyl, amino and methylamino.
  • the second aspect of the present invention relates to phosphate-modified nucleosides represented by the structural formula (I) wherein R 1 , R 3 and W have any of the values as described herein-above, wherein R 2 is represented by the structural formula (II), wherein n, R 4 and R 5 have any of the values as described herein-above, and wherein B is a pyrimidine base substituted with one or two substituents independently selected from the group consisting of halogen, hydroxyl, sulfhydryl, methyl, ethyl, amino and methylamino.
  • the second aspect of the present invention relates to phosphate-modified nucleosides represented by the structural formula (I) wherein R 1 , R 3 and W have any of the values as described herein-above, wherein R 2 is represented by the structural formula (II), wherein n, R 4 and R 5 have any of the values as described herein-above, and wherein B is a purine base substituted with one or two substituents independently selected from the group consisting of halogen, hydroxyl, sulfhydryl, methyl, ethyl, amino and methylamino.
  • the second aspect of the present invention relates to the phosphate-modified nucleosides represented by the structural formula (I) wherein B, R 1 , R 2 and W have any of the values as described herein, and wherein R 3 is hydrogen.
  • the second aspect of the present invention relates to the phosphate-modified nucleosides represented by the structural formula (I) wherein B, R 1 , R 2 and R 3 have any of the values as described herein, and wherein W is O.
  • the second aspect of the present invention relates to the phosphate-modified nucleosides represented by the structural formula (I) wherein B, R 2 , R 3 and W have any of the values as described herein, and wherein R 1 is hydrogen.
  • the second aspect of the present invention relates to phosphate-modified nucleoside represented by the structural formula (I) wherein B, R 2 , R 3 and W have any of the values as described herein, and wherein R 1 is OH.
  • the se aspect of the present invention also relates to phosphate-modified nucleosides represented by the structural formula (I) wherein B, R 1 , R 3 and W have any of the values described herein-above, wherein R 2 is represented by the structural formula (V), and wherein n is O.
  • the se aspect of the present invention also relates to phosphate-modified nucleosides represented by the structural formula (I) wherein B, R 1 , R 3 and W have any of the values described herein-above, wherein R 2 is represented by the structural formula (V), and wherein n is 1 .
  • the se aspect of the present invention also relates to phosphate-modified nucleosides represented by the structural formula (I) wherein B, R 1 , R 3 and W have any of the values described herein-above, wherein R 2 is represented by the structural formula (II), and wherein n is 0.
  • the se aspect of the present invention also relates to phosphate-modified nucleosides represented by the structural formula (I) wherein B, R 1 , R 3 and W have any of the values described herein-above, wherein R 2 is represented by the structural formula (II), and wherein n is 1 .
  • the second aspect of the present invention relates to phosphate-modified nucleosides represented by the structural formula (I) wherein B, R 1 , R 3 and W have any of the values described herein, wherein R 2 is represented by the structural formula (II), wherein R 5 is represented by the structural formula (III), and wherein m is 1.
  • the second aspect of the present invention relates to phosphate-modified nucleosides represented by the structural formula (I) wherein B, R 1 , R 3 and W have any of the values described herein, wherein R 2 is represented by the structural formula (II), wherein R 2 is represented by the structural formula (IV), and wherein p is 0.
  • the second aspect of the present invention relates to phosphate-modified nucleosides represented by the structural formula (I) wherein R 1 , R 2 , R 3 and W have any of the values described herein, and wherein B is a pyrimidine or purine base analogue as described in the Definitions section below, in particular 5-azapyrimidine, 5-azacytosine, 7-deazapurine, 7-deazaadenine, 7- deazaguanine or 7-deaza-8-azapurine.
  • the second aspect of the present invention relates to phosphate-modified nucleosides represented by the structural formula (I) wherein B, R 1 , R 3 and W have any of the values as described herein, and wherein R 2 is a nitrogen-linked carboxylic acid-containing group coupled by a phosphoramide binding, and in a more particular embodiment of the foregoing said carboxylic acid containing group comprises two carboxylic acid groups linked via 0, 1 , 2 or 3 CH 2 -groups to the N of the phosphoramide binding, and in an even more particular embodiment of the foregoing R 2 is N(CH 2 -COOH) 2 .
  • the first aspect of the present invention relates to phosphate-modified nucleosides represented by the structural formula (A) wherein Nuc, R 1 , R 3 and W have any of the meanings as described herein, and wherein R 2 is a nitrogen-linked carboxylic acid-containing group coupled to the nucleoside by a phosphoramide binding moiety,
  • said carboxylic acid-containing group comprises two carboxylic acid groups linked to the nitrogen atom of the phosphoramide binding moiety either directly or via a short alkylene chain such as 1 , 2 or 3 methylene groups.
  • R 2 is N(CH 2 COOH) 2 .
  • Z is O; NH or NCH 3 .
  • R 4 is COOH and R 5 is CH 2 -COOH or CH 2 -imidazolyl.
  • an aryl group is a C 6 aryl (i.e. phenyl) group optionally substituted with one or more substituents independently selected from the group consisting of halogen, amino, trifluoromethyl, hydroxyl, sulfhydryl, nitro, Ci -6 alkoxy, trifluoro-methoxy, cyano and (CH 2 ) q -COOR 6 , wherein R 6 is hydrogen or Ci -6 alkyl or benzyl, and q is O, 1 or 2.
  • said C 6 aryl i.e.
  • phenyl) group is substituted with 1 , 2 or 3 (CH 2 ) q -COOR 6 , wherein R 6 is hydrogen or Ci -6 alkyl or benzyl, and q is selected from O, 1 , and 2.
  • R 3 is hydrogen and each aryl group may be a C 6 aryl (i.e. phenyl) group substituted with 1 , 2 or 3 (CH 2 ) q -COOR 6 , wherein R 6 is hydrogen or benzyl or Ci -6 alkyl, and q is 0, 1 or 2.
  • R 3 is hydrogen and each aryl group may be a C 6 aryl (i.e. phenyl) group substituted with two carboxylic acid groups such as 1 ,2-dicarboxyphenyl or 1 ,3- dicarboxyphenyl.
  • R 3 is hydrogen and R 2 is represented by the structural formula (V), wherein Z is O, S, NH or NCH 3 , and Ar is phthalic acid or isophthalic acid.
  • R 3 is hydrogen and R 2 is represented by the structural formula (II), wherein n is 1 , R 4 is COOH and R 5 is CH 2 -COOH.
  • R 3 is hydrogen and R 2 is represented by the structural formula (II), wherein n is 1 , R 4 is COOCH 3 and R 5 is CH 2 -COOCH 3 .
  • the present invention relates to phosphate-modified nucleoside represented by the structural formula (I) wherein the pyrimidine analogue B is represented by the structural formula (C):
  • R 7 is selected from the group consisting of -OH, -SH, -NH 2 , -
  • R 8 is selected from the group consisting of hydrogen, methyl, ethyl, isopropyl, amino, ethylamino, trifluoromethyl, cyano and halogen;
  • X is CH or N.
  • the present invention relates to phosphate-modified nucleosides represented by the structural formula (I) wherein the purine analogue is represented by the structural formula (D):
  • - R 9 is selected from the group consisting of H, -OH, -SH, -NH 2 , and -NHCH 3 ;
  • - R 10 is selected from the group consisting of hydrogen, methyl, ethyl, hydroxyl, amino and halogen; and - Y is CH or N.
  • novel phosphate- modified nucleoside may be selected from the group consisting of:
  • IA-dAMP 2'-deoxy-adenosine-5'-iminodiacetate-phosphoramidate
  • IA-dCMP 2'-deoxy- cytidine-5'-iminodiacetate-phosphoramidate
  • IA-dGMP 2'-deoxy-guanosine-5'- iminodiacetate-phosphoramidate
  • IA-dTMP 2'-deoxy-thymidine-5'-iminodiacetate- phosphoramidate
  • IA-dUMP 2'-deoxy-uridine-5'-iminodiacetate- phosphoramidate
  • the novel phosphate-modified nucleoside may be selected from the group consisting of: - adenosine- ⁇ '-iminodiacetate-phosphoramidate (IA-AMP); cytidine-5'-iminodiacetate- phosphoramidate (IA-CMP); guanosine-5'-iminodiacetate-phosphoramidate (IA- GMP); S-methyluridine- ⁇ '-iminodiacetate-phosphoramidate (IA-m5uMP), thymidine-5'- iminodiacetate-phosphoramidate (IA-TMP) and uridine-5'-iminodiacetate- phosphoramidate (IA-UMP), i.e.
  • IA-AMP adenosine- ⁇ '-iminodiacetate-phosphoramidate
  • IA-CMP cytidine-5'-iminodiacetate- phosphoramidate
  • IA- GMP guanosine-5'-iminodiacetate-phosphoramidate
  • novel phosphate-modified nucleoside may be selected from the group consisting of:
  • the novel phosphate-modified nucleoside may be selected from the group consisting of: - .2'-deoxy-adenosine-5'-(dimethyl iminodiacetate)-phosphoramidate; 2'-deoxy- cytidine-5'-(dimethyl iminodiacetate)-phosphoramidate; 2'-deoxy-guanosine-5'- (dimethyl iminodiacetate)-phosphoramidate; 2'-deoxy-thymidine-5'-(dimethyl iminodiacetate)-phosphoramidate; 2'-deoxy-uridine-5'-(dimethyl iminodiacetate)- phosphoramidate, adenosine-5'-(dimethyl iminodiacetate)-phosphoramidate; cytidine- 5'-(dimethyl iminodiacetate)-phosphoramidate; guanosine-5'-(dimethyl iminodiacetate)-phosphoramidate; thymidine-5'
  • the novel phosphate-modified nucleoside may be selected from the groups consisting of 5-0- IsoPhthalicAcid-dAMP, 5-NH-lsoPhthalicAcid-dAMP, 4-O-PhthalicAcid-dAMP, 5-0- IsoPhthalicAcid-dCMP, 5-NH-lsoPhthalicAcid-dCMP, 4-0-PhthalicAcid-dCMP, 5-0- IsoPhthalicAcid-dGMP, 5-NH-lsoPhthalicAcid-dGMP, 4-0-PhthalicAcid-dGMP, 5-0- IsoPhthalicAcid-dTMP, 5-NH-lsoPhthalicAcid-dTMP, 4-0-PhthalicAcid-dTMP, 5-0- IsoPhthalicAcid-dUMP, 5-NH-lsoPhthalicAcid-dUMP, or 4-0-
  • the novel phosphate-modified nucleoside may be selected from the groups consisting of 5-0- IsoPhthalicAcid-AMP, 5-NH-lsoPhthalicAcid-AMP, 4-0-PhthalicAcid-AMP, 5-0- IsoPhthalicAcid-CMP, 5-NH-lsoPhthalicAcid-CMP, 4-0-PhthalicAcid-CMP, 5-0- IsoPhthalicAcid-GMP, 5-NH-lsoPhthalicAcid-GMP, 4-0-PhthalicAcid-GMP, 5-0- IsoPhthalicAcid-TMP, 5-NH-lsoPhthalicAcid-TMP, 4-0-PhthalicAcid-TMP, 5-0- IsoPhthalicAcid-UMP, 5-NH-lsoPhthalicAcid-UMP, or 4-0-PhthalicAcid-UMP (in each of these ab
  • Another aspect of the present invention relates to the use of the phosphate- modified nucleosides represented by the structural formulae (A) and (I), including any one of the above-referred specific embodiments thereof, as a substrate for DNA- or RNA-polymerases, these polymerases being either wild-type (naturally occurring) or mutated according to common knowledge in the art.
  • said DNA- or RNA-polymerases are selected from Therminator DNA polymerase, KF (exo ' ) DNA polymerase, or Reverse Transcriptase (e.g. HIV-RT) or mutated forms of these enzymes.
  • the enzymes as described herein above can be mutated, using common knowledge in the art, in order to better adapt to the novel phosphate- modified nucleoside disclosed in this invention.
  • the present invention relates to the use of the phosphate-modified nucleosides of the invention, as a substrate for DNA- or RNA-polymerases in bacteriae or in vitro.
  • said DNA- or RNA-polymerase originates from a micro-organism or from bacterial or viral origin.
  • the phosphate-modified nucleosides of this invention being represented by the structural formulae (A) and (I), including any one of the above-referred specific embodiments thereof, can be used to build in at least 1 , 2 or 3 nucleotides in a growing DNA- or RNA-strand.
  • the phosphate-modified nucleosides of this invention being represented by the structural formulae (A) and (I), including any one of the above- referred specific embodiments thereof, can be used to build in at most 1 , 2 or 3 nucleotides in a growing DNA- or RNA-strand.
  • the phosphate-modified nucleosides of this invention being represented by the structural formulae (A) and (I), including any one of the above-referred specific embodiments thereof, can be used to build in at most 300 nucleotides in a growing DNA- or RNA-strand.
  • the phosphate-modified nucleosides of this invention being represented by the structural formulae (A) and (I), including any one of the above- referred specific embodiments thereof, can be used with a mixture of natural dNTPs or NTPs (ATP,CTP,GTP, UTPJTP) as a substrate for DNA/RNA- polymerases, more in particular to build in 1 -300 (e.g. 2-300) nucleotides in a growing DNA- or RNA-strand.
  • a mixture of natural dNTPs or NTPs ATP,CTP,GTP, UTPJTP
  • the present invention also relates to the use of the phosphate-modified nucleoside represented by the structural formulae (A) and (I), including any one of the above-referred specific embodiments thereof, for the enzymatic production of oligonucleotides, peptides or proteins.
  • the phosphate-modified nucleosides of the invention being represented by the structural formulae (A) and (I), including any one of the above- referred specific embodiments thereof, can be used for in vitro production of DNA or RNA.
  • the phosphate- modified nucleosides of the invention being represented by the structural formulae (A) and (I), including any one of the above-referred specific embodiments thereof, can also be used for in vitro production of peptides or proteins.
  • the phosphate-modified nucleosides of the invention being represented by the structural formulae (A) and (I), including any one of the above-referred specific embodiments thereof, can be used for PCR (polymerase chain reaction).
  • the phosphate-modified nucleosides of the invention being represented by the structural formulae (A) and (I), including any one of the above-referred specific embodiments thereof, can be used as a substrate for the growth of wild type and/or mutated bacteriae.
  • the phosphate-modified nucleotides of the invention being represented by the structural formulae (A) and (I), including any one of the above-referred specific embodiments thereof, can be used as a substrate for the growth of bacteriae with mutated DNA/RNA polymerase, preferably wherein the mutation is suitable to better adapt better to the new substrate.
  • a pharmaceutical, veterinary or non-pharmaceutical composition comprising an anti-virally effective amount of a phosphate-modified nucleoside of the invention being represented by the structural formulae (A) and (I), including any one of the above-referred specific embodiments thereof.
  • said pharmaceutical, veterinary or non-pharmaceutical composition may further comprise an aqueous solution and optionally one or more buffering agents.
  • said pharmaceutical, veterinary or non- pharmaceutical composition may further comprise one or more natural NTPs or dNTPs (e.g. ATP, CTP, GTP, UTP or TTP).
  • Another aspect of the invention relates to the use of the non-pharmaceutical composition of the invention as a substrate to build in at least 1 , 2 or 3 nucleotides in a growing DNA- or RNA-strand.
  • Yet another aspect of the invention relates to the use of the phosphate- modified nucleosides of the invention, being represented by the structural formulae (A) and (I), including any one of the above-referred specific embodiments thereof, in a non-human living organism for sustaining growth, survival or proliferation of said organism.
  • said organism is selected from the group consisting of a virus, a bacterium, an archaeon and an eukaryote, and in a more particular embodiment said eukaryote is selected from the group consisting of yeast, mold, fungus, microalga, multicellular plant and protist.
  • Yet another aspect of the invention relates to a method for the production of oligonucleotides, RNA, DNA, peptides and/or proteins by using the phosphate- modified nucleotides of the invention, being represented by the structural formulae (A) and (I), including any one of the above-referred specific embodiments thereof.
  • Another aspect of the present invention relates to compounds represented by the structural formulae (A) and (I), including any one of the above- referred specific embodiments thereof, having antiviral activity, specifically to these compounds that inhibit the replication of viruses (such as, but not limited to, viruses belonging to the order Herpesvirales, in particular the family Herpesviridae, the family Alloherpesviridae or the family Malacoherpesviridae), in particular retroviruses, and even more specifically to these compounds that inhibit the replication of HIV-1 or HIV- 2.
  • viruses such as, but not limited to, viruses belonging to the order Herpesvirales, in particular the family Herpesviridae, the family Alloherpesviridae or the family Malacoherpesviridae
  • Another aspect of the present invention relates to the compounds represented by the structural formulae (A) and (I), including any one of the above-referred specific embodiments thereof, for use as a medicine, more particularly for use to treat or prevent a viral infection in a mammal, even more particularly to treat or prevent HIV infection in a mammal such as a human being.
  • compositions comprising an anti-virally effective amount of at least one compound being represented by the structural formulae (A) and (I), including any one of the above- referred specific embodiments thereof, in combination with one or more pharmaceutically acceptable excipients being well known in the art for the formulation of phosphate nucleosides.
  • the invention further relates to the use of the compounds represented by the structural formulae (A) and (I), including any one of the above- referred specific embodiments thereof, in the manufacture of a medicament useful for the treatment of viral infections (e.g. from a virus belonging to the order Herpesvirales), more specifically for the treatment of a retroviral infection such as a HIV-1 or HIV-2 infection.
  • the present invention also relates to a method of treatment or prevention of a viral infection in a mammal, comprising the administration of a therapeutically effective (e.g. anti-virally effective or replication-inhibiting) amount of a compound of this invention represented by the structural formulae (A) and (I), including any one of the above-referred specific embodiments thereof, optionally in combination with one or more pharmaceutically acceptable excipients.
  • said viral infection is a HIV infection.
  • said mammal is a human being.
  • Still another aspect of the invention relates to processes and methods for the preparation of the phosphate-modified nucleosides of the invention represented by the structural formulae (A) and (I), including any one of the above- referred specific embodiments thereof.
  • the method comprises the steps of interacting a nucleoside monophosphate (NMP) with an ester, e.g. a methyl, ethyl or benzyl ester of the carboxylic acid to be introduced as a leaving group into the compound of this invention to produce the ester of a phosphoramidate nucleoside analogue, as depicted in any one of schemes 2, 3, 4 and 5 below.
  • a deprotecting agent such as, but not limited to, an alkali hydroxide, e.g. 0.04 M NaOH, provides the desired phosphoramidate nucleoside of this invention.
  • the process for the preparation of the phosphate-modified nucleosides of the invention comprises a synthetic step as shown in the following scheme 1 :
  • (a) schematically represents the presence in the reaction mixture of an effective amount of a suitable catalyst for the condensation of the 5'-OH and phosphate acid groups.
  • Suitable such catalysts are well known in the art and include, but are not limited to, an arylsulfonyl halide, e.g. an optionally substituted phenylsulfonyl chloride.
  • Phosphates, phosphorothioates and phosphoramidates wherein R 2 and R 3 are as defined in the structural formulae (A) and (I), including any one of the above- referred specific embodiments thereof, as shown on the left part of scheme 1 to be used as starting materials of this method may be known in the art or may be produced according to one or more of the synthetic procedures as described by Scheit in Nucleotide analogs, J Wiley and sons, New York (1980) or by Vaghefi in Nucleoside Triphosphate and their analogs, Taylor and Francis, CRC Press (2005), the content of which is incorporated by reference.
  • Figure 1 schematically shows a synthetic procedure for producing a modified nucleoside according to one embodiment of the present invention including a phosphoramidate moiety wherein R 2 and R 5 are represented by the structural formulae (II) and (III) respectively, and R 4 and R 11 are both carboxylic acid groups.
  • Figure 2 shows a representation of the chemical structures of 5-O-isophthalic acid-dAMP 2, 5-NH-isophthalic acid-dAMP 3, and 4-O-phthalic acid-dAMP 4 respectively.
  • Figure 3 shows the incorporation of the illustrative compounds 2 5-O-iPA- dAMP, 3 5-NH-iPA-dAMP, and 4 4-O-PA-dAMP of this invention into P1T1 by HIV
  • FIG. 4 shows the incorporation of 5-H-iPA-dAMP (compound 2) at different concentrations into P1T1 by HIV Reverse Transcriptase. Aliquots were taken at 5, 10,
  • Figure 5 shows the elongation of P1T2 with IA-dAMP by HIV-1 Reverse Transcriptase. Aliquots were taken at 15, 30, 60, 90 and 120 minutes, indicated as 1 ,
  • Figure 6 shows the elongation of P1T3 with IA-dAMP by HIV-1 Reverse
  • FIGS. 7A and 7B respectively show the incorporation of the illustrative compounds 5 and 6 of this invention into P1T1 by HIV-1 Reverse Transcriptase.
  • Figures 8 and 9 show the data of elongation capacity of P1T2 with the illustrative compound 5 of this invention by HIV-1 Reverse Transcriptase.
  • Figure 10 shows activation of AZT monophosphate by a novel leaving group moiety of this invention (top) and an application of iminodiacetate phosphoramidate moiety for in vivo delivery and activation of AZT monophosphate (bottom).
  • nucleoside conventionally refers to natural glycosylamines consisting of a nucleobase bound to a ribose or deoxyribose sugar via a ⁇ -glycosidic linkage such as cytidine, urisine, adenosine, guanosine and thymidine.
  • nucleoside analogue refers to known nucleosides consisting of a sugar linked to a pyrimidine or purine base, including modifications wherein the sugar ring is modified and/or wherein the nucleobase is further substituted.
  • Nucleoside analogues wherein the natural sugar moiety is modified include but are not limited to: nucleoside analogues wherein the ribose sugar is replaced with another monocyclic sugar such as, but not limited to, arabinofuranose, arabinopyranose, xylofuranose, xylopyranose, lyxofuranose, lyxopyranose, ⁇ -D-threofuranose; threose nucleic acids (TNA) also referred as ⁇ -threofuranosyl nucleosides are described for example by Orgel in Science 290 (5495) 1306-1307 and by Schong et al in Science 290 (5495) 1347-1351 , the content of which is incorporated herein by reference; nucleoside analogues wherein the ribose sugar is replaced with a bicyclic or tricyclic sugar, such as locked nucleic acids (LNA) wherein the ribose moiety is modified with at least
  • sugar as used herein includes ribose and deoxyribose linked by the oxygen atom at position 5 of the ribose or deoxyribose moiety to the phosphorus atom P in the structural formula (A) or the structural formula (I), but is not limited to, i.e. also includes modifications and variants of the ribose or deoxyribose moiety. Such modifications are well known to those skilled in the art, examples being pentofuranoses such as listed above, unsaturated and/or substituted monocyclic sugars such as, but not limited to, 2,3-deoxy-3-azido-ribose, and also bicyclic or tricyclic sugars such as present in locked nucleic acids (LNA).
  • LNA locked nucleic acids
  • pyrimidine or purine base includes, but is not limited to, adenine, thymine, cytosine, uracyl, guanine and 2,6-diaminopurine and analogues and derivatives thereof.
  • a purine or pyrimidine base as used herein includes a purine or pyrimidine base found in naturally occurring nucleosides as mentioned above.
  • An analogue thereof is a base which mimics such naturally occurring bases in such a way that their structures (the kinds of atoms and their arrangement) are similar to the naturally occurring bases but may either possess additional or lack certain of the functional properties of the naturally occurring bases.
  • Such analogues include those derived by replacement of a CH moiety by a nitrogen atom (e.g. 5-azapyrimidines such as 5-azacytosine) or vice versa (e.g., 7- deazapurines, such as 7-deazaadenine or 7-deazaguanine) or both (e.g., 7-deaza, 8- azapurines).
  • 5-azapyrimidines such as 5-azacytosine
  • 7- deazapurines such as 7-deazaadenine or 7-deazaguanine
  • 7-deaza, 8- azapurines e.g., 7-deaza, 8- azapurines.
  • derivatives of such bases or analogues are meant those bases wherein one or more ring substituents are either incorporated, removed, or modified by conventional substituents known in the art, e.g. halogen, hydroxyl, amino, C 1-6 alkyl and other non-reactive and biocompatible substituents.
  • purine and pyrimidine analogues B for the purpose of the present invention may be selected from the group comprising pyrimidine bases represented by the structural formula (C):
  • R 7 and R 9 are independently selected from the group consisting of H, -OH, -
  • R 8 and R 10 are independently selected from the group consisting of H, methyl, ethyl, isopropyl, hydroxyl, amino, ethylamino, trifluoromethyl, cyano and halogen; and - X and Y are independently selected from CH and N.
  • C 1-6 alkyl refers to normal, secondary, or tertiary hydrocarbon chains having from 1 to 6 carbon atoms.
  • Examples thereof are methyl, ethyl, 1 -propyl, 2-propyl, 1 -butyl, 2-methyl-1 -propyl(i-Bu), 2-butyl (s-Bu) 2-methyl-2- propyl (t-Bu), 1 -pentyl (n-pentyl), 2-pentyl, 3-pentyl, 2-methyl-2-butyl, 3-methyl-2- butyl, 3-methyl-1 -butyl, 2-methyl-1 -butyl, 1 -hexyl, 2-hexyl, 3-hexyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl, 3-methyl-3-pentyl, 2-methyl-3-pentyl, 2,3- dimethyl-2-butyl, 3,3-dimethyl-2-butyl, n-pentyl, n-hexyl.
  • cycloalkyl means a monocyclic saturated hydrocarbon monovalent group having from 3 to 10 carbon atoms, such as for instance cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, or a C 7-I 0 polycyclic saturated hydrocarbon monovalent groupl having from 7 to 10 carbon atoms such as, for instance, norbornyl, fenchyl, trimethyltricycloheptyl or adamantyl.
  • d- 6 alkoxy refers to substituents wherein a carbon atom of a Ci -6 alkyl group (such as defined herein), is attached to an oxygen atom through a single bond such as, but not limited to, methoxy, ethoxy, propoxy, isopropoxy, butoxy, iso-butoxy, sec-butoxy, te/t-butoxy, pentoxy, 3-pentoxy, or n- hexyloxy.
  • aryl designates any mono- or polycyclic aromatic monovalent hydrocarbon groupl having from 6 up to 30 carbon atoms such as, but not limited to phenyl, naphthyl, anthracenyl, phenantracyl, fluoranthenyl, chrysenyl, pyrenyl, biphenylyl, terphenyl, picenyl, indenyl, biphenyl, indacenyl, benzocyclobutenyl, benzocyclooctenyl and the like, including benzo-fused cycloalkyl radicals (the latter being as defined above) such as, for instance, indanyl, tetrahydronaphthyl, fluorenyl and the like, all of the said groups being optionally substituted with one or more substituents independently selected from the group consisting of halogen, amino, trifluoromethyl, hydroxyl, sulfhydry
  • aryl-C 1-6 alkyl refers to an aliphatic saturated hydrocarbon monovalent group (preferably a C 1-6 alkyl group such as defined above) onto which an aryl group (such as defined above) is linked via a carbon atom, and wherein the said aliphatic group and/or the said aryl group may be optionally substituted with one or more substituents independently selected from the group consisting of halogen, amino, hydroxyl, sulfhydryl, C 1-6 alkyl, C 1 -6 alkoxy, trifluoromethyl, trifluoromethoxy, nitro and carboxylic acid, such as but not limited to benzyl, 4-chlorobenzyl, 4- fluorobenzyl, 2-fluorobenzyl, 3,4-dichlorobenzyl, 2,6-dichlorobenzyl, 3-methylbenzyl, 4-methylbenzyl, 4-te/t-butoxy
  • amino acid refers to any "natural amino acid
  • This term also comprises natural and non-natural amino acids being protected at their carboxylic terminus (e.g. as a d- 6 alkyl, phenyl or benzyl ester or as an amide, such as for example, a mono-Crealkyl or CN-(CVe alkyl) amide.
  • carboxylic terminus e.g. as a d- 6 alkyl, phenyl or benzyl ester or as an amide, such as for example, a mono-Crealkyl or CN-(CVe alkyl) amide.
  • Other suitable carboxy protecting groups are known to those skilled in the art (see for example, T.W. Greene, Protecting Groups In Organic Synthesis; Wiley: New York, (1981 ) and references cited therein, the content of which is incorporated herein by reference).
  • modified nucleosides of this invention having a chiral center may exist in, and be isolated in, optically active and racemic forms. Some modified nucleosides may exhibit polymorphism. It is to be understood that the present invention encompasses any racemic, optically-active, polymorphic, or stereoisomeric form, or mixtures thereof in any proportions, of a modified nucleoside of this invention, which may possess the useful properties described herein. It is well known in the art how to prepare optically active forms (for example, by resolution of the racemic form by recrystallization techniques, by synthesis from optically-active starting materials, by chiral synthesis, or by chromatographic separation using a chiral stationary phase).
  • stereoisomer refers to all possible different isomeric as well as conformational forms which the compounds of formula (I) may possess, in particular all possible stereochemical ⁇ and conformational ⁇ isomeric forms, all diastereomers, enantiomers and/or conformers of the basic molecular structure. Some compounds of the present invention may exist in different tautomeric forms, all of the latter being included within the scope of the present invention.
  • enantiomer means each individual optically active form of a compound of the invention, having an optical purity or enantiomeric excess (as determined by methods standard in the art) of at least 80% (i.e.
  • phosphate-modified nucleotides herein when substituted with appropriate selected functionalities are capable of acting as pro-drugs. These are labile functional groups which separate from an active inhibitory phosphate-modified nucleotide during metabolism, systemically, inside a cell, by hydrolysis, enzymatic cleavage, or by some other process (Bundgaard "Design and Application of Pro-drugs” in Textbook of Drug Design and Development (1991 ), Eds. Harwood Academic Publishers, pp. 1 13-191 , the content of which is incorporated herein by reference).
  • pro-drug moieties can serve to enhance solubility, absorption and lipophilicity to optimize drug delivery, bioavailability and efficacy.
  • a "pro-drug” is thus a covalently modified analogue of a therapeutically-active modified nucleoside of this invention.
  • a pro-drug moiety can also be therapeutically active in its own right.
  • Suitable pro-drug moieties include, but are not limited to, esters of nucleosides like the POM (pivaloyloxymethyl), POC (isopropyloxycarbonyloxymethyl) and SATE (S-acyl-2-thioethyl) esters.
  • salts of the compounds having structural formula I may be formed at any acid or base functionality within the compound, in particular, R 3 , R 4 and R 5 may represent or comprise an acid or base functionality.
  • salts of the compounds represented by the structural formula (I) or the structural formula (A) may be formed as follows.
  • R 3 is hydrogen, it is acidic and may therefore engage in salt formation with an inorganic or organic base.
  • R 4 and R 5 comprise acid functionalities such as carboxylic groups (i.e. -COOH), which can equally engage in salt formation with an organic or inorganic base.
  • R 4 and R 5 may comprise base functionalities such as the imidazolyl, which in turn can engage in salt formation with an organic or inorganic acid.
  • pro-drug relates to an inactive or active derivative of a compound represented by the structural formula (I) or the structural formula (A) as defined herein above or any one of their specific embodiments, which undergoes spontaneous or enzymatic transformation within the body of an animal, e.g. a mammal such as a human being, in order to release the pharmacologically active form of the compound.
  • an animal e.g. a mammal such as a human being
  • Rautio J. et al. Pro-drugs: design and clinical applications” in Nature Reviews Drug Discovery (2008) doi: 10.1038/nrd2468, the content of which is incorporated herein by reference).
  • pro-drugs of the compounds represented by the structural formula (A) or the structural formula (I), including any one of the above-described specific embodiments thereof may be formed as follows.
  • R 3 is H
  • a free phosphate acid function is available for prodrug formation as described in detail by Hecker et al. ("Prodrugs of phosphates and phosphonates” Journal of Medicinal Chemistry (2008) doi: 10.1021/jm701260b, the content of which is incorporated herein by reference).
  • R 4 and R 5 comprise acid functionalities such as carboxylic acid groups (i.e. -COOH) which may be used for the formation of a pro-drug.
  • Such carboxylic acid pro-drug may occur in the form of an ester, in particular acyloxyalkyl esters (e.g. pivaloyloxymethyl ester (POM)) or S- acylthioethyl (SATE) esters, a carbonate, a carbamate or an amide, such as amino acid pro-drugs.
  • ester in particular acyloxyalkyl esters (e.g. pivaloyloxymethyl ester (POM)) or S- acylthioethyl (SATE) esters, a carbonate, a carbamate or an amide, such as amino acid pro-drugs.
  • acyloxyalkyl esters e.g. pivaloyloxymethyl ester (POM)
  • SATE S- acylthioethyl
  • peptide refers to a sequence of 2 to 50 amino acids (e.g. as defined hereinabove) or peptidyl residues.
  • the sequence may be linear or cyclic.
  • a peptide comprises 2 to 25, or 5 to 20 amino acids.
  • oligonucleotide refers to a polynucleotide formed by a plurality of linked nucleotide units.
  • the nucleotide units each include a nucleoside unit linked together via a phosphate linking group.
  • These nucleotides can be natural or modified in their phosphate, sugar or nucleobase group.
  • the oligonucleotide may be naturally occurring or non naturally occurring.
  • polymerase refers to an enzyme that can synthesize DNA or RNA from a DNA or RNA template and includes but is not limited to Therminator DNA polymerase, KF(exo )DNA polymerase and HIV Reverse Transcriptase. BIOLOGICAL APPLICATIONS OF THE INVENTION
  • Novel phosphoramidates, phospho-esters and phospho-thioesters according to this invention may be used as alternative substrates and biotechnology tools.
  • modified nucleosides as biotechnology tools also require new and efficient ways to synthesize DNA and RNA building blocks such as nucleoside triphosphates and amidites for the use, for example, in PCR, labeling, or enzymatic incorporation of nucleotides, and in the automated DNA synthesis, respectively.
  • some biotechnology applications require incorporation of a nucleotide by enzymatic means using DNA or RNA polymerases.
  • triphosphate synthesis is not always feasible and/or provides insufficient and low yields.
  • carboxyl-containing groups coupled to a nucleoside monophosphate through a phosphoramidate (P-N) bond can serve as an alternative or substitute group to a pyrophosphate moiety.
  • P-N phosphoramidate
  • fitting into an active site and the subsequent nucleotidyl transfer may be less efficient for such carboxyl-containing phosphoramidate (e.g. IA-dAMP) compared to the natural substrates/dNTPs (e.g. dATP) for the natural polymerase/enzyme.
  • mutated polymerases can be used to increase the efficiency of recognition and incorporation of the compounds of this invention.
  • Such an embodiment of the invention with mutated polymerases can be used to specifically select or grow bacteriae by using these carboxyl-containing phosphoramidate nucleosides as a substrate.
  • An additional advantage of this application is that polymerases that demonstrated efficient recognition and incorporation of carboxyl-containing phosphoramidate nucleosides in our studies are also shown to tolerate various sugar modifications and unnatural nucleobases quite well. Therefore, the enzymatic synthesis of DNA and, RNA sequences containing unnatural nucleobases can be accomplished whilst avoiding at times cumbersome nucleoside triphosphate synthesis and purification.
  • the phosphoramidates, phospho-esters and phospho-thioesters of this invention are also useful as antiviral compounds
  • the compounds of the invention can be efficiently used for the treatment of viral infections, particularly retroviral infections, more particularly Human Immunodeficiency Virus (HIV) infections, in particular of Human Immunodeficiency Virus type 1 (HIV-1 ).
  • HIV Human Immunodeficiency Virus
  • the active ingredients of the compound(s) may be administered to the mammal (including a human being) to be treated by any means well known in the art, i.e.
  • the therapeutically effective amount of the preparation of the compound(s), especially for the treatment of viral infections in humans and other mammals preferably is a HIV enzyme inhibiting amount. More preferably, it is a HIV replication inhibiting amount or a HIV enzyme (in particular reverse transcriptase) inhibiting amount of the derivative(s) of formula I as defined herein corresponds to an amount which ensures a plasma level of between 1 ⁇ g/ml and 100 mg/ml, optionally of 10 mg/ml.
  • the said effective amount may be divided into several sub-units per day or may be administered at more than one day intervals.
  • the present invention further relates to a method for preventing or treating a viral infection, e.g. a retroviral infection, in a subject or patient by administering to the patient in need thereof a therapeutically effective amount, e.g. an anti-virally effective amount, of a compounds of the present invention.
  • the therapeutically effective amount of the compound(s), especially for the treatment of viral infections in humans and other mammals preferably is a HIV enzyme inhibiting amount. More preferably, it is a HIV replication inhibiting amount or a HIV enzyme (in particular reverse transcriptase) inhibiting amount of the derivative(s) of represented by the structural formula (I) or the structural formula (A) as defined herein.
  • the said effective amount may be divided into several sub-units per day or may be administered at more than one-day intervals.
  • the present invention also relates to a combination of different antiviral drugs of the invention or to a combination of the antiviral drugs of the invention with other drugs that exhibit anti-HIV.
  • the invention also relates to a combined preparation of antiviral drugs which may be either:
  • composition comprising
  • Suitable anti-viral agents (c) for inclusion into the antiviral combined preparations of this invention include for instance, inhibitors of BVDV or HCV replication respectively, such as interferon-alpha (pegylated or not), ribavirin and other selective inhibitors of the replication of HCV, such as a compound disclosed in EP-1 , 162,196, WO 03/010141 , WO 03/007945, WO 03/010140 or WO 00/204425 (the contents of which are incorporated herein by reference) and/or an inhibitor of flaviviral protease and/or one or more additional flavivirus polymerase inhibitors.
  • inhibitors of BVDV or HCV replication respectively such as interferon-alpha (pegylated or not), ribavirin and other selective inhibitors of the replication of HCV, such as a compound disclosed in EP-1 , 162,196, WO 03/010141 , WO 03/007945, WO 03/010140 or WO 00
  • the pharmaceutical composition or combined preparation with activity against viral infection according to this invention may contain a compound of the present invention, including any one of the specific embodiments thereof, over a broad content range depending on the contemplated use and the expected effect of the preparation.
  • the content of the compound of the present invention including any one of the specific embodiments thereof, in the combined preparation is within the range of 0.1 to 99.9% by weight, preferably from 1 to 99% by weight, more preferably from 5 to 95% by weight.
  • the active ingredients may be administered to the mammal (including a human) to be treated by any means well known in the art, i.e. orally, intranasally, subcutaneously, intramuscularly, intradermal ⁇ , intravenously, intra-arterially, parenterally or by catheterization; and/or the therapeutically effective amount of each of the active agents, especially for the treatment of viral infections in humans and other mammals, particularly is a HIV enzyme inhibiting amount.
  • the active ingredients may be administered simultaneously but it is also beneficial to administer them separately or sequentially, for instance within a relatively short period of time (e.g. within about 24 hours) in order to achieve their functional fusion in the body to be treated.
  • the invention also relates to the compounds of the formulae described herein, including any one of the above-described specific embodiments thereof, for use in the inhibition of the proliferation of other viruses than HIV-1 , particularly for the inhibition of other members of the family of the retroviruses.
  • the present invention further provides veterinary compositions comprising at least one active ingredient as above defined together with a veterinary carrier therefor.
  • Veterinary carriers are materials useful for the purpose of administering the composition and may be solid, liquid or gaseous materials which are otherwise inert or acceptable in the veterinary art and are compatible with the active ingredient. These veterinary compositions may be administered orally, parenterally or by any other desired route.
  • the invention relates to the compounds represented by the structural formula (I) or the structural formula (A), including any one of the above- described specific embodiments thereof, being useful as agents having biological activity (particularly antiviral activity) or as diagnostic agents.
  • the compounds of the present invention may for instance be bound covalently to an insoluble matrix and used for affinity chromatography separations, depending on the nature of the groups of the compounds, for example compounds with pendant aryl are useful in hydrophobic affinity separations.
  • the compounds of the invention may exist in many different protonation states, depending on, among other things, the pH of their environment. While the structural formulae provided herein depict the compounds in only one of several possible protonation states, it will be understood that these structures are illustrative only, and that the invention is not limited to any particular protonation state - any and all protonated forms of the compounds are intended to fall within the scope of the invention.
  • the term "pharmaceutically acceptable salts" as used herein means the therapeutically active non-toxic salt forms which the compounds represented by the structural formula (I) or the structural formula (A), including any one of the above- described specific embodiments thereof, are able to form. Therefore, the compounds of this invention optionally comprise salts of the compounds herein, especially pharmaceutically acceptable non-toxic salts containing, for example, Na + , Li + , K + , Ca 2+ and Mg 2+ . Such salts may include those derived by combination of appropriate cations such as alkali and alkaline earth metal ions or ammonium and quaternary amino ions with an acid anion moiety, typically a carboxylic acid.
  • the compounds of the invention may bear multiple positive or negative charges.
  • the net charge of the compounds of the invention may be either positive or negative.
  • Any associated counter ions are typically dictated by the synthesis and/or isolation methods by which the compounds are obtained.
  • Typical counter ions include, but are not limited to ammonium, sodium, potassium, lithium, halides, acetate, trifluoroacetate, etc., and mixtures thereof. It will be understood that the identity of any associated counter ion is not a critical feature of the invention, and that the invention encompasses the compounds in association with any type of counter ion.
  • the invention is intended to encompass not only forms of the compounds that are in association with counter ions (e.g., dry salts), but also forms that are not in association with counter ions (e.g., aqueous or organic solutions).
  • Metal salts typically are prepared by reacting the metal hydroxide with a compound of this invention. Examples of metal salts which are prepared in this way are salts containing Li + , Na + , and K + . A less soluble metal salt can be precipitated from the solution of a more soluble salt by addition of the suitable metal compound.
  • salts may be formed from acid addition of certain organic and inorganic acids to basic centers, typically amines, or to acidic groups.
  • acids include, for instance, inorganic acids such as hydrohalic acids, e.g. hydrochloric or hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like; or organic acids such as, for example, acetic, propanoic, hydroxyacetic, 2- hydroxypropanoic, 2-oxopropanoic, lactic, pyruvic, oxalic (i.e. ethanedioic), malonic, succinic (i.e.
  • inorganic acids such as hydrohalic acids, e.g. hydrochloric or hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like
  • organic acids such as, for example, acetic, propanoic, hydroxyacetic, 2- hydroxypropanoic, 2-oxopropanoic, lactic, pyruvic, oxalic (i.e. ethanedioic), malonic, succinic (i.e.
  • compositions herein comprise compounds of the invention in their unon-ionized, as well as zwitterionic form, and combinations with stoichiometric amounts of water as in hydrates.
  • the salts of the parental compounds with one or more amino acids especially the naturally-occurring amino acids found as protein components.
  • the amino acid typically is one bearing a side chain with a basic or acidic group, e.g., lysine, arginine or glutamic acid, or a neutral group such as glycine, serine, threonine, alanine, isoleucine, or leucine.
  • the compounds of the invention also include physiologically acceptable salts thereof.
  • physiologically acceptable salts of the compounds of the invention include salts derived from an appropriate base, such as an alkali metal (for example, sodium), an alkaline earth (for example, magnesium), ammonium and NA 4 + (wherein A is CrC 4 alkyl).
  • an appropriate base such as an alkali metal (for example, sodium), an alkaline earth (for example, magnesium), ammonium and NA 4 + (wherein A is CrC 4 alkyl).
  • Physiologically acceptable salts of an hydrogen atom or an amino group include salts of organic carboxylic acids such as acetic, benzoic, lactic, fumaric, tartaric, maleic, malonic, malic, isethionic, lactobionic and succinic acids; organic sulfonic acids, such as methanesulfonic, ethanesulfonic, benzenesulfonic and p-toluenesulfonic acids; and inorganic acids, such as hydrochloric, sulfuric, phosphoric and sulfamic acids.
  • organic carboxylic acids such as acetic, benzoic, lactic, fumaric, tartaric, maleic, malonic, malic, isethionic, lactobionic and succinic acids
  • organic sulfonic acids such as methanesulfonic, ethanesulfonic, benzenesulfonic and p-toluenesulfonic acids
  • Physiologically acceptable salts of a compound containing a hydroxy group include the anion of said compound in combination with a suitable cation such as Na + and NA 4 + (wherein A typically is independently selected from H or a CrC 4 alkyl group).
  • a suitable cation such as Na + and NA 4 + (wherein A typically is independently selected from H or a CrC 4 alkyl group).
  • salts of acids or bases which are not physiologically acceptable may also find use, for example, in the preparation or purification of a physiologically acceptable compound. All salts, whether or not derived form a physiologically acceptable acid or base, are within the scope of the present invention.
  • stereogenic centers may have either the R- or S-configuration, and multiple bonds may have either cis- or trans-configuration.
  • Pure isomeric forms of the said compounds are defined as isomers substantially free of other enantiomeric or diastereomeric forms of the same basic molecular structure.
  • stereoisomerically pure or “chirally pure” relates to compounds having a stereoisomeric excess of at least about 80% (i.e. at least 90% of one isomer and at most 10% of the other possible isomers), preferably at least 90%, more preferably at least 94% and most preferably at least 97%.
  • enantiomerically pure and diastereomerically pure should be understood in a similar way, having regard to the enantiomeric excess, respectively the diastereomeric excess, of the mixture in question. Separation of stereoisomers is accomplished by standard methods known to those in the art. One enantiomer of a compound of the invention can be separated substantially free of its opposing enantiomer by a method such as formation of diastereomers using optically active resolving agents ("Stereochemistry of Carbon Compounds," (1962) by E. L. ENeI, McGraw Hill; Lochmuller, C. H., (1975) J. Chromatogr., 1 13:(3) 283-302).
  • Separation of isomers in a mixture can be accomplished by any suitable method, including: (1 ) formation of ionic, diastereomeric salts with chiral compounds and separation by fractional crystallization or other methods, (2) formation of diastereomeric compounds with chiral derivatizing reagents, separation of the diastereomers, and conversion to the pure enantiomers, or (3) enantiomers can be separated directly under chiral conditions.
  • diastereomeric salts can be formed by reaction of enantiomerically pure chiral bases such as brucine, quinine, ephedrine, strychnine, a- methyl-b-phenylethylamine (amphetamine), and the like with asymmetric compounds bearing acidic functionality, such as carboxylic acid and sulfonic acid.
  • the diastereomeric salts may be induced to separate by fractional crystallization or ionic chromatography.
  • the substrate to be resolved may be reacted with one enantiomer of a chiral compound to form a diastereomeric pair (EMeI, E. and Wilen, S. (1994) Stereochemistry of Organic Compounds, John Wiley & Sons, Inc., p. 322).
  • Diastereomeric compounds can be formed by reacting asymmetric compounds with enantiomerically pure chiral derivatizing reagents, such as menthyl derivatives, followed by separation of the diastereomers and hydrolysis to yield the free, enantiomerically enriched xanthene.
  • a method of determining optical purity involves making chiral esters, such as a menthyl ester or Mosher ester, a-methoxy-a- (trifluoromethyl)phenyl acetate (Jacob III. (1982) J. Org. Chem. 47:4165) of the racemic mixture, and analyzing the NMR spectrum for the presence of the two atropisomeric diastereomers.
  • Stable diastereomers can be separated and isolated by normal- and reverse-phase chromatography following methods for separation of atropisomeric naphthyl-isoquinolines (e.g. WO96/151 1 1 ).
  • a racemic mixture of two asymmetric enantiomers is separated by chromatography using a chiral stationary phase.
  • Suitable chiral stationary phases are, for example, polysaccharides, in particular cellulose or amylose derivatives.
  • Commercially available polysaccharide based chiral stationary phases are ChiralCelTM CA, OA, OB5, OC5, OD, OF, OG, OJ and OK, and ChiralpakTM AD, AS, 0P(+) and 0T(+).
  • Appropriate eluents or mobile phases for use in combination with said polysaccharide chiral stationary phases are hexane and the like, modified with an alcohol such as ethanol, isopropanol and the like.
  • the terms cis and trans are used herein in accordance with Chemical Abstracts nomenclature and include reference to the position of the substituents on a ring moiety.
  • the absolute stereochemical configuration of the compounds of formula I may easily be determined by those skilled in the art while using well-known methods such as, for example, X-ray diffraction.
  • the compounds of the invention may be formulated with conventional carriers and excipients, which will be selected in accord with ordinary practice. Tablets will contain excipients, glidants, fillers, binders and the like.
  • Aqueous formulations are prepared in sterile form, and when intended for delivery by other than oral administration generally will be isotonic.
  • Formulations optionally contain excipients such as those set forth in the "Handbook of Pharmaceutical Excipients” (1986) and include ascorbic acid and other antioxidants, chelating agents such as EDTA, carbohydrates such as dextrin, hydroxyalkylcellulose, hydroxyalkylmethylcellulose, stearic acid and the like.
  • pharmaceutically acceptable carrier means any material or substance with which the active ingredient is formulated in order to facilitate its application or dissemination to the locus to be treated, for instance by dissolving, dispersing or diffusing the said composition, and/or to facilitate its storage, transport or handling without impairing its effectiveness.
  • the pharmaceutically acceptable carrier may be a solid or a liquid or a gas which has been compressed to form a liquid, i.e. the compositions of this invention can suitably be used as concentrates, emulsions, solutions, granulates, dusts, sprays, aerosols, suspensions, ointments, creams, tablets, pellets or powders.
  • Suitable pharmaceutical carriers for use in the said pharmaceutical compositions and their formulation are well known to those skilled in the art, and there is no particular restriction to their selection within the present invention. They may also include additives such as wetting agents, dispersing agents, stickers, adhesives, emulsifying agents, solvents, coatings, antibacterial and antifungal agents (for example phenol, sorbic acid, chlorobutanol), isotonic agents (such as sugars or sodium chloride) and the like, provided the same are consistent with pharmaceutical practice, i.e. carriers and additives which do not create permanent damage to mammals.
  • additives such as wetting agents, dispersing agents, stickers, adhesives, emulsifying agents, solvents, coatings, antibacterial and antifungal agents (for example phenol, sorbic acid, chlorobutanol), isotonic agents (such as sugars or sodium chloride) and the like, provided the same are consistent with pharmaceutical practice, i.e. carriers and additives which do not create permanent damage to mammals.
  • compositions of the present invention may be prepared in any known manner, for instance by homogeneously mixing, coating and/or grinding the active ingredients, in a one-step or multi-steps procedure, with the selected carrier material and, where appropriate, the other additives such as surface- active agents. They may also be prepared by micronisation, for instance in view to obtain them in the form of microspheres usually having a diameter of about 1 to 10 ⁇ m, namely for the manufacture of microcapsules for controlled or sustained release of the active ingredients.
  • Suitable surface-active agents, also known as emulgent or emulsifier, to be used in the pharmaceutical compositions of the present invention are non-ionic, cationic and/or anionic materials having good emulsifying, dispersing and/or wetting properties.
  • Suitable anionic surfactants include both water-soluble soaps and water- soluble synthetic surface-active agents.
  • Suitable soaps are alkaline or alkaline-earth metal salts, non-substituted or substituted ammonium salts of higher fatty acids (C 10 - C 22 ), e.g. the sodium or potassium salts of oleic or stearic acid, or of natural fatty acid mixtures obtainable form coconut oil or tallow oil.
  • Synthetic surfactants include sodium or calcium salts of polyacrylic acids; fatty sulfonates and sulfates; sulfonated benzimidazole derivatives and alkylarylsulfonates.
  • Fatty sulfonates or sulfates are usually in the form of alkaline or alkaline-earth metal salts, non-substituted ammonium salts or ammonium salts substituted with an alkyl or acyl radical having from 8 to 22 carbon atoms, e.g. the sodium or calcium salt of lignosulfonic acid or dodecylsulfonic acid or a mixture of fatty alcohol sulfates obtained from natural fatty acids, alkaline or alkaline-earth metal salts of sulfuric or sulfonic acid esters (such as sodium lauryl sulfate) and sulfonic acids of fatty alcohol/ethylene oxide adducts.
  • Suitable sulfonated benzimidazole derivatives preferably contain 8 to 22 carbon atoms.
  • alkylarylsulfonates are the sodium, calcium or alcanolamine salts of dodecylbenzene sulfonic acid or dibutyl-naphthalenesulfonic acid or a naphthalene-sulfonic acid/formaldehyde condensation product.
  • corresponding phosphates e.g. salts of phosphoric acid ester and an adduct of p- nonylphenol with ethylene and/or propylene oxide, or phospholipids.
  • Suitable phospholipids for this purpose are the natural (originating from animal or plant cells) or synthetic phospholipids of the cephalin or lecithin type such as e.g. phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerine, lysolecithin, cardiolipin, dioctanylphosphatidyl-choline, dipalmitoylphosphatidyl -choline and their mixtures.
  • cephalin or lecithin type such as e.g. phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerine, lysolecithin, cardiolipin, dioctanylphosphatidyl-choline, dipalmitoylphosphatidyl -choline and their mixtures.
  • Suitable non-ionic surfactants include polyethoxylated and polypropoxylated derivatives of alkylphenols, fatty alcohols, fatty acids, aliphatic amines or amides containing at least 12 carbon atoms in the molecule, alkylarenesulfonates and dialkyl- sulfosuccinates, such as polyglycol ether derivatives of aliphatic and cycloaliphatic alcohols, saturated and unsaturated fatty acids and alkylphenols, said derivatives preferably containing 3 to 10 glycol ether groups and 8 to 20 carbon atoms in the (aliphatic) hydrocarbon moiety and 6 to 18 carbon atoms in the alkyl moiety of the alkylphenol.
  • non-ionic surfactants are water-soluble adducts of polyethylene oxide with polypropylene glycol, ethylenediamino-polypropylene glycol containing 1 to 10 carbon atoms in the alkyl chain, which adducts contain 20 to 250 ethyleneglycol ether groups and/or 10 to 100 propyleneglycol ether groups.
  • Such compounds usually contain from 1 to 5 ethyleneglycol units per propyleneglycol unit.
  • non-ionic surfactants are nonylphenol polyethoxyethanol, castor oil polyglycolic ethers, polypropylene/polyethylene oxide adducts, tributylphenoxypolyethoxyethanol, polyethyleneglycol and octylphenoxypoly- ethoxyethanol.
  • Fatty acid esters of polyethylene sorbitan such as polyoxyethylene sorbitan trioleate
  • glycerol glycerol
  • sorbitan sucrose and pentaerythritol are also suitable non-ionic surfactants.
  • Suitable cationic surfactants include quaternary ammonium salts, particularly halides, having 4 hydrocarbon radicals optionally substituted with halo, phenyl, substituted phenyl or hydroxy; for instance quaternary ammonium salts containing as N-substituent at least one C 8 - 22 alkyl radical (e.g. cetyl, lauryl, palmityl, myristyl, oleyl and the like) and, as further substituents, unsubstituted or halogenated lower alkyl, benzyl and/or hydroxy-lower alkyl radicals.
  • quaternary ammonium salts particularly halides, having 4 hydrocarbon radicals optionally substituted with halo, phenyl, substituted phenyl or hydroxy
  • quaternary ammonium salts containing as N-substituent at least one C 8 - 22 alkyl radical (e.g. cetyl, lauryl, palmityl
  • Compounds of the invention and their physiologically acceptable salts may be administered by any route appropriate to the condition to be treated, suitable routes including oral, rectal, nasal, topical (including ocular, buccal and sublingual), vaginal and parenteral (including subcutaneous, intramuscular, intravenous, intradermal, intrathecal and epidural).
  • suitable routes including oral, rectal, nasal, topical (including ocular, buccal and sublingual), vaginal and parenteral (including subcutaneous, intramuscular, intravenous, intradermal, intrathecal and epidural).
  • the preferred route of administration may vary with for example the condition of the recipient.
  • the formulations both for veterinary and for human use, of the present invention comprise at least one active ingredient, as above described, together with one or more pharmaceutically acceptable carriers therefore and optionally other therapeutic ingredients.
  • the carrier(s) optimally are "acceptable” in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.
  • the formulations include those suitable for oral, rectal, nasal, topical (including buccal and sublingual), vaginal or parenteral (including subcutaneous, intramuscular, intravenous, intradermal, intrathecal and epidural) administration.
  • the formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy.
  • Such methods include the step of bringing into association the active ingredient with the carrier which constitutes one or more accessory ingredients.
  • the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.
  • Formulations of the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets or tablets each containing a predetermined amount of the active ingredient; as a powder or granules; as solution or a suspension in an aqueous liquid or a non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion.
  • the active ingredient may also be presented as a bolus, electuary or paste.
  • a tablet may be made by compression or molding, optionally with one or more accessory ingredients.
  • Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with a binder, lubricant, inert diluent, preservative, surface active or dispersing agent.
  • Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.
  • the tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active ingredient therein.
  • For infections of the eye or other external tissues e.g.
  • the formulations are optionally applied as a topical ointment or cream containing the active ingredient(s) in an amount of, for example, 0.075 to 20% w/w (including active ingredient(s) in a range between 0.1% and 20% in increments of 0.1% w/w such as 0.6% w/w, 0.7% w/w, etc), preferably 0.2 to 15% w/w and most preferably 0.5 to 10% w/w.
  • the active ingredients may be employed with either a paraffinic or a water-miscible ointment base.
  • the active ingredients may be formulated in a cream with an oil-in-water cream base.
  • the aqueous phase of the cream base may include, for example, at least 30% w/w of a polyhydric alcohol, i.e. an alcohol having two or more hydroxyl groups such as propylene glycol, butane 1 ,3-diol, mannitol, sorbitol, glycerol and polyethylene glycol (including PEG400) and mixtures thereof.
  • the topical formulations may desirably include a compound which enhances absorption or penetration of the active ingredient through the skin or other affected areas. Examples of such dermal penetration enhancers include dimethylsulfoxide and related analogs.
  • the oily phase of the emulsions of this invention may be constituted from known ingredients in a known manner.
  • the phase may comprise merely an emulsifier (otherwise known as an emulgent), it desirably comprises a mixture of at least one emulsifier with a fat or an oil or with both a fat and an oil.
  • a hydrophilic emulsifier is included together with a lipophilic emulsifier which acts as a stabilizer. It is also preferred to include both an oil and a fat.
  • the emulsifier(s) with or without stabilizer(s) make up the so-called emulsifying wax, and the wax together with the oil and fat make up the so-called emulsifying ointment base which forms the oily dispersed phase of the cream formulations.
  • oils or fats for the formulation is based on achieving the desired cosmetic properties, since the solubility of the active compound in most oils likely to be used in pharmaceutical emulsion formulations is very low.
  • the cream should optionally be a non-greasy, non-staining and washable product with suitable consistency to avoid leakage from tubes or other containers.
  • Straight or branched chain, mono- or dibasic alkyl esters such as di-isoadipate, isocetyl stearate, propylene glycol diester of coconut fatty acids, isopropyl myristate, decyl oleate, isopropyl palmitate, butyl stearate, 2-ethylhexyl palmitate or a blend of branched chain esters known as Crodamol CAP may be used, the last three being preferred esters. These may be used alone or in combination depending on the properties required. Alternatively, high melting point lipids such as white soft paraffin and/or liquid paraffin or other mineral oils can be used.
  • Formulations suitable for topical administration to the eye also include eye drops wherein the active ingredient is dissolved or suspended in a suitable carrier, especially an aqueous solvent for the active ingredient.
  • the active ingredient is optionally present in such formulations in a concentration of 0.5 to 20%, advantageously 0.5 to 10% particularly about 1.5% w/w.
  • Formulations suitable for topical administration in the mouth include lozenges comprising the active ingredient in a flavored basis, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert basis such as gelatin and glycerin, or sucrose and acacia; and mouthwashes comprising the active ingredient in a suitable liquid carrier.
  • Formulations for rectal administration may be presented as a suppository with a suitable base comprising for example cocoa butter or a salicylate.
  • Formulations suitable for nasal administration wherein the carrier is a solid include a coarse powder having a particle size for example in the range 20 to 500 microns (including particle sizes in a range between 20 and 500 microns in increments of 5 microns such as 30 microns, 35 microns, etc), which is administered in the manner in which snuff is taken, i.e. by rapid inhalation through the nasal passage from a container of the powder held close up to the nose.
  • Suitable formulations wherein the carrier is a liquid, for administration as for example a nasal spray or as nasal drops include aqueous or oily solutions of the active ingredient.
  • Formulations suitable for aerosol administration may be prepared according to conventional methods and may be delivered with other therapeutic agents.
  • Formulations suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or spray formulations containing in addition to the active ingredient such carriers as are known in the art to be appropriate.
  • Formulations suitable for parenteral administration include aqueous and non- aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents.
  • the formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use.
  • Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.
  • Preferred unit dosage formulations are those containing a daily dose or unit daily sub-dose, as herein above recited, or an appropriate fraction thereof, of an active ingredient.
  • formulations of this invention may include other agents conventional in the art having regard to the type of formulation in question, for example those suitable for oral administration may include flavoring agents.
  • This invention includes controlled release pharmaceutical formulations containing as active ingredient one or more compounds of the invention ("controlled release formulations") in which the release of the active ingredient can be controlled and regulated to allow less frequency dosing or to improve the pharmacokinetic or toxicity profile of a given invention compound.
  • Controlled release formulations adapted for oral administration in which discrete units comprising one or more compounds of the invention can be prepared according to conventional methods.
  • Control release compositions may thus be achieved by selecting appropriate polymer carriers such as for example polyesters, polyamino acids, polyvinyl pyrrolidone, ethylene-vinyl acetate copolymers, methylcellulose, carboxymethylcellulose, protamine sulfate and the like.
  • the rate of drug release and duration of action may also be controlled by incorporating the active ingredient into particles, e.g. microcapsules, of a polymeric substance such as hydrogels, polylactic acid, hydroxymethylcellulose, polyniethyl methacrylate and the other above-described polymers.
  • Such methods include colloid drug delivery systems like liposomes, microspheres, microemulsions, nanoparticles, nanocapsules and so on.
  • the pharmaceutical composition may require protective coatings.
  • Pharmaceutical forms suitable for injectionable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation thereof. Typical carriers for this purpose therefore include biocompatible aqueous buffers, ethanol, glycerol, propylene glycol, polyethylene glycol and the like and mixtures thereof.
  • each active ingredient may therefore be formulated in a way suitable for an administration route different from that of the other ingredient, e.g. one of them may be in the form of an oral or parenteral formulation whereas the other is in the form of an ampoule for intravenous injection or an aerosol.
  • This invention shows that a modified nucleoside such as, but not limited to; 2'- deoxy-adenosine-5'-iminodiacetate-phosphoramidate (IA-dAMP) is successfully recognized and efficiently incorporated into a growing DNA strand by HIV RT.
  • IA-dAMP 2'- deoxy-adenosine-5'-iminodiacetate-phosphoramidate
  • an iminodiacetate-phosphoramidate moiety can mimic a pyrophosphate group and behave as a good leaving group in a nucleotidyl transfer.
  • Incorporation of phosphor-amidate or diester analogues although to a lesser extent, was also observed for phtalic acid phosphoramidates and phosphodiesters, respectively.
  • a better leaving group capacity of an oxygen linked moiety may compensate for a loss in electrostatic effects. This is confirmed when using leaving groups with an aromatic character.
  • the difference between the phthalic acid and isophthalic acid embodiments described below may be related to a different way that these molecules can be accommodated in the active site of the polymerase.
  • IA-dAMP iminodiacetate dAMP as phosphoramidate
  • nucleoside analogues of this invention may be accomplished according to the method illustrated by scheme 2 below, starting from a nucleoside monophosphate, which itself can be tailor-made by phosphorylation of a suitable nucleoside.
  • the compounds according to the invention may be synthesized by derivatisation of the 5'-mono-phosphate nucleoside precursor molecule as illustrated in Scheme 2.
  • a first step (a) the phosphate group of the 5'-mono-phosphate nucleoside is coupled with the Z-group of a reagent represented by the structural formula (E) for producing compounds according to the structural formula (II):
  • alkoxy-anilines suitable as starting materials for the first step reaction of the method of scheme 2 include, but are not limited to, 2- methoxyaniline, 3-methoxyaniline, 4-methoxyaniline, 2-ethoxyaniline, 3-ethoxyaniline, 4-ethoxyaniline, 4-bromo-3-ethoxyaniline hydrochloride, 2-propoxyaniline, 3-propoxy- aniline, 4-propoxyaniline, 3-isopropoxyaniline, 4-isopropoxyaniline, 2,5- diethoxyaniline, 3,4-diethoxyaniline, 4-n-butoxyaniline, 3-n-butoxyaniline, 2-n- butoxyaniline, 4-iso-butoxyaniline, 3-isobutoxyaniline, 2-isobutoxyaniline, 2-methyl-4- methoxyaniline, 2-(methylthio)aniline, 3-(methylthio)aniline, 4-(methylthio)aniline, 2- trifluoromethoxyaniline, 3-trifluoromethoxyaniline, 4-trifluoromethyani
  • Halo-substituted anilines suitable as starting materials for the first step reaction of the method of scheme 2 include, but are not limited to, 2-fluoroaniline, 3- fluoroaniline, 4-fluoroaniline, 2,3-difluoroaniline, 2,4-difluoroaniline, 2,5-difluoroaniline, 2,6-difluoroaniline, 3,4-difluoroaniline, 2,3,4-trifluoroaniline, 2,3,5-trifluoroaniline, 2,3,6-trifluoroaniline, 2,4,6-trifluoroaniline, 2,4,5-trifluoroaniline, 3,4,5-trifluoroaniline, 3-chloro-2-fluoroaniline, 4-chloro-2-fluoroaniline, 5-chloro-2-fluoroaniline, 2-chloro-3- fluoroaniline, 2-chloro-4-fluoroaniline, 2-chloro-6-fluoroaniline, 3-chloro-5- fluoro
  • Alkyl-substituted anilines suitable as starting materials for the first step reaction of the method of scheme 2 include, but are not limited to, 4-methylaniline, 3- methylaniline, 2-methylaniline, 2,3-dimethylaniline, 2,4-dimethylaniline, 2,5- dimethylaniline, 2,6-dimethylaniline, 3,4-dimethylaniline, 3,5-dimethylaniline, 2,4,6- trimethylaniline, 3,4,5-trimethylaniline, 2,4,5-trimethylaniline, 2,4,6-triethylaniline, 4- ethylaniline, 3-ethylaniline, 2-ethylaniline, 2-n-propylaniline, 4-n-propylaniline, 2- isopropylaniline, 3-isopropylaniline, 4-isopropylaniline, 2,6-diisopropylaniline, 2-n- butylaniline, 4-n-butylaniline, 2-sec-butylaniline, 4-sec-buty
  • substituted anilines suitable as starting materials for the first step reaction of the method of scheme 2 include, but are not limited to, 4-cyclohexylaniline; 2- nitroaniline, 3-nitroaniline, 4-nitroaniline, 2-trifluoromethylaniline, 3- trifluoromethylaniline, 4-trifluoromethylaniline, and the like.
  • Benzyl alcohols suitable as starting materials for the first step reaction of the method of scheme 2 include, but are not limited to, 2-nitrobenzyl alcohol, 3- nitrobenzyl alcohol, 4-nitrobenzyl alcohol, 4-methylbenzyl alcohol, 4-isopropylbenzyl alcohol, 4-tert-butylbenzyl alcohol, 4-methoxybenzyl alcohol, 3-methoxybenzyl alcohol, 2-methoxybenzyl alcohol, 3-isopropoxybenzyl alcohol, 4-n-butoxybenzyl alcohol, 2-bromobenzyl alcohol, 3-bromobenzyl alcohol, 4-bromobenzyl alcohol, 3,5- dibromobenzyl alcohol, 3-chlorobenzyl alcohol, 2-chlorobenzyl alcohol, 3,4- dichlorobenzyl alcohol, 3,5-dichlorobenzyl alcohol, 2,4-dichlorobenzyl alcohol, 2,6- dichlorobenzyl alcohol, 2,3-dichlorobenzyl alcohol, 4-fluorobenzyl alcohol, 3- fluoro
  • Suitable commercial benzylamines for use in the first step of scheme 2 include, but are not limited to, 2-chlorobenzylamine, 4-chlorobenzylamine, 2,4- dichlorobenzylamine, 3,4-dichlorobenzylamine, 4-methoxybenzylamine, 4- methylbenzylamine, piperonylamine, 3,4-dimethoxybenzylamine, 3- methylbenzylamine, 3-fluorobenzylamine, 2-methylbenzylamine, 2- methoxybenzylamine, 3-methoxybenzylamine, 2-fluorobenzylamine, 4-fluorobenzylamine, 3,4-dihydroxybenzylamine, 3-chlorobenzylamine,
  • Said coupling reaction may be performed using any coupling agent (also referred to as dehydrating agent) known in the art for esterification or amide formation, in particular using a carbodiimide coupling agent, more in particular using dicyclohexylcarbodiimide (DCC).
  • the coupling reaction is performed at a temperature between room temperature and reflux temperature of the solvent the reaction is perfomed in.
  • reagent (E) may be transiently protected to prevent these functionalities from interfering with the condensation reaction between the phosphate acid and Z. Therefore, the synthetic route provides for an optional subsequent step (b) of deprotecting such functionalities.
  • This deprotection step can be carried out with potassium carbonate in methanol-water solution.
  • Schemes 3 and 4 below illustrate a synthetic route for the synthesis of pyrimidine and purine derived compounds according to the present invention respectively.
  • esters of the phosphate-modified nucleosides of the invention were accomplished according to the general principles of the method described by Wagner et al. in Mini- Rev. Med. Chem. (2004) 4:409, starting from a nucleoside monophosphate. Deprotection of the esters was carried out with potassium carbonate in methanol- water solution.
  • NMR spectral analyses were carried out on a Brucker AvanceTM Il 300 MHz or 500 MHz with PAXTI probe.
  • the Bruker TopspinTM 2.1 software was used to process spectra.
  • Standard mass spectra were measured with a Finnigan LCQ DuO (Thermo Fischer Scientific) using the ionisation by electron impact technique (ESI); data were acquired with the LAC/E 32 system (Waters). Exact mass spectra were obtained with a Q-Tof 2TM (Micromass Ltd.) coupled to a CapLCTM system (Waters). Chemicals of analytical or synthetic grade were obtained from cornm ⁇ rciai sources and were used as received (deoxyadenosine monophosphate: Sigma Aldrich; dicyciohexylcarbodiimide (DCC), dimethyl iminodiacetic acid hydrochloride: Fluka; te/t-butano! and triethylamine: Acros).
  • Oligodeoxyribonucleotides P1 , T1 , T2 and T3 were purchased from Sigma Genosys. The concentrations were determined with a Varian Cary-300-Bio UV Spectrophotometer. The lyophilized oligonucleotides were dissolved in diethylpyrocarbonate (DEPC)-treated water and stored at -2CO. The primer oligonucleotides were 5'- 33 P-labeled with 5'-[ ⁇ 33 P]-ATP (Perkin Elmer) using T4 polynucleotide kinase (New England Biolabs) according to standard procedures. The labeled oligonucleotide was further purified using IHustraTM MicrospinTM G-25 columns (GE Healthcare).
  • DNA polymerase reactions end-labeled primer was annealed to its template by combining primer and template in a molar ratio of 1 :2 and heating the mixture to 70 9 C for 10 minutes followed by slow cooling to room temperature over a period of 1.5 hour.
  • a series of 20 ⁇ L-batch reactions was performed with the enzyme HIV-1 RT (Ambion, 10 U/ ⁇ L stock soln, specific activity 8.095 U/mg, concentration 1 .2 mg/mL).
  • the final mixture contained 125 nM primer template complex, RT buffer (250 mM Tris.HCI, 250 mM KCI, 50 mM MgCI 2 , 2.5 mM spermidine, 50 mM dithiothreitol (DTT); pH 8.3), 0.025 U/ ⁇ L HIV-1 RT , and different concentrations of phosphoramidate or phosphodiester building blocks (1 mM, 500 ⁇ M, 200 ⁇ M and 100 ⁇ M). In the case of the aromatic analogues 3 and 4, the range of concentrations was limited to 1 mM. In the control reaction with the natural nucleotide, a 10 ⁇ M dATP concentration was used. The mixture was incubated at 37°C and 2.5 ⁇ L aliquots were quenched after 5, 10, 20, 30 , 60 and 120 minutes.
  • RT buffer 250 mM Tris.HCI, 250 mM KCI, 50 mM MgCI 2 , 2.5 mM spermidine, 50 m
  • the steady-state kinetics of single nucleotide incorporation of the iminodiacetate phosphoramidate 1 (IA-dAMP) and of a natural nucleoside triphosphate (dATP) was determined by gel-based polymerase assay.
  • the template T1 and the primer P1 were used.
  • the primer and template in 1 :2 molar ratio were hybridised in a buffer containing 20 mM Tris.HCI, 10 mM KCI, 2 mM MgSO 4 , 0.1 % Triton X-100, pH 8.3 and used in an amount to provide 125 nM concentration of the primer in each 20 ⁇ l_ reaction.
  • the range of concentrations for iminodiacetate dAMP was optimized according to a K M value for the incorporation of an individual nucleotide.
  • reaction mixtures containing the enzyme in 0.025 U/ ⁇ L concentration and appropriate substrate concentration to attain 5-25 % conversion were incubated at 37°C and run for 8 different time intervals.
  • the reactions were quenched by addition of the buffer 80% formamide, 2 mM EDTA, 1X TBE buffer.
  • the analysis of polymerase reaction was performed by polyacrylamide gel electrophoresis (see detailed protocol below).
  • V incorporation rates
  • Electrophoresis all polymerase reaction aliquots (2.5 ⁇ l_) were quenched by the addition of 10 ⁇ L of loading buffer (90% formamide, 0.05% bromophenol blue, 0.05% xylene cyanol and 50 mM ethylenediaminetetraacetic acid (EDTA)). Samples were heated at 85 °C for 5 minutes prior to analysis by electrophoresis for 2.5 hours at 2000 V on a 30 cm x 40 cm x 0.4 mm 20% (19:1 mono:bis) denaturing gel in the presence of a 100 mM Tris-borate, 2.5 mM EDTA buffer; pH 8.3. Products were visualized by phosphorimaging. The amount of radioactivity in the bands corresponding to the products of enzymatic reactions was determined by using the imaging device Cyclone® and the Optiquant image analysis software (Perkin Elmer).
  • Figure 1 shows the structure of a representative example of this class (IA- dAMP) and a synthetic scheme including an easy-to-perform two steps reaction to produce it.
  • the synthesis starts from an iminodiacetic acid diester (dimethyl ester shown in figure 1 , but the skilled person understands that the same reaction may be performed starting from the commercially available diethyl ester or dibenzyl erster as well).
  • iminodiacetic acid diester dimethyl ester shown in figure 1 , but the skilled person understands that the same reaction may be performed starting from the commercially available diethyl ester or dibenzyl erster as well.
  • the usage of naming amino-acids containing a secondary amine group as "imino acids” is disputed by some, and the IUPAC name for "iminodiacetic acid” is 2-(carboxymethylamino)acetic acid.
  • EXAMPLE 3 single incorporation HIV-1 Reverse Transcriptase serves, in the HIV-1 viral replication process, as a catalyst and uses deoxynucleotides as substrates. This polymerase is error-prone and thus has a high mutation rate.
  • the initial screening was carried out using a template with an overhang of one thymidine nucleotide followed by three non- pyrimidine bases (Table 1 ). Incorporation efficiency was analysed by the polyacrylamide gel-based single nucleotide incorporation assay.
  • the isophthalic acid-derived phosphodiester (2) was recognized by HIV-1 RT and efficiently incorporated into a growing primer strand (as shown in figure 3) with a conversion to an n+1 strand 90-92 % (2) over a period of 2 hours at 1 mM concentration.
  • the corresponding anilino-derived phosphate nucleoside (3) was less well recognized as substrate.
  • little incorporation (13% n+1 product after 2 hours) was observed with the phthalic acid dAMP derivative (4).
  • the one carrying both carboxyl substituents in meta position (2) is more successful than the one carrying the carboxyl substituents in meta and para positions respectively (4).
  • Compound (2) was further evaluated at different concentrations (as shown in figure 4). At 500 ⁇ M compound (2) displayed 75% of n+1 formation, which represents 88% of L-Asp-dAMP capacity. We also tested the possibility of a polymerase independent incorporation, but no compound was incorporated in the absence of the enzyme.
  • Oligodeoxyribonucleotides P1 , T1 , T2 and T3 were purchased from Sigma
  • Genosys The concentrations were determined with a Varian Cary-300-Bio UV Spectrophotometer.
  • the lyophilized oligonucleotides were dissolved in diethylpyrocarbonate (DEPC)-treated water and stored at -2CO.
  • the primer oligonucleotides were 5'- 33 P- labeled with 5'-[ ⁇ 33 P]-ATP (Perkin Elmer) using T4 polynucleotide kinase (New England Biolabs) according to Standard procedures.
  • the labeled oligonucleotide was further purified using IHustraTM MicrospinTM G-25 columns (GE Healthcare).
  • End-labeled primer was annealed to its template by combining primer and template in a molar ratio of 1 :2 and heating the mixture to 70°C for 10 minutes followed by slow cooling to room temperature over a period of 1.5 hour.
  • a series of 20 ⁇ L-batch reactions was performed with the enzyme HIV-1 RT (Ambion, 10 U/ ⁇ L stock solution, specific activity 8.095 U/mg, concentration 1.2 mg/mL).
  • the final mixture contained 125 nM primer template complex, RT buffer (250 mM Tris.HCI, 250 mM KCI, 50 mM MgCI 2 , 2.5 mM spermidine, 50 mM dithiothreitol (DTT); pH 8.3), 0.025 U/ ⁇ L HIV-1 RT, and different concentrations of phosphoramidate or phosphodiester building blocks (1 mM, 500 ⁇ M, 200 ⁇ M and 100 ⁇ M respectively).
  • the range of concentrations was limited to 1 mM.
  • a 10 ⁇ M dATP concentration was used in the control reaction with the natural nucleotide.
  • the mixture was incubated at 37°C and 2.5 ⁇ L aliquots were quenched after 5, 10, 20, 30 , 60 and 120 minutes. Results are shown in figure 3.
  • the increased elongation efficiency compared to Asp-dAMP might also be due to the constitutional change i.e. that the leaving nitrogen atom of IA-dAMP is a secondary amine while that of L-Asp-dAMP is a primary amine, and to the better chelating ability of the N-diacetate group of IA-dAMP when compared with the Asp group of L-Asp-dAMP.
  • one magnesium ion is coordinated by the 3' end of the primer and the ⁇ -phosphate, while the second magnesium ion is thought to facilitate the leaving group properties of the pyrophosphate moiety by chelating with the ⁇ - and ⁇ -phosphates of the nucleotide.
  • Iminodiacetic acid and aspartic acid display dissociation constants (K D 25° c) for the divalent magnesium ion of 2.98 and 2.43, respectively, while the dissociation constant of pyrophosphate equals 5.45.
  • K D 25° c dissociation constant for the divalent magnesium ion of 2.98 and 2.43, respectively
  • dissociation constant of pyrophosphate equals 5.45.
  • Compound 1 (IA-dAMP) was further investigated as follows. Kinetic parameters for the incorporation of both the natural and the modified substrate by HIV-1 RT were determined on the basis of the single completed hit model; P1 and T1 were used as the priming and templating DNA strands, respectively. The kinetic values K M and V Max corresponding to the substrate efficiency of the nucleotides are given in Table 3.
  • the incorporation efficiency of IA-dAMP for HIV-1 RT is approximately 10 4 times lower than that of dATP.
  • the K M of IA-dAMP is much higher than for the natural substrate, whereas the V Max is 1 .3 folds lower.
  • R 1 CH 2 COOCH 3 2
  • R 1 CH 2 COOH 2b
  • R 2 COOCH 3 3
  • R 2 COOH
  • step 1 DCC, 1 ,4-dioxane/DMF at 80 ⁇ O; step 2) 0.4M NaOH (MeOH/H 2 O).
  • HIV-1 reverse transcriptase to incorporate phosphoramidate analogues 5-6 was analysed by gel-based single-nucleotide-incorporation assays using primer-template complex PiT 1
  • Compound 5 shows the most remarkable result at 50 ⁇ M concentration ( Figure 7A).
  • Efficient substrate incorporation can also be detected when substrate concentration decreased to 5 ⁇ M or 10 ⁇ M, with 17.8% and 33% incorporation, respectively.
  • the corresponding analogue 6 is less well recognized under the same condition, compound 6 can only get 63% incorporation within 60 minutes with a 500 ⁇ M substrate concentration ( Figure 7B).
  • Figure 7A In order to investigate the strand elongation capacity of compound 5, a template dependent incorporation of more than one nucleotide experiment was carried out. In this experiment HIV-1 RT and PiT 2 duplex, where seven thymidine bases overhang of the template is flanked by four non-thymidine units at the 3'-end were used.

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