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
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/04—Antibacterial agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
  • A61P31/14—Antivirals for RNA viruses
  • A61P31/18—Antivirals for RNA viruses for HIV
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • 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
  • C07H19/14—Pyrrolo-pyrimidine radicals
• • 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

Definitions

• HCV Hepatitis C virus
• a major cause of viral hepatitis infects more than 200 million people worldwide.
• Current treatment for HCV infection is restricted to immunotherapy with interferon- ⁇ , alone or in combination with Ribavirin, a nucleoside analog. This treatment is effective in only about half of the patients.
• Hepatitis B virus (HBV) acutely infects almost a third of the world's human population, and about 5% of the infected are chronic carriers of the virus.
• Chronic HBV infection causes liver damage that frequently progresses to cirrhosis and liver cancer later in life. Despite the availability and widespread use of effective chemotherapy and vaccines, the number of chronic carriers approaches 400 million worldwide.
• HIV Human immunodeficiency virus
• a number of drugs have been used clinically, including HIV nucleoside and non-nucleoside reverse transcriptase inhibitors and protease inhibitors.
• combination therapies are the accepted standard of treatment of AIDS in order to reduce viral load and the emergence of drug resistance.
• anti-HIV drugs AIDS is still one of the leading epidemic diseases. Therefore, there is still an urgent need for new, more effective HCV, HBV, and HIV drugs.
• the treatment of viral infections caused by other viruses such as HSV, CMV, influenza viruses, West Nile virus, small pox, EBV, VZV, and RSV also needs better medicines.
• Bacterial infections long have been the sources of many infectious diseases. The widespread use of antibiotics results in the emergence of drug-resistant life-threatening bacteria. Fungal infections are another type of infectious disease, some of which also can be life- threatening. There is an increasing demand for the treatment of bacterial and fungal infections. Antimicrobial drugs based on new mechanisms of action and new chemical classes are especially important.
• alkylating agents such as cisplatin
• antimetabolites such as 5-Fluorouracil, Gemcitabine, Cytarabine, Fludarabine, and Cladarabine.
• nucleoside analogs have been used clinically for the treatment of viral infections and proliferative disorders for decades. Most of the nucleoside drugs are classified as antimetabolites. After they enter cells, nucleoside analogs are successively phosphorylated to nucleoside 5'-monophosphates, 5'-diphosphates, and 5'-triphosphates. In most cases, nucleoside triphosphates, e.g.
• 3'-azido-3'-deoxythymidine (AZT, an anti-HIV drug) triphosphate and arabinosylcytosine (Cytarabine, an anticancer drug) triphosphate
• ZT an anti-HIV drug
• Cytarabine an anticancer drug triphosphate
• Nucleosides may act also as their diphosphates.
• 2'-deoxy-2',2'-difluorocytidine (Gemcitabine, an anticancer drug) 5'-diphosphate has been shown to inhibit human ribonucleotide reductase.
• nucleoside kinases nucleoside to nucleotide
• nucleotide kinases nucleotides to di- and tri-nucleotides. Efficient phosphorylation at each step is required for a nucleoside to be an effective drug.
• nucleoside mono-phosphate prodrugs have been intensively investigated as an alternative drug form (Wagner et al, Med. Res. Rev. 2000, 20, 417-451; Jones et al, Antiviral Res. 1995, 27, 1-17; Perigaud et al. Adv. in Antiviral Drug Des. 1995, 2, 147-172; Huynh-Dinh, Curr. Opin. Invest.
• nucleoside mono-phosphate prodrugs which mask the negative charges on the phosphate by reversible chemical modifications, now being much more lipophilic, would transverse cell membranes and liberate the nucleoside mono-phosphate intracellularly. Cleavage of the prodrug moiety from the nucleoside mono-phosphate would proceed enzymatically via a variety of ubiquitous, non-specific enzymes, like esterases or hydrolytically. Having now bypassed the first kinase step, which is often the most difficult of the three steps with unnatural nucleoside, higher concentration of the required, active species, the nucleoside tri-phosphate was expected.
• phosphate prodrugs of anti-HIV nucleosides have been explored for their use as antiviral drugs.
• the di-and tri-phosphates of 3'-deoxy-3'- azidothymidine (AZT) and 2',3'-didehydro-2',3'-dideoxythymidine (D4T) were converted to their acyl prodrugs (Bonnaffe et al. J. Org. Chem. 1996, 61, 895-902).
• AZT di- and triphosphate prodrugs demonstrated similar inhibition of HIV-infected cells as AZT itself, while the corresponding D4T di-and tri-phosphate prodrugs exhibited lower, but still significant anti- HIV activity. Since the acylphosphate moiety of the prodrugs is sensitive to chemical hydrolysis, it is assumed that the prodrugs had been converted to AZT and D4T before they enter cells. Phospholipids also have been as the masking moiety of nucleoside mono- and di-phosphates. AZT di-phosphate tethered with a thioether lipid showed potent inhibition of HIV-infected CEM cells (Hong et al. J. Med. Chem. 1996, 39, 1771-1777).
• lipid-tethered nucleoside di- and tri-phosphates also have been studied (Hostetler et al, J. Biol. Chem. 1990, 265, 6112-6117). Some antitumor nucleosides were also converted to the corresponding nucleotide prodrugs aimed at enhancing antitumor activities. Treatment with lipid-tethered Ara-C di-phosphates demonstrated longer life-span of p388 -infected mice than that with Ara-C itself (Hong et al, J. Med. Chem. 1986, 29, 2038-2044; 1990, 33, 1380-1386).
• nucleotide prodrugs may, in certain cases, deliver negatively-charged nucleotides into cells better than the parent nucleotides, but are not significantly stabile towards enzymatic and hydrolytic degradation.
• nucleoside phosphates bearing natural phosphates when released from their prodrugs intracellularly may not be anabolized to the required active species (nucleoside di- or tri- phosphates), but may be catabolized back to the inactive parent nucleoside, which is resistant to phosphoylation.
• an intermediate nucleoside phosphate that is formed may be a toxic species.
• AZT mono-phosphate accumulates in cells because the nucleoside mono-phosphate is a poor substrate for thymidylate kinase and is thought to be responsible for cellular toxicity.
• nucleoside phosphates bearing di-phosphate or tri-phosphate mimics have been prepared and some of them have been evaluated various biological assays.
• Many nucleotide mimics or their biological use have been disclosed (Eckstein et al. U. S. Patent No. 3,846,402 issued November 1974; Horwitz et al, U.S. Patent No. 4,266,048 issued May 1981; Schinazi et al, U.S. Patent No. 5,118,672 issued June 1992; Ingels et al, U.S. Patent No. 5,721,219 February 1998; Bottaro, et al, U.S. Patent No.
• nucleoside di- and tri-phosphate mimic has modifications at the bridging positions of nucleoside diphosphates and triphosphates (Yount et al, Biochemistry 1971, 10, 2484-2489; Ma et al, J. Med. Chem. 1992, 35, 1938-1941; Ma et al, Bioorg. Chem. 1989, 17, 194-206; Li et al, Bioorg. Chem. 1996, 24, 251-261; Trowbridge et al, J. Am. Chem. Soc. 1972, 94, 3816-3824; Stock, J. Org. Chem. 1979, 44, 3997-4000; Blackburn etal, J. Chem. Soc. Chem. Comm.
• phosphate mimics are the ⁇ , ⁇ - imidotriphosphates, ⁇ , ⁇ -methylimidotriphosphates, ⁇ , ⁇ -imidotriphosphates, ⁇ , ⁇ : ⁇ , ⁇ - diimidotriphosphates, ⁇ , ⁇ -methylenetriphosphates, ⁇ , ⁇ -methylenetriphosphates, ⁇ , ⁇ : ⁇ , ⁇ - bismethylenetriphosphates, ⁇ , ⁇ -dihalomethylenetriphosphates, ⁇ , ⁇ - dihalomethylenetriphosphates, ⁇ , ⁇ -halomethylenetriphosphates, and ⁇ , ⁇ - halomethylenetriphosphates.
• phosphate mimics usually enhance the stability of the nucleotide towards hydrolysis by cellular enzymes.
• Methylene and halomethylenes render the nucleoside di- and tri-phosphate mimics considerable more stable to both chemical and enzymatic hydrolysis.
• nucleoside di-phosphate and tri-phosphate mimic is the substitution of one or more phosphate non-bridging oxygen with other heteroatoms or functional group (Ludwig et al, J. Org. Chem. 1991, 56, 1777-1783; Dineva, Nucleosides Nucleotides 1996, 15, 1459-1467; Dyatkina et al Nucleosides Nucleotides 1995, 14, 91-103; He et al, J. Org. Chem. 1998, 63, 5769-5773; He et al, Nucleic Acids Res.
• phosphate mimics are ⁇ -O-alkyltriphosphate, ⁇ -O-aryltriphosphate, -P- alkyltriphosphate, ⁇ -E-aryltriphosphate, ⁇ -P-alkylaminotriphosphate, ⁇ -P-thiotriphosphate, ⁇ - P-boranotriphosphate, ⁇ -O-alkyltriphosphate, ⁇ -O-aryltriphosphate, ⁇ -P-alkyltriphosphate, y-P- aryltriphosphate, ⁇ -R-alkylaminotriphosphate, ⁇ -E-thiotriphosphate.
• nucleoside phosphate mimics generally are more stable to cellular nucleases than natural nucleoside phosphates.
• nucleoside di- and tri-phosphate mimics include modifications at the 5'- position of nucleosides. For instance, 3'-azido'-3',5'-dideoxy-5'-methylenethymidine 5'-C- triphosphate in which the 5 '-oxygen is replaced with methylene was synthesized and evaluated for anti-HIV activity (Freeman et al, J. Med. Chem. 1992, 35, 3192-3196). The nucleotide mimics in which the 5 '-oxygen is replaced by sulfur or amino also were reported (Trowbridge et al, J. Am. Chem. Soc. 1972, 94, 3816-3824; Letsinger et al, J. Am. Chem.
• nucleotide mimics comprising combinations of two or more phosphate modifications. So far, only nucleotide mimics containing ⁇ , ⁇ : ⁇ , ⁇ -diimidotriphosphate, ⁇ , ⁇ : ⁇ , ⁇ - bismethylenetriphosphate, and ⁇ -E-borano- ⁇ -P-thiotriphosphate were reported, which contain two modifications each.
• nucleosides for the preparation of nucleotide mimics in the reported work are generally selected from natural nucleosides and a few well known antiviral nucleosides such as AZT, D4T, and 3'-deoxythymidine. [0012] Some of these nucleotide mimics have been evaluated for their biological activity. AZT 5'- ⁇ -P-boranotriphosphate and D4T 5'- ⁇ -P-boranotriphosphate exhibited very potent inhibition of HIV reverse transcriptase (RT) with Ki values in the low nM range in assays using homopolymer templates.
• RT HIV reverse transcriptase
• AZT 5'- ⁇ , ⁇ -difluoromethylenetriphosphate and AZT 5'- ⁇ , ⁇ - imidotriphosphates also exhibited significant inhibition of DNA polymerase or HIV RT.
• the negatively-charged nucleotide mimics are not likely to be taken up intact by cells, and no meaningful cell-based antiviral data for di- and tri-nucleotide mimics has ever been published.
• These nucleotide mimics that are active in cell free biochemical assays contain only one modification each, either at the triphosphate bridging position or simply as a substitution of a phosphate oxygen and, thus, are ready substrates for enzymatic hydrolysis. Therefore, the mimics rapidly are degraded extracellularly to provide the parent nucleoside. Any biological activity would result from the parent nucleoside being taken into cells and anabolized to an active nucleotide.
• Nucleotide di- and tri-phosphate mimics that are resistant to cellular enzymes and demonstrate significant biological activities have not been disclosed.
• the several known nucleotide mimics are constructed from natural nucleosides, such as adenosine, or from known biologically active nucleosides, such as AZT.
• novel nucleoside di- and tri-phosphate mimics that are resistant to enzymatic degradation possess one or more prodrugs to allow effective intracellular transport.
• Nucleoside di- and tri-phosphate mimics with attached prodrugs have not been disclosed in the literature.
• this invention overcomes the need for anabolic enzymatic activation of nucleosides to active nucleotide species by providing the nucleosides as their di- and tri-phosphates.
• the di- and tri-phosphate moieties have been modified one or more times to provide novel nucleotide mimics that are resistant to degrading enzymes.
• novel nucleotide mimics are provided that contain a modified di- or tri-phosphate mimic that is stable to chemical and enzymatic degradation via hydrolysis, phosphorolysis and other possible reaction types.
• the novel nucleoside di- and tri-phosphates are converted into prodrugs to enhance drug absorption and/or drug delivery into cells.
• Another aspect of the present invention is to provide a combination of the first two aspects and provide prodrugs of novel nucleotide mimics to enhance drug absorption and/or drug delivery into cells.
• Another aspect of the present invention is to provide novel nucleotide mimics that are a combination of a novel nucleoside and a di- or tri-phosphate mimic.
• An additional aspect of the present invention is to provide novel nucleotide mimics as a composition for therapeutic use for treatment of viral infections, microbial infections, parasite infections and cellular proliferative disorders.
• a further aspect of the present invention is to provide a method for treatment of viral infections, microbial infections, parasite infections and proliferative disorders comprising administrating a nucleotide mimic or its prodrug of the present invention.
• Such compounds of the invention include prodrugs (i.e., one of X 7 -10 x must be the prodrug substituent R*) such as a compound of Formula (I) which may be a D- or L- nucleotide:
• A is O, S, CY 2 , NH or R;
• R 4' is -L-R 5
• L is selected from the group consisting of O, S, NH, NR, CY 2 O, CY 2 S, CY 2 NH, CY 2 , CY 2 CY 2 , CY 2 OCY 2 , CY 2 SCY 2 , and CY 2 NHCY 2 , wherein Y is selected from the group consisting of H, F, Cl, Br, alkyl, alkenyl, and alkynyl, wherein alkyl, alkenyl, and alkynyl may each optionally contain one or more heteroatoms;
• R 5 is a di- or tri-phosphate moiety of Formula (II):
• X 2 , X 3 , and X 4 are selected independently from the group consisting of O, S, Se,
• X 5 and X are selected independently from the group consisting of O, S, Se, O 2 ,
• CY 2 CO, CHOH, C(OH) 2 , CH 2 O, CH 2 CH 2 , CH 2 CHNH 2 , CH 2 CH 2 CHNH 2 , CY 2 OCY 2 , CY 2 , CRY, CY 2 C Y 2 , CHR, CC, HC CH, NH, NR, NOH, NOR, NNH 2 , and NNHR;
• X 7 , X 8 , X 9 , and X 10 are selected independently from the group consisting of H, F,
• R* may be conjugated to one or more X -X positions
• R is selected from the group consisting of alkyl, alkenyl, alkynyl, aryl, acyl, and aralkyl, each optionally containing one or more heteroatoms;
• M + is a cation; n is 0 or 1 ;
• D is a nucleoside base of Formula (III) or (IV):
• Z Z 2 , and Z 3 are independently N, CH or C-G 3 ;
• G 1 , G 2 , and G 3 are selected independently from the group consisting of H, F, Cl, Br, I, OH, SH, NH 2 , NHOH, NHNH 2 , N 3 , NO, NO 2 , CHO, COOH, CN, CONH 2 , CONHR, C(S)NH 2 , C(S)NHR, COOR, R, OR, SR, NHR, and NR 2 ; when two of G 3 are present on a molecule they may be same as or different from each other.
• Such prodrugs of the invention also include (i.e., one of X 7 -X 10 must be the prodrug substituent R*) also include compound of Formula (XVI):
• XVI wherein X°, X 5 and X 6 are selected independently from the group consisting of O, S, NH, and CY 2 ; wherein Y is selected from the group consisting of H, F, Cl, Br, alkyl, alkenyl, and alkynyl, wherein alkyl, alkenyl, and alkynyl may each optionally contain one or more heteroatoms; wherein X 2 , X 3 , and X 4 are selected independently from the group consisting of O, S, and Se; wherein X , X , X , and X are selected independently from the group consisting of H,
• R is selected from the group consisting of alkyl, alkenyl, alkynyl, aryl, acyl, and aralkyl each optionally containing one or more heteroatoms.
• nucleotide mimics of the invention i.e., X 7 -X 10 do not require the presence of a prodrug substituent R*
• nucleotide mimics of the invention include compounds of Formula (I) which may be a D- or L-nucleotide: wherein
• A is O, S, CY 2 , NH orNR;
• R 4' is -L-R 5
• L is selected from the group consisting of O, S, NH, NR, CY 2 O, CY 2 S, CY 2 NH, CY 2 , CY 2 CY 2 , CY 2 OCY 2 , CY 2 SCY 2 , and CY 2 NHCY 2 , CY 2 SCY 2 , and wherein Y is selected from the group consisting of H, F, Cl, Br, alkyl, alkenyl, and alkynyl, wherein alkyl, alkenyl, and alkynyl optionally contain one or more heteroatoms;
• R 5 is a di- or tri-phosphate moiety of Formula (II):
• X 2 , X 3 , and X 4 are selected independently from the group consisting of O, S, Se,
• X 7 , X 8 , X 9 , and X 10 are selected independently from the group consisting of H, F,
• n 0 or 1 ;
• D is a nucleoside base of Formula (III) or (IV):
• Z 1 , Z 2 , and Z 3 are independently N, CH or C-G 3 ;
• G 1 , G 2 , and G 3 are selected independently from the group consisting of H, F, Cl, Br, I, OH, SH, NH 2 , NHOH, NHNH 2 , N 3 , NO, NO 2 , CHO, COOH, CN, CONH 2 , CONHR, C(S)NH 2 , C(S)NHR, COOR, R, OR, SR, NHR, and NR 2 ; when two of G 3 are present on a molecule they may be same as or different from each other; and
• R is selected from the group consisting of alkyl, alkenyl, alkynyl, aryl, acyl, and aralkyl each optionally containing one or more heteroatoms; with provisos that:
• nucleotide mimics of the invention include compound of Formula (XVI):
• X 2 , X 3 , and X are each independently O, S, or Se; wherein X 7 , X 8 , X 9 , and X 10 are selected independently from the group consisting of H,
• Nu is selected from the group consisting of natural nucleosides, sugar-modified nucleosides, base-modified nucleosides, and nucleosides with both sugar and base modifications; wherein Nu is linked to X° through CH 2 of the sugar moiety of Nu; and
• R is selected from the group consisting of alkyl, alkenyl, alkynyl, aryl, acyl, and aralkyl each optionally containing one or more heteroatoms; with provisos that:
• Preferred embodiments of the prodrug of Formula (I) described above include prodrugs of the following formulas:
• L is selected from the group consisting of O, S, NH, CY 2 , CY 2 CY 2 , and CH 2 CY 2 where Y is H, F, Cl, or Br; wherein X 2 , X 3 , and X 4 are O, S, Se ; wherein X 5 and X 6 are selected independently from the group consisting of O, S, NH, NR, CY 2 ; and wherein X 7 , X 8 , X 9 , and X 10 are selected independently from the group consisting of H,
• X 2 is O and X 7 is SH, NH 2 ,
• X 9 and X 10 are selected independently from the group consisting of OH, SH, alkyl, alkoxy, aryl, and aryloxy, wherein X 9 and X 10 are selected independently from the group consisting of OH, alkoxy, acylthioethoxy, acyloxymethoxy, 1,2-O-diacylglyceryloxy, and 1- O-alkyl-2-O-acylglyceryloxy; and wherein X 6 is selected from a group consisting of NH, CHF, CCI 2 and CF 2. Most preferably, at least one of X 9 or X 10 is 1, 2,-O-dialkylglyceryloxy.
• X y and X ⁇ are selected independently from the group consisting of OH, alkoxy, and aryloxy, or wherein X 9 and X 10 are selected independently from the group consisting of OH, alkoxy, acylthioethoxy, acyloxymethoxy, 1,2-O-diacylglyceryloxy, and l-O-alkyl-2-O-acylglyceryloxy; and wherein X 6 is selected from a group consisting of NH, CHF, CC1 2 and CF 2.
• R 2 is methyl, ethyl, vinyl, ethynyl, or hydroxymethyl; wherein R 2 is H, F, OH, or OMe, alkyl, methoxyethoxy, or NH 2 ; wherein R 3 is H, F, OH, or N 3 ; wherein G 1 is OH or NH 2 ; wherein G is H or Me; wherein X 9 and X 10 are selected independently from the group consisting of OH, alkoxy, and aryloxy, or wherein X 9 and X 10 are selected independently from the group consisting of acylthioethoxy, acyloxymethoxy, 1,2-O-diacylglyceryloxy, 1,2-O-dialkylglyceryloxy, and 1-0- alkyl-2-O-acylglyceryloxy.
• R 2' is H, F, OH, or OMe; wherein R 3 is methyl, ethyl, vinyl, ethynyl, or hydroxymethyl; wherein R 3' is H, F, OH, or N 3 ; wherein G 1 is OH or NH 2 ; wherein G 2 is H or Me; and wherein X 9 and X 10 are selected independently from the group consisting of OH, alkoxy, and aryloxy, or wherein X 9 and X 10 are selected independently from the group consisting of acylthioethoxy, acyloxymethoxy, 1,2-O-diacylglyceryloxy, 1,2-O-dialkylglyceryloxy, and 1-O- alkyl-2-O-acylglyceryloxy.
• R 2 is methyl, ethyl, vinyl, ethynyl, hydroxymethyl or haloalkyl; wherein R is H, F, OH, OCH 3 , or methoxyethoxy; wherein R 3' is H, F, OH, N 3 , NH 2 , or CH 2 OH; wherein G 1 is OH, NH 2 , Cl, OMe, NH-cyclopropyl, SH, or S-alkyl; wherein G 2 is H, NH 2 , NHR, F, Cl, Br, or I; wherein Z 1 is N or CH; and wherein X 9 and X 10 are selected independently from the group consisting of OH, alkoxy and aryloxy, or wherein X 9 and X 10 are selected independently from the group consisting of acylthioethoxy, acyloxymethoxy, 1,2-O-diacylglyceryloxy, 1,2-O-dialkylglyceryl
• R >2 is H, F, OH or OCH 3 ; wherein R is methyl, ethyl, vinyl, ethynyl, or hydroxymethyl; wherein R 3' is H, F, OH, or N 3 ; wherein G 1 is OH or NH 2 ; wherein G is H orNH 2 ; wherein Z 1 is N or CH; and wherein X 9 and X 10 are selected independently from the group consisting of OH, alkoxy and aryloxy, or wherein X 9 and X 10 are selected independently from the group consisting of acylthioethoxy, acyloxymethoxy, 1 ,2-O-diacylglyceryloxy, 1,2-O-dialkylglyceryloxy, and 1-0- alkyl-2-O-acylglyceryloxy.
• R 2 is H, F, OH or OCH 3 ; wherein R is H, F, OH, or N 3 ; wherein R 4 is methyl, ethyl, vinyl, ethynyl, or hydroxymethyl; wherein G 1 is OH or NH 2 ; wherein G is H or NH2; wherein Z 1 is N or CH; and wherein X 9 and X 10 are selected independently from the group consisting of OH, alkoxy and aryloxy, or wherein X 9 and X 10 are selected independently from the group consisting of acylthioethoxy, acyloxymethoxy, 1,2-O-diacylglyceryloxy, 1,2-O-dialkylglyceryloxy, and 1-0- alkyl-2-O-acylglyceryloxy.
• Preferred embodiments of the compound of Formula (XVI) include prodrugs of the following formulas:
• X° and X 6 are selected independently from the group consisting of O, S, NH, CH 2 , CHC1, CHBr, CHF, CC1 2 , CBr 2 , and CF 2 wherein X 2 is O or S; wherein X 7 is selected from the group consisting of OH, F, SH, NH 2 , NHOH, " BH 3 M + , R, SR, and NHR; and wherein X 9 and X 10 are selected independently from the group consisting of OH, SH,
• X° and X 6 are selected independently from the group consisting of O, S, NH, CH 2 , CHC1, CHBr, CHF, CC1 2 , CBr 2 , and CF 2 wherein X 2 is O or S; wherein X 7 is selected from the group consisting of OH, F, SH, NH 2 , NHOH " B ⁇ 1" , R, SR, and NHR; and wherein X 9 and X 10 are selected independently from the group consisting of acylthioethoxy, acyloxymethoxy, 1,2-O-diacylglyceryloxy, 1,2-O-dialkylglyceryloxy, and l-O- alkyl-2- -acylglyceryloxy.
• X 6 is selected from the group consisting of NH, CH 2 , CHCl, CHBr, CHF, CC1 2 , CBr 2 , and CF 2; and wherein X 9 and X 10 are selected independently from the group consisting of OH, SH, alkyl, alkoxy, aryl and aryloxy.
• X 6 is selected from the group consisting of NH, CH 2 , CHCl, CHBr, CHF, CC1 2 , CBr 2 , and CF 2 ; and wherein X 9 and X 10 are selected independently from the group consisting of acylthioethoxy, acyloxymethoxy, 1,2-O-diacylglyceroxy, 1,2-O-dialkylglyceroxy, and 1-0- alkyl-2-O-acylglyceroxy.
• X 9 and X 10 are selected independently from the group consisting of OH, SH, alkyl, alkoxy, aryl and aryloxy, or wherein X 9 and X 10 are selected independently from the group consisting of acylthioethoxy, acyloxymethoxy, 1,2- -diacylglyceryloxy, 1,2-0- dialkylglyceryloxy, and l-O-alkyl-2- -acylglyceryloxy.
• Nu is preferably selected from:
• the invention also relates to a pharmaceutical composition
• a pharmaceutical composition comprising a therapeutically effective amount of any of the above compounds, a pharmaceutically acceptable salt thereof, optionally in combination with one or more other active ingredients and/or with a pharmaceutically acceptable carrier.
• the above any of the compounds may be used in a method for the treatment of a microbial infection or proliferative disorder comprising administering a therapeutically effective amount of any of the above compounds to a subject in need thereof.
• nucleotide mimics of the compound of Formula (I) include:
• X is selected from the group consisting of SH, NH 2 , " BH 3 M , alkyl, aryl, alkylamino, and arylamino, or wherein X is selected from the group consisting of SH,
• R is methyl, ethyl, vinyl, ethynyl, or hydroxymethyl; wherein R is H, F, OH, or OMe, alkyl, methoxyethoxy, or ML; wherein R is H, F, OH, or N ; wherein G 1 is OH or NH 2 ; wherein G 2 is H or Me; and wherein X and X ° are selected independently from the group consisting of OH, alkoxy, and aryloxy.
• R 2' is H, F, OH, or OMe; wherein R 3 is methyl, ethyl, vinyl, ethynyl, or hydroxymethyl; wherein R 3' is H, F, OH, or N 3 ; wherein G 1 is OH or NH 2 ; wherein G is H or Me; and wherein X 9 and X 10 are selected independently from the group consisting of OH, alkoxy, and aryloxy.
• R 2' is H, F, OH, or OMe
• R 3' is H, F, OH, or N 3
• R 4 is methyl, ethyl, vinyl, ethynyl, or hydroxymetliyl
• G 1 is OH or NH 2
• G is H or Me
• X 9 and X 10 are selected independently from the group consisting of OH, alkoxy and aryloxy.
• R 2 is methyl, ethyl, vinyl, ethynyl, hydroxymethyl, or haloalkyl; wherein R 2 is H, F, OH, OCH 3 , or methoxyethoxy; wherein R 3' is H, F, OH, N 3 , NH 2 , or CH 2 OH; wherein G 1 is OH, NH 2 , Cl, OMe, NH-cyclopropyl, SH, or S-alkyl; wherein G 2 is H, NH 2 , NHR, F, Cl, Br, or I; wherein Z 1 is N or CH; and wherein X 9 and X 10 are selected independently from the group consisting of OH, alkoxy and aryloxy.
• R 2 is H, F, OH or OCH 3 ; wherein R 3' is H, F, OH, or N 3 ; wherein R 4 is methyl, ethyl, vinyl, ethynyl, or hydroxymethyl; wherein G 1 is OH or NH 2 ; wherein G 2 is H or NH 2 ; wherein Z 1 is N or CH; and wherein X 9 and X 10 are selected independently from the group consistir f OH, alkoxy and aryloxy.
• preferred nucleotide mimics of the compound of Formula (XVI) include:
• XVII wherein X° and X 6 are selected independently from the group consisting of O, S, NH, CY 2 ; wherein X is O or S; wherein X 7 is selected from the group consisting of OH, F, SH, NH 2 , NHOH “ B ⁇ M " , R, SR, and NHR; and wherein X 9 and X 10 are selected independently from the group consisting of OH, SH,
• XIX wherein X 9 and X 10 are selected independently from the group consisting of OH, SH, alkyl, alkoxy, aryl and aryloxy.
• the invention also includes a pharmaceutical composition comprising a therapeutically effective amount of any of the above nucleotide mimic compounds, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable prodrug thereof, optionally in combination with one or more other active ingredients and/or with a pharmaceutically acceptable carrier.
• a pharmaceutical composition comprising a therapeutically effective amount of any of the above nucleotide mimic compounds, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable prodrug thereof, optionally in combination with one or more other active ingredients and/or with a pharmaceutically acceptable carrier.
• any of the above mimics may be used in a method for the treatment of microbial infects or a proliferative disorder comprising administering a therapeutically effective amount of any of the above nucleotide mimic compounds to a subject in need thereof.
• moiety refers to a portion of a molecule. Moiety may be, but is not limited to, a functional group, an acyclic chain, a phosphate mimic, an aromatic ring, a carbohydrate, a carbocyclic ring, a heterocycle or a prodrug
• a "natural" nucleoside is one that occurs in nature.
• the following nucleosides are defined as the natural nucleosides: adenosine, cytidine, guanosine, uridine, 2'-deoxyadenosine, 2 '-deoxy cytidine, 2 '-deoxy guanosine, thymidine, and inosine.
• base refers to the base moiety of a nucleoside or nucleotide (a nucleobases).
• the base moiety is the heterocycle portion of a nucleoside or nucleotide.
• the base moiety of a nucleotide mimic of Formula (I) or (XVI) may be a pyrimidine derivative or analog, a purine derivative or analog, or other heterocycle.
• the nucleoside base may contain two or more nitrogen atoms and may contain one or more peripheral substituents.
• the nucleoside base is attached to the sugar moiety of the nucleotide mimic in such ways that both ⁇ -D- and ⁇ -L-nucleoside and nucleotide can be produced.
• sugar refers to the ribofuranose of deoxyribofuranose portion of a nucleoside or nucleotide.
• the sugar moiety of nucleotide mimics of Formula (I) may contain one or more substituents at the C1-, C2-, C3-, C4, and C-5-position of the ribofuranose. Substituents may direct to either the ⁇ - or ⁇ -face of the ribofuranose.
• the nucleoside base that can be considered as a substituent at the C-1 position of the ribofuranose directs to the ⁇ -face of the sugar.
• the ⁇ -face is the side of a ribofuranose on which a purine or pyrimidine base of natural ⁇ -D-nucleosides is present.
• the -face is the side of the sugar opposite to the ⁇ -face.
• the sugar moiety of nucleotide mimics (XVI) of the present invention is not limited to a ribofuranose and its derivatives, instead, it may be a carbohydrate, a carbohydrate analog, a carbocyclic ring, or other ribofuranose analog.
• sugar-modified nucleoside refers to a nucleoside containing a modified sugar moiety.
• base-modified nucleoside refers to a nucleoside containing a modified base moiety, relative to a base moiety found in a natural nucleoside.
• phosphate mimic refers to a phosphate analog, which may be a phosphonate, phosphothioate, phosphoselenate, selenophosphate, thiophosphate, P-boranophosphate, or phosphoramidate, or a combination thereof.
• diphosphate mimic and triphosphate mimic specifically refer to a diphosphate analog and a triphosphate analog, respectively, which comprises at least one of the phosphate mimics, one of the modifications at the bridging site of diphosphate and triphosphate, or replacements of non- bridging phosphate oxygens.
• Non-bridging phosphate oxygens e.g., in X 7 -X 10 positions of Formula (II) can be replaced by a variety of substituents including H, F, OH, SH, NH 2 , NHOH, NHOR, NHNH 2 , NHNHR, CN, N 3 , " BH 3 M + , R, OR, SR, SeH, SeR, NHR, NR 2 , and R* where R is as defined above, and wherein R* is a prodrug substituent.
• substituents including H, F, OH, SH, NH 2 , NHOH, NHOR, NHNH 2 , NHNHR, CN, N 3 , " BH 3 M + , R, OR, SR, SeH, SeR, NHR, NR 2 , and R* where R is as defined above, and wherein R* is a prodrug substituent.
• M “1" is a cation preferably a pharmaceutically acceptable cation such as Ca 2+ , ammonium, trialkylammonium or tertaalkylammonium, e.g., NH 4 + , Et 3 NH + , Bu 3 NH + , and BI N".
• the ⁇ -P, ⁇ -P, and ⁇ -P in the diphosphate mimics and triphosphate mimics may independently adopt either R or S configurations when they become a chiral phosphorus.
• nucleotide mimic refers to a nucleoside di-phosphate mimic and/or a nucleoside tri-phosphate mimic. They may be termed as NDP mimic and NTP mimic, respectively.
• alkyl refers to a saturated straight, branched, or cyclic hydrocarbon of Ci to C 18 .
• Alkyls may include, but are not limited to, methyl, ethyl, n- propyl, isopropyl, cyclopropyl, n-butyl, isobutyl, t-butyl, cyclobutyl, n-pentyl, isopentyl, neopentyl, cyclopentyl, n-hexyl, cyclohexyl, dodecyl, tetradecyl, hexadecyl, and octadecyl.
• alkenyl refers to an unsaturated hydrocarbon of C2 to C 18 that contains at least one carbon-carbon double bond and may be straight, branched or cyclic.
• Alkenyls may include, but are not limited to, olefinic, propenyl, allyl, 1-butenyl, 3- butenyl, 1-pentenyl, 4-pentenyl, 1-hexenyl, and cyclohexenyl.
• alkynyl refers to an unsaturated hydrocarbon of C 2 to C 18 that contains at least one carbon-carbon triple bond and may be straight, branched or cyclic.
• Alkynyls may include, but are not limited to, ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, and 3-butynyl.
• aryl refers to an aromatic or heteroaromatic hydrocarbon moiety.
• Aryls may include, but are not limited to, phenyl, biphenyl, naphthyl, pyridinyl, pyrrolyl, and imidazolyl, optionally containing one or more substituents.
• the substituents may include, but are not limited to, hydroxy, amino, thio, halogen, cyano, nitro, alkoxy, alkylamino, alkylthio, hydroxycarbonyl, alkoxycarbonyl, and carbamoyl.
• aralkyl refers to a moiety that contains both an aryl and an alkyl, an alkenyl, or an alkynyl.
• Aralkyls can be attached through either the aromatic portion or the non-aromatic position.
• Aralkyls may include, but are not limited to, benzyl, phenethyl, phenylpropyl, methylphenyl, ethylphenyl, propylphenyl, butylphenyl, phenylethenyl, phenylpropenyl, phenylethynyl, and phenylpropynyl.
• acyl refers to alkylcarbonyl.
• Acyls may include, but are not limited to, formyl, acetyl, fluoroacetyl, difluoroacetyl, trifluoroacetyl, chloroacetyl, dichloroacetyl, trichloroacetyl, propionyl, benzoyl, toluoyl, butyryl, isobutyryl, pivaloyl, acylthioethoxy, acyloxymethoxy, 1,2-O-diacylglyceryloxy, 1,2-O-dialkylglyceryloxy, and l-O-alkyl-2-O-acylglyceryloxy.
• heteroatom refers to oxygen, sulfur, nitrogen, selenium, borane, or halogen.
• heteroatoms When one or more heteroatoms are attached to alkyl, alkeneyl, alkynyl, acyl, aryl, or arakyl, a new functional group may be produced.
• substituted alkyls may be produced, including, but not limited to, fluoroalkyl, chloroalkyl, bromoalkyl, iodoalkyl, alkoxy, hydroxyalkyl, alkylamino, aminoalkyl, alkylthio, thioalkyl, azidoalkyl, cyanoalkyl, nitroalkyl, carbamoylalkyl, carboxylalkyl, acylalkyl, acylthioethoxy, acyloxymethoxy, 1,2-O-diacylglyceryloxy, 1 ,2-O-dialkylglyceryloxy, and l-O- alkyl-2- -acylglyceryloxy.
• halogen or halo refers to fluorine, chlorine, bromine, or iodine.
• function refers to a substituent. Functions may include, but are not limited to, hydroxy, amino, sulfhydryl, azido, cyano, halo, nitro, hydroxycarbonyl, alkoxycarbonyl, or carboxyl, either protected or unprotected.
• R 4 of Formula (I) represents a combination (-L-R 5 ) of a linker moiety (L) and a phosphate moiety (R 5 ).
• L is usually either a one-atom or a two-atom linker, which may, through either side, attach to the sugar and phosphate mimic moiety.
• R 5 represents a diphosphate mimic or a triphosphate mimic.
• R of Formula (I) and (XVI) is a univalent substituent and present on the base, sugar and phosphate mimic moieties.
• R is selected from the group consisting of alkyl, alkenyl, alkynyl, aryl, acyl, and aralkyl, each optionally containing one or more heteroatoms, which are as defined above.
• R* is a prodrug substituent.
• prodrug refers to a masked (protected) form of a nucleotide mimic of Formula (I) or (XVI) when one or more of X 7 -X 10 is R*.
• the prodrug of the nucleotide mimic can mask the negative charges of the diphosphate (X 7 , X 8 , X 10 ) mimic or tri-phosphate (X 7 -X ) mimic moiety or phosphate moiety, entirely or partially, or mask a heteroatom substituted alkyl, aryl or aryalkyl (W, see below) attached to a phosphate or phosphate mimic moiety in order to enhance drug absorption and/or drug delivery into cells.
• the prodrug can be liberated either by cellular enzymes such as Upases, esterases, reductases, oxidases, nucleases or by chemical cleavage such as hydrolysis to release (liberate) the nucleotide mimic, preferably intracellularly.
• Prodrugs are often referred to as cleavable prodrugs.
• Prodrug substituents include, but are not limited to: proteins, antibiotics (and antibiotic fragments), D- and L-amino acids attached to a phosphate moiety or a phosphate mimic moiety via a carbon atom (phosphonates), a nitrogen atom (phosphoamidates), or an oxygen atom (phosphoesters), peptides (up to 10 amino acids ) attached to a phosphate moiety or a phosphate mimic moiety via a carbon atom (phosphonates), a nitrogen atom (phosphoamidates), or an oxygen atom (phosphoesters), drug moieties attached to a phosphate moiety or a phosphate mimic moiety via a carbon atom (phosphonates), a nitrogen atom (phosphoamidates), or an oxygen atom (phosphoesters), cholesterols, folic acids, vitamins, polyamines, carbohydrates, polyethylene glycols (PEGs), cyclosaligenyls
• a combination of prodrug moieties may be attached (conjugated) to one or more X 7 -X 10 positions on a nucleoside di- or tri-phosphate mimic.
• W is alkyl, aryl, aralkyl as described above or a heterocycle.
• microbial infections refer to the infections caused by bacteria, parasite, virus or fungus.
• microbes that cause such infections include: Acanthamoeba, African Sleeping Sickness (Trypanosomiasis), amebiasis, American Trypanosomiasis (Chagas Disease), Bilharzia (Schistosomiasis), cryptosporidiosis (diarrheal disease, Cryptosporidium Parvum), Giardiasis (diarrheal disease, Giardia lamblia), hepatitis A, B, C, D, E, leishmaniasis (skin sores and visceral), malaria (Plasmodium falciparum), Salmonella enteritides infection (stomach cramps, diarrhea and fever), tuberculosis (mycobacterium tuberculosis), varicella (chicken pox), yellow fever, pneumonias, urinary tract infections (Chlamydia and Mycoplasma), meningitis
• pharmaceutically acceptable carrier refers to a pharmaceutical formulation which serves as a carrier to deliver negatively-charged nucleotide mimics of the present invention into cells.
• Liposomes, polyethylenimine, and cationic lipids are examples of those carriers.
• nucleotide mimics of the present invention can be prepared using known methodologies in the practice of nucleoside and nucleotide chemistry by those who are ordinarily skilled in the art. The following descriptions are served as representative demonstrations of the preparation of the nucleotide mimics of the present invention.
• novel nucleosides that are used to prepare the nucleotide mimics of the present invention can be synthesized either according to published, known procedures or can be prepared using well-established synthetic methodologies (Chemistry of Nucleosides and Nucleotides Vol. 1, 2, 3, edited by Townsend, Plenum Press, 1988, 1991, 1994); Handbook of Nucleoside Synthesis by Vorbrtiggen Ruh-Pohlenz, John Wiley & Sons, Inc., 2001; The Organic Chemistry of Nucleic Acids by Yoshihisa Mizuno, Elsevier, 1986).
• the nucleosides can be converted to their corresponding diphosphate mimics, and triphosphate mimics by established phosphorylation methodologies.
• nucleosides One of the general approaches for the preparation of novel nucleosides is as follow: 1. properly protected, modified sugars including 1-, 2-, 3-, 4-, 5 -substituted furanose derivatives and analogs which are not commercially available need to be synthesized; 2. the modified sugars are condensed with properly substituted purine or pyrimidine derivatives and analogs to yield modified nucleosides; and 3. the resulting nucleosides can be further derivatized at nucleoside level through reactions on the base and/or sugar moieties. Also quite often, natural nucleosides are modified directly to obtain base-modified and sugar-modified nucleosides. There are nucleoside analogs which may have to be synthesized using alternative approaches.
• the nucleosides may be prepared through solution or solid-phase parallel synthesis.
• a representative example below (Scheme 1) can demonstrate the process.
• the 1-O- acetylribofuranose i is condensed with 6-chloro-2-iodopurine to yield the nucleoside ii, which is, at first, subject to a nucleophilic displacement of 6-chloro under a mild condition, then a nucleophilic substitution under a vigorous condition to replace iodo.
• the resulting compound iii is deprotected to yield nucleoside iv.
• a partial deprotection at the 5'-position may be more favorable.
• R', R", and R"' represent a substituent that may be halogen, R, NHR, NR 2 , OR, or SR where R is alkyl, alkenyl, alkynyl optionally with one or more heteroatoms.
• the sugar moieties of the nucleotide mimics of the present invention may be selected from the group consisting of l,2-(dihydroxymethyl)-l- ethylcycloprop-1-yl (a, b), 3,4-(dihydroxymethyl)-2-oxacyclobut-l-yl (c), 2,3- (dihydroxymethyl)cyclobut-l-yl (d), 3-hydroxymethyl-2,4-dioxacyclopent-l-yl (e), 3- hydroxymethyl-2-oxa-4-thiacyclopent-l-yl (f), 3-hydroxymethyl-2-thia-4-oxacyclopent-l-yl (g), 3-hydroxymethyl-2-methylenecyclopent-l-yl (h),
• nucleosides A large number of known nucleosides are prepared from the modifications of purine and pyrimidine nucleosides. The modifications can be done on the sugars and/or nucleoside bases.
• a simple, widely-used reaction is the nucleophilic substitution of halopurine or halopyrimidine base by a variety of nucleophiles such as hydroxide, ammonia, hydrogen sulfide, alkoxides, amines, alkylthiol, hydrazine, hydroxyamines, azide, cyanide, and hydride.
• This type of reactions can be very useful for preparation of 2-substituted purine nucleoside, 6-substituted purine nucleosides, 8-susbstituted purine nucleosides, 2,6-disubstituted purine nucleosides, 2,8- disubstituted purine nucleosides, 6,8-disubstituted purine nucleosides, 2,6,8-trisubstituted purine nucleosides (Halbfmger et al, J. Med. Chem. 1999, 42, 5323-5337, Lin et al, J. Med. Chem. 1985, 28, 1481-1485; Bressi et al, J. Med. Chem.
• the sugar moieties of synthesized nucleosides can be further modified.
• the reactions frequently used include deoxygenation, oxidation/addition, substitution, and halogenation.
• the deoxygenations are useful for the preparation of 2'-deoxy-, 3'-deoxy-, and 2',3'-dideoxy- nucleosides.
• a widely-used reagent is phenyl chlorothionoformate, which reacts with the hydroxy of nucleosides to yield a thionocarbonate.
• the treatment of the thionocarbonate with tributyltin hydride and AIBN yields deoxygenated nucleosides.
• the oxidation addition includes the conversion of a hydroxy group to a carbonyl group, followed by a nucleophilic addition, resulting in C-alkylated nucleosides and C-substituted nucleosides.
• the substitution may be just a simple displacement of a hydroxyl proton by alkyl, or may be a conversion of a hydroxyl to a leaving group, followed by a nucleophilic substitution.
• the leaving group is usually a halogen, mesylate, tosylate, nisylate, or a triflate.
• nucleophiles can be used, resulting in 2-, or 3-substituted nucleosides.
• Halogenation can be used to prepare 1 '-halo-, 2'-halo-, 3'-halo-, or 4'-halonucleosides. Chlorination and fluorination are commonly used and result in important fluoro-sugar and chloro-sugar nucleosides.
• Clinically-used nucleoside drugs can be phosphorylated by cellular enzymes and maintain Watson-Crick hydrogen bonding between base pairs.
• a large number of novel nucleosides synthesized may not be phosphorylated by a cellular enzyme and may not show in vitro or in vivo biological activities. It would be of a great value to convert some inactive nucleosides into active nucleotide mimics and to convert some moderately-active ones into potent nucleotide mimics.
• One approach provided in the present invention is to convert the nucleosides to the nucleotide diphosphate mimics or nucleoside triphosphate mimics, which themselves can be active chemical entities since cellular phsosphorylation is no longer necessary.
• nucleosides including, but not limited to, the following: adenosine, cytidine, guanosine, uridine,
• nucleosides Conversion of nucleosides to nucleoside diphosphates or nucleoside triphosphates can be achieved according to published procedures. Certain nucleoside diphosphate mimics and nucleoside triphosphate mimics also have been reported. Many nucleotide mimics of the present invention can be prepared by similar approaches as published or by using well-known knowledge of organic phosphorus chemistry. Scheme 3 and 4 show the general methodologies for the preparation of nucleoside diphosphate mimics and nucleoside triphosphate mimics, respectively.
• nucleotide diphosphate mimics and nucleoside triphosphate mimics of the present invention can be prepared using the methodologies shown in Scheme 2 and 3 where a protected ribonucleoside v is used as an example.
• the ribonucleoside v is converted to the nucleoside derivative vi having a leaving group such as an iodo, tosylate or triflate at the 5'- position.
• the reaction of vi with a tri- or tetraalkylammonium salt of pyrophosphate or a diphosphate mimic yields the nucleoside diphosphate mimic vii, which is subject to a deprotection to yield the nucleoside diphosphate mimic viii.
• vii can be prepared from the reaction of the nucleoside v with diphosphate mimic chloridate such as methylenediphosphonate tetrachloridate and a subsequent hydrolysis.
• compound v is converted to an activated form of nucleoside phosphate or phosphite ix, which is treated with a tri- or tetraalkylammonium salt of pyrophosphate or a diphosphate mimic to yield nucleoside triphosphate mimic x.
• x can be prepared from the reaction of vii with a tri- or teraalkylammonium salt of monophosphate or a monophosphate mimic.
• R' represents a protecting group such as acetyl, benzoyl, benzyl, isopropylidene, a silyl group, trityl, or dimethoxytrityl
• X' is a leaving group including halogen, and sulfonates
• AP represent an activated phosphate such as monophosphate imidazolate, an active phosphine such as bis(diisopropylamino)phosphine or an activated phosphite
• Y' is O, S, Mi, substituted amino, methylene, ethylene, and halomethylene
• Y" can be a variety of univalent substituents including H, OH, SH, M ⁇ 2 , F, N , CN, and " BH 3 M + , or selected from alkyl, alkoxy, alkenyl, and alkynyl optionally
• the compounds were purified by anion exchange (AX) chromatography using a 10 x 160 mm Mono Q column (Pharmacia). Initial conditions were typically 0 to 35 mM NaCI. Elution gradients were typically a linear gradient from 0 to 35 mM to 350 mM NaCI to 1 M NaCI in two to three column volumes at 6.5 ml/min. A constant concentration of 50 mM Tris, pH 8 was maintained throughout the purification. Fractions from anion exchange chromatography containing the target compound were collected and desalted by reversed phase (RP) chromatography using a Luna C18 250 x 21 mm column (Phenomenex) with a flow rate of 10 ml/min.
• RP reversed phase
• Elution gradients were generally from 0-20% to 95% methanol in 20-60 min at a constant concentration of triethylammonium acetate (50 mM).
• Compounds that did not require AX-HPLC purification were purified by Reverse-phase HPLC only. However, most nucleotide mimics in the example section, unless specified, were purified using both ion-exchange HPLC and reverse-phase HPLC as indicated simply by HPLC.
• Mass Spectra and purity of the nucleotide mimics and their prodrugs were determined using on-line HPLC mass spectrometry on a ThermoFinnigan (San Jose, CA) Deca XP plus.
• a Phenomenex Luna (Cl 8(2) or c5), 75 x 2 mm, 3-um particle size was used for RP HPLC.
• a 0 to 50% linear gradient (15 min) of AcCN in 10 mM NNDMHA, pH 7 was performed in series with mass spectra detection in the negative ionization mode. Nitrogen gas and a pneumatic nebulizer were used to generate the electrospray. The mass range of 150 -1500 was typically sampled.
• 3 '-azido-3 '-deoxythymidine serves as an example to illustrate various approaches and reactions.
• the approaches and methodologies shown below are general and can be applied to the preparation of other nucleotide mimics of the present invention although in some cases nucleosides may need appropriate protection.
• the detailed procedures are given in the section of Examples.
• nucleoside 5 '-diphosphate mimics were prepared through similar reactions, including 2'-deoxy-2',2'-difluorocytidine 5 '-diphosphate mimics (5-7, 81) and 3 '-azido-3 '-deoxythymidine 5'-imidodiphosphate (8).
• An alternative approach is to convert the 5 '-hydroxy of a nucleoside to a leaving group, which is reacted with the tetrabutylammonium salt of diphosphate mimics to give nucleoside diphosphate mimics.
• 3 '-azido-3 '-deoxythymidine was converted to the 5'-tosylate 9, which was treated with the tetrabutylammonium salt of difluoromethylenediphosphonate to give 3 '-azido-3' -deoxythymidine 5'- (difluoromethylene)diphosphonate (l ⁇ ).
• nucleoside 5'- ⁇ -P-substituted diphosphates can be prepared.
• nucleoside triphosphate mimics are more diverse than diphosphate mimics
• One approach includes the preparation of an activated phosphate or phosphite linked to the 5 '-position of a nucleoside and a subsequent condensation with diphosphate mimics.
• Many nucleoside triphosphate mimics of the present invention were prepared through this approach. For example, the reaction of 3 '-azido-3 '-deoxythymidine with 2-chloro-4H-l,3,2-benzodioxaphosphorin-4- one gives the intermediate 14, which is condensed with diphosphate mimics to form a cyclic triphosphate intermediate 15.
• X is O, S, NH, methylene, or halomethylene
• Y is O, S, N 3 , alkoxy, aryloxy, alkylamino, arylamino, or " B ⁇ M "1"
• P N is an ammonium, trialkylammonium, or tetraalkylammonium cation such as NH 4 + , Et 3 NH + , Bu M ⁇ + , and B ⁇ N " .
• nucleoside 5'- ⁇ -F- boranotriphosphate mimics were prepared.
• This methodology is a general method and can be applied to a variety of nucleosides, however, an appropriate protection is necessary for nucleosides bearing reactive groups.
• a representative example is shown below. 7-Deaza-2'-C- methyladenosine (18) was converted to 7-deaza-2'-C-methyl-2',3'-O-6-triacetyladenosine (20), which was subject to the reactions shown above to give the triacetyl nucleoside triphosphate mimic 20.
• a deprotection with aqueous ammonia or methanolic ammonia yielded the nucleoside triphosphate mimic 22.
• nucleoside 5 '- ⁇ -P-boranotriphosphate mimics 23-28 containing different ⁇ , ⁇ -bridging modifications were synthesized through the same approach and are shown below.
• (27) (28)
• the treatment of 15 with elemental sulfur and subsequent reaction with water gave the ⁇ -P-thiotriphosphate mimics 29.
• Compound 30 was also prepared through the same method.
• Compound 31 was obtained from the treatment of a cyclic triphosphate equivalent to 15 with sulfur and subsequent reaction with lithium sulfide.
• compound 32 was prepared from the treatment of the cyclic triphosphate withborane-diisopropylethylamine complex and subsequent reaction with lithium sulfide.
• the treatment of the cyclic triphosphate intermediate 15 with iodine followed by reaction with a nucleophile can yield a variety of nucleoside 5'- ⁇ -P-substituted triphosphate mimics.
• the reaction of 15 with iodine, followed by treatment with ethylamine and aniline yielded the ⁇ -P-aminotriphosphate mimics 33 and 34, respectively.
• potassium fluoride, sodium azide, sodium phenoxide, and sodium methoxide were chosen as the nucleophile, compounds 35-38 were prepared, respectively.
• Compound 39 was also formed when the nucleophile was hydroxyamine.
• Nucleoside 5'- ⁇ -P-substituted triphosphate mimics can be prepared through a few different approaches.
• One approach is the alkylation of nucleoside triphosphate mimics with a reagent bearing a leaving group.
• the alkylating reagent may be a halide such as a bromide and iodide or a sulfonate such as a tosylate, nisylate, and triflate. This type of reaction can be used to introduce a small aliphatic hydrocarbon or a large moiety such as lipids and other phosphate- masking moieties.
• the reaction of the tetrabutylammonium salt of the nucleotide mimic 27 with methyl triflate yielded the ⁇ -P-methyltriphosphate mimic 40.
• the ⁇ -P- palmityltriphosphate mimic 41 was prepared from the reaction of 27 with palmityl p- nitrobenzenesulfonate.
• nucleoside 5 '- ⁇ -P-substituted triphosphate mimics is the reaction of nucleoside diphosphate mimics with substituted phosphonodichloridate as shown below.
• nucleoside diphosphate mimics with substituted phosphonodichloridate as shown below.
• the treatment of 3'-azido-3'- deoxythymidine 5'-(difluoromethylene)diphosphonate (10) with methylphosphonodichloridate yields, after hydrolysis, the triphosphate mimics 44.
• the compound 45 containing ⁇ -P- phenyl- ⁇ , ⁇ -(difuoromethylene)triphosphate was prepared.
• nucleoside 5 '- ⁇ -P-substituted triphosphate mimics 47 is the reaction of nucleoside 5'-phosphodichloridate 46 or 5'- phophonodichloridate with substituted pyrophosphate or substituted diphosphate mimics. Also, the reaction of the nucleoside cyclic phosphate 14 with substituted pyrophosphate or substituted diphosphate mimics, followed by treatment with oxidizing reagents such as sulfur, iodine, and borane and then with nucleophiles, can yield a variety of nucleoside 5'- ⁇ -P-(and/or ⁇ -P)- substituted triphosphate mimics 48.
• the present invention also provides approaches for the preparation of nucleoside triphosphate mimics in which the modifications are present at the 5 '-position of the nucleotide mimics.
• the 5 '-oxygen of nucleotide mimics can be replaced with other heteroatoms, methylene, halomethylene or dihalomethylene. Alternatively, the 5 '-oxygen can be eliminated.
• the first scheme shows the synthesis of 3' -azido-3' -deoxythymidine 5'-deoxy- ⁇ , ⁇ - (difluoromethylene)triphosphate.
• l-(2-Deoxyxylofuranosyl)thymine was converted to a 5'-iodo derivative, which was subjected to acetylation.
• the resulting intermediate 49 was converted to the 5'-deoxy-5'-phosphonate ester 50.
• the 3'-acetyl was removed and the resulting intermediate was converted to the mesylate 51.
• the treatment of 51 with sodium azide at elevated temperature gave the 3'-azido derivative 52.
• the triethylammonium salt of the nucleoside 5'-phosphonate 61 was treated with carbonyldiimidazole, and then condensed with the tributylammonium salts of difluoromethylenediphosphonate to give 3',5'-dideoxy-5'-difluoromethylenethymidine 5'- ⁇ , ⁇ - (difluoromethylene)triphosphate (62).
• Compounds 63 and 64 were prepared through similar procedures.
• Another general approach for the preparation of ⁇ -P-substituted nucleoside triphosphates can be used for the preparation of the nucleotide mimics of the present invention, involving the reaction of an activated nucleoside monophosphonate with pyrophosphate or diphosphate mimics.
• the activation is usually achieved by attaching an imidazole or a morpholino group to the phosphate, as shown by the example given below.
• the resulting intermediate 66 is condensed with the tetrabutylammonium salt of difluoromethylenediphosphonate to give 3'-azido-3'- deoxythymidine 5'- ⁇ -P-substituted ⁇ , ⁇ -(difluoromethylene)triphosphate (67).
• the prodrug approach is one of the efficient methods to deliver polar, negatively- charged nucleotide mimics into cells.
• a number of prodrug approaches for nucleoside 5'- monophosphates have been developed and potentially can be applied to the nucleotide mimics of the present invention.
• the nucleotide mimic prodrugs may include, but are not limited to, alkyl phosphate esters, aryl phosphate ester, acylthioethyl phosphate esters, acyloxymethyl phosphate esters, 1 ,2-O-diacylglyceryl phosphate esters, 1,2-O-dialkylglyceryl phosphate esters, and phosphoramidate esters. These masking groups were also successfully attached to the nucleoside mimics of the present invention. The resulting compounds can serve as the prodrugs of the nucleotide mimics.
• nucleotide mimic prodrugs One of the straightforward methods for the preparation of nucleotide mimic prodrugs is the reaction of the nucleotide mimics with a masking reagent bearing a leaving group.
• the nucleoside 5'-imidodiphosphate 8 is treated with tributylstannyl methoxide to convert the diphosphate 8 to a stannic salt, which was reacted with pivaloxymethyl iodide to give compound 68.
• the pivaloxymethyl group (POM) can be cleaved by a variety of cellular esterases. In cells compound 68 can be converted to compound 8.
• Compound 69 was prepared from the reaction of the corresponding triphosphate 27 with pivaloxymethyl iodide through a similar procedure.
• Compound 70 was also prepared through this type of reaction, but the reagent is S-pivaloyl-2-thioethyl (SATE) yp-nitrobenzenesulfonate.
• S-Pivaloyl-2-thioethyl and pivaloxymethyl as the making groups of phosphate have been intensively studied and usually abbreviated as SATE and POM, respectively.
• SATE can also be readily cleaved by cellular esterases.
• nucleotide mimics Another approach for the preparation of the prodrugs from nucleotide mimics is the condensation of nucleotide mimics with a masking group bearing a hydroxyl group.
• compound 8 was treated with l-(mesitylene-2-sulfonyl)-3-nitro-l,2,4-triazole and then reacted with S-pivaloxy-2-thioethanol to give compounds 71 and 72, which were separated by chromatography.
• the prodrugs of certain nucleotide mimics can be prepared from nucleosides.
• 2'-deoxy-2',2'-difluorocytidine 73 was converted to the ⁇ -O-(S- pivaloyl-2-thioethyl)-, di-O-(S-pivaloyl-2-thioethyl)- and tri-O ⁇ (S-pivaloyl-2- thioethoxy)methylenediphosphonate 75-77 in a one-pot reaction.
• the nucleoside 73 was first reacted with methylenediphosphonotetrachloridate to yield the trichloro intermediate 74, which was then treated with excess S-pivaloyl-2-thioethanol.
• the resulting three S-pivaloyl-2-thioethyl derivatives 75-77 were separated on a reverse-phase HPLC.
• lipid-masked nucleotide mimics in which a lipid is attached to the terminal phosphorus of a nucleotide mimic directly or through a biologically-cleavable linker.
• compound 79 was prepared through the condensation of the nucleoside triphosphate mimic 27 with the lipid reagent 78, which was synthesized from the 1,2-O-dipalmitoylglycerol. (79) C. Biological applications and administration
• nucleoside diphosphate mimics and nucleoside triphosphate mimics of the present mvention may be useful for the inhibition of a variety of enzymes including, but not limited to, DNA or RNA polymerases, helicases, ribonucleotide reductases, protein kinases, and telomerases and for the modulation of G-proteins, P2 purinergic receptors and the allosteric sites of a variety of enzymes.
• nucleotide mimics of the present invention are useful as human therapeutics for the treatment of infectious diseases caused by viruses including, but not limited to, HIV, HBV, HCV, HDV, HSV, HCMV, small pox, West Nile virus, influenza viruses, measles, rhinovirus, RSV. VZV, EBV, vaccinia virus, and papilloma virus.
• viruses including, but not limited to, HIV, HBV, HCV, HDV, HSV, HCMV, small pox, West Nile virus, influenza viruses, measles, rhinovirus, RSV. VZV, EBV, vaccinia virus, and papilloma virus.
• nucleoside diphosphate mimics and nucleoside triphosphate mimics of the present invention are useful for the treatment of infectious diseases caused by bacteria and fungi.
• nucleotide mimics that have potent cytotoxicities to fast-dividing cancerous cells are useful for the treatment of proliferative disorders, including, but not limited to, lung cancer, liver cancer, prostate cancer, colon cancer, breast cancer, ovarian cancer, melanoma, and leukemia.
• the nucleostide mimics of the present invention are useful for the treatment of a wide range of other diseases and disorders such as inflammatory diseases, autoimmune diseases, Type 2 diabetes, and cardiovascular diseases.
• nucleotide mimics or their prodrugs of the present invention may be therapeutically administered as a single drug, or alternatively may be administered in combination with one or more other active chemical entities to form a combination therapy.
• the other active chemical entities may be a small molecule, a polypeptide, or a polynucleotide.
• the pharmaceutical composition of the present invention comprises at least one of the compounds represented by Formula (I) and (XVI) or pharmaceutically acceptable salts, esters or prodrugs thereof as active ingredients.
• the compositions include those suitable for oral, topical, intravenous, subcutaneous, nasal, ocular, pulmonary, and rectal administration.
• the compounds of the invention can be administered to mammalian individuals, including humans, as therapeutic agents.
• the compounds of the invention are useful as antiviral agents.
• the present invention provides a method for the treatment of a patient afflicted with a viral infection comprising administering to the patient a therapeutically effective antiviral amount of a compound of the invention.
• viral infection refers to an abnormal state or condition characterized by viral transformation of cells, viral replication and proliferation.
• Viral infections for which treatment with a compound of the invention will be particularly useful include the virues mentioned above.
• a “therapeutically effective amount” of a compound of the invention refers to an amount which is effective, upon single or multiple dose administration to the patient, in controlling the growth of e.g., the microbe or tumor or in prolonging the survivability of the patient beyond that expected in the absence of such treatment.
• controlling the growth refers to slowing, interrupting, arresting or stopping the microbial or proliferative transformation of cells or the replication and proliferation of the microbe and does not necessarily indicate a total elimination of e.g., the microbe or tumor.
• the present invention includes pharmaceutical compositions comprising, as an active ingredient, at least one of the compounds of the invention in association with a pharmaceutical carrier.
• the compounds of this invention can be administered by oral, parenteral (intramuscular, intraperitoneal, intravenous (IV) or subcutaneous injection), topical, transdermal (either passively or using iontophoresis or electroporation), transmucosal (e.g., nasal, vaginal, rectal, or sublingual) or pulmonary (e.g., via dry powder inhalation) routes of administration or using bioerodible inserts and can be formulated in dosage forms appropriate for each route of administration.
• parenteral intramuscular, intraperitoneal, intravenous (IV) or subcutaneous injection
• transdermal either passively or using iontophoresis or electroporation
• transmucosal e.g., nasal, vaginal, rectal, or sublingual
• pulmonary e.g., via dry powder inhalation
• Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules.
• the active compound is admixed with at least one inert pharmaceutically acceptable carrier such as sucrose, lactose, or starch.
• Such dosage forms can also comprise, as is normal practice, additional substances other than inert diluents, e.g., lubricating, agents such as magnesium stearate.
• the dosage forms may also comprise buffering agents. Tablets and pills can additionally be prepared with enteric coatings.
• Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, with the elixirs containing inert diluents commonly used in the art, such as water. Besides such inert diluents, compositions can also include adjuvants, such as wetting agents, emulsifying and suspending agents, and sweetening, flavoring, and perfuming agents.
• Preparations according to this invention for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, or emulsions.
• non-aqueous solvents or vehicles are propylene glycol polyethylene glycol, vegetable oils, such as olive oil and corn oil, gelatin, and injectable organic esters such as ethyl oleate.
• Such dosage forms may also contain adjuvants such as preserving, wetting, emulsifying, and dispersing agents. They may be sterilized by, for example, filtration through a bacteria retaining filter, by incorporating sterilizing agents into the compositions, by irradiating the compositions, or by heating the compositions. They can also be manufactured using sterile water, or some other sterile injectable medium, immediately before use.
• compositions for rectal or vaginal administration are preferably suppositories which may contain, in addition to the active substance, excipients such as cocoa butter or a suppository wax.
• Compositions for nasal or sublingual administration are also prepared with standard excipients well known in the art.
• Topical formulations will generally comprise ointments, creams, lotions, gels or solutions.
• Ointments will contain a conventional ointment base selected from the four recognized classes: oleaginous bases; emulsif ⁇ able bases; emulsion bases; and water-soluble bases.
• Lotions are preparations to be applied to the skin or mucosal surface without friction, and are typically liquid or semiliquid preparations in which solid particles, including the active agent, are present in a water or alcohol base. Lotions are usually suspensions of solids, and preferably, for the present purpose, comprise a liquid oily emulsion of the oil-in- water type.
• Creams are viscous liquid or semisolid emulsions, either oil-in-water or water-in-oil. Topical formulations may also be in the form of a gel, i.e., a semisolid, suspension- type system, or in the form of a solution.
• formulations of these drugs in dry powder form for delivery by a dry powder inhaler offer yet another means of administration. This overcomes many of the disadvantages of the oral and intravenous routes.
• the dosage of active ingredient in the compositions of this invention maybe varied; however, it is necessary that the amount of the active ingredient shall be such that a suitable dosage form is obtained.
• the selected dosage depends upon the desired therapeutic effect, on the route of administration, and on the duration of the treatment desired. Generally, dosage levels of between 0.001 to 10 mg/kg of body weight daily are administered to mammals.
• nucleotide mimics of the present invention are given in this section.
• the examples herein are not intended to limit the scope of the present invention in any way.
• the nucleotide mimics of the present invention can be prepared by those skilled in the art of nucleoside and nucleotide chemistry.
• the nucleotide mimics of the present invention exist as the salts of nucleotide mimics.
• the amounts of the products given below in the examples are based on UV absorptions and calculated from the molecular weights of H-forms of nucleotide mimics.
• Step 1 Tetraisopropyl difluoromethylenediphosphonate (2.1 g, 5.51 mmol) was dissolved in methylene dichloride (20 mL) and treated with trimethylsilyl bromide (3.6 mL, 27.6 mmol). The mixture was heated at reflux for 16 h under an argon atmosphere, cooled and then evaporated in vacuo. The residue was coevaporated with carbon tetrachloride (20 mL), resuspended in carbon tetrachloride (20 mL) and added to phosphorus pentachloride (2.,9 g, 13.2 mmol). After stirring for 16 h at room temperature under an argon atmosphere difluoromethylenebis(diphosphonic dichloride) was isolated by vacuum distillation (65°C, 0.5 mmHg) and used directly in the next step.
• Step 2 2'-Deoxy-2',2'-difluorocytidine (276 mg, 1.05 mmol) was dissolved in anhydrous trimethyl phosphate (5 mL) and stirred with molecular sieves under an argon atmosphere for 16 h. The mixture was added to difluoromethylenebis(phosphonic dichloride) (300 mg, 1 mmol) from Step 1 at -40°C under an argon atmosphere. Stirring continued for 2 h, then the reaction mixture was poured into ice-cold TEAB solution (1.0 M, 5 mL) and stirred for 30 minutes. The aqueous portion was purified by HPLC to give 41.8 mg of the titled compound.
• Step A The preparation of tetraisopropyl difluoromethylenediphosphonate
• reaction mixture was stirred at -78°C for 1 h, quenched with saturated aqueous ammonium chloride (20 mL), warmed to room temperature, diluted with ether, washed with 10% aqueous sodium bicarbonate and then with brine, dried over sodium sulfate, and concentrated.
• Cliromatography on silica gel with 5-30% ethyl acetate in hexanes gave 2.18 g of tetraisopropyl difluoromethylenediphosphonate and 0.32 g of tetraisopropyl monofluoromethylenediphosphonate.
• Step B The preparation of difluoromethylenediphosphonic acid bis(tributylammuniun ⁇ ) salts
• Tributylamine (1.8 mL, 16.5 mmol) was added, followed by addition of difluoromethylenediphosphonic acid bis(tributylammunium) salt (2.28 g, 3.9 mmol) in DMF (6 mL). The reaction mixture was stirred at room temperature for 1 h and cooled with ice. Borane-diisopropylethylamine complex (12 mL) was added and the resulting mixture was stirred at room temperature overnight. The reaction mixture was cooled with ice and quenched by slow addition of water (30 mL). The mixture was stirred at room temperature for 2 h, diluted with water (30 mL), and extracted with chloroform three times.
• aqueous solution was loaded on a reverse-phase (C18) HPLC and eluted with TEAA buffer (pH 7.0), and the purified product was desalted by repeated lyophilization.
• 3 '-azido-3 '-deoxythymidine -P-borano- ⁇ , ⁇ -(difluoromethylene)triphosphate (501 mg, triethylammonium salts, a mixture of two diastereomers) was obtained as a white foam.
• Step A The preparation of 7-deaza-2'-C-methyl-2',3'-O-N 6 -triacetyladenosine (18)
• Step B The preparation of 7-deaza-2' -C-methyladenosine 5'- ⁇ -P-borano- ⁇ , ⁇ - (difluoromethylene)triphosphate (22)
• Tributylamine (0.1 mL) was added, followed by addition of difluoromethylenediphosphonic acid bis(tributylammunium) salt (140 mg, 0.24 mmol) in DMF (0.4 mL). The reaction mixture was stirred at room temperature for 1 h and cooled with ice. Borane diisopropylethylamine complex (0.6 mL) was added and the resulting mixture was stirred at room temperature for 6 h, cooled with ice and quenched by slow addition of water (1.5 mL). The mixture was stirred at room temperature for
• 3 '-Azido-3' -deoxythymidine 134 mg, 0.5 mmol was dissolved in 1 mL of anhydrous DMF in a 10 mL flask kept under argon. To this solution was added 0.25 mL of anhydrous pyridine. A freshly prepared solution of 2-chloro-4 ⁇ l,3,2-benzodioxaphosphorin-4- one (101 mg, 0.495 mmol) in anhydrous DMF (0.5 mL) was added via syringe.
• tributylamine 0.3 mL was added followed by a mixture of methylenediphosphonic acid (88 mg, 0.495 mmol) and 3 eq. tributylamine in 0.5 mL of anhydrous DMF. The mixture was stirred for 1 hour and 2 mL of borane-diisopropylethylamine complex was added. After 6 hours stirring at room temperature 5 mL of deionized water was added and stirred at room temperature for 3 hour. Purification by HPLC yielded 46 mg of the titled compound 24.
• Step A The preparation of fluoromethylenediphosphonic acid bis(tributylammunium) salts
• Step B The preparation of 3 '-azido-3 '-deoxythymidine ⁇ -P-borano- ⁇ , ⁇ - (fluoromethylene)triphosphate (25)
• reaction mixture was stirred at room temperature for 1 h and cooled with ice.
• Borane diisopropylethylamine complex (1.20 mL) was added and the resulting mixture was stirred at room temperature for 6 h, cooled with ice and quenched by slow addition of water (3 mL). The mixture was stirred at room temperature overnight, diluted with water (10 mL), and extracted with chloroform three times.
• Step B The preparation of 3' -azido-3' -deoxythymidine ⁇ -P-borano- ⁇ , ⁇ - (dichloromethylene triphosphate (26)
• 3 '-Deoxythymidine (66.3 mg, 0.150 mmol) was dissolved in 0.5 mL of anhydrous DMF in a 10 mL round-bottom flask kept under argon. To this was added 0.1 mL of anhydrous pyridine. A freshly prepared solution of 2-chloro-4H-l,3,2-benzodioxaphosphorin-4-one (36.4 mg, 0.180 mmol) in anhydrous DMF (0.5 mL) was added via syringe at 0°C.
• 3 '-Azido-3 '-deoxythymidine (103.8 mg, 0.388 mmol) was dissolved in 1 mL of anhydrous DMF in a 10 mL round-bottom flask kept under argon. To this was added 0.20 mL of anhydrous pyridine. A freshly prepared solution of 2-chloro-4H-l,3,2-benzodioxaphosphorin-4- one (101.2 mg, 0.495 mmol) in anhydrous DMF (1 mL) was added via syringe at 0°C.
• 3 '-Deoxythymidine (66.3 mg, 0.150 mmol) was dissolved in 0.5 mL of anhydrous DMF in a 10 mL round-bottom flask kept under argon. To this was added 0.1 mL of anhydrous pyridine. A freshly prepared solution of 2-chloro-4H- 1,3,2 -benzodioxaphosphorin-4-one (36.4 mg, 0.180 mmol) in anhydrous DMF (0.5 mL) was added via syringe at 0°C.
• 3 '-Deoxythymidine (66.3 mg, 0.150 mmol) was dissolved in 0.5 mL of anhydrous DMF in a 10 mL round-bottom flask kept under argon. To this was added 0.1 mL of anhydrous pyridine. A freshly prepared solution of 2-chloro-4H-l,3,2-benzodioxaphosphorin-4-one (36.4 mg, 0.180 mmol) in anhydrous DMF (0.5 mL) was added via syringe at 0°C.
• 3 '-Deoxythymidine (2.0 mg, 0.186 mmol) was dissolved in 0.5 mL of anhydrous DMF in a 10 mL round-bottom flask kept under argon. To this was added 0.1 mL of anhydrous pyridine. A freshly prepared solution of 2-chloro-4H-l,3,2-benzodioxaphosphorin-4-one (45.1 mg, 0.223 mmol) in anhydrous DMF (0.5 mL) was added via syringe at 0°C.
• Example 19 3 ' - Azido-3 ' -deoxythymidine 5 ' - ⁇ -P-ethylamino- ⁇ , ⁇ -(difluoromethylene triphosphate (33)
• 3 '-Azido-3 '-deoxythymidine (133.6 mg, 0.495 mmol) was dissolved in 1 mL of anhydrous DMF in a 10 mL round-bottom flask kept under argon. To this was added 0.25 mL of anhydrous pyridine. A freshly prepared solution of 2-chloro-4H- 1,3,2- benzodioxaphosphorin-4-one (101.2 mg, 0.495 mmol) in anhydrous DMF (0.5 mL) was added via syringe.
• 3 '-Azido-3 '-deoxythymidine (103.8 mg, 0.388 mmol) was dissolved in 1 mL of anhydrous DMF in a 10 mL round-bottom flask kept under argon. To this was added 0.20 mL of anhydrous pyridine. A freshly prepared solution of 2-chloro-4H- 1,3,2- benzodioxaphosphorin-4-one (101.2 mg, 0.495 mmol) in anhydrous DMF (1 mL) was added via syringe at 0°C.
• Example 21 3 '-Deoxythymidine 5 '- ⁇ -P-fluoro- ⁇ , ⁇ -(difluoromethylene)triphosphate (35)
• 3 '-Deoxythymidine (47.0 mg, 0.208 mmol) was dissolved in 0.5 mL of anhydrous DMF in a 10 mL round-bottom flask kept under argon. To this was added 0.125 mL of anhydrous pyridine. A freshly prepared solution of 2-chloro-4H-l,3,2-benzodioxaphosphorin-4- one (50.6 mg, 0.250 mmol) in anhydrous DMF (0.5 mL) was added via syringe at 0°C.
• 3 '-Azido-3 '-deoxythymidine (40.09 mg, 0.150 mmol) was dissolved in 0.5 mL of anhydrous DMF in a 10 mL round-bottom flask kept under argon. To this was added 0.10 mL of anhydrous pyridine. A freshly prepared solution of 2-chloro-4H-l,3,2- benzodioxaphosphorin-4-one (33.4 mg, 0.165 mmol) in anhydrous DMF (0.5 mL) was added via syringe at 0°C.
• 3 '-Azido-3 '-deoxythymidine (40.09 mg, 0.150 mmol) was dissolved in 0.5 mL of anhydrous DMF in a 10 mL round-bottom flask kept under argon. To this was added 0.10 mL of anhydrous pyridine. A freshly prepared solution of 2-chloro-4H-l,3,2-benzodioxaphosphorin-4- one (33.4 mg, 0.165 mmol) in anhydrous DMF (0.5 mL) was added via syringe at 0°C.
• 3 '-Deoxythymidine (66.3 mg, 0.150 mmol) was dissolved in 0.5 mL of anhydrous DMF in a 10 mL round-bottom flask kept under argon. To this was added 0.1 mL of anhydrous pyridine. A freshly prepared solution of 2-chloro-4H- 1,3,2 -benzodioxaphosphorin-4-one (36.4 mg, 0.180 mmol) in anhydrous DMF (0.5 mL) was added via syringe at 0°C.
• Example 26 3 ' - Azido-3 ' -deoxythymidine 5 ' - ⁇ -P-b orano- ⁇ , ⁇ -difluoromethylene- ⁇ -O-palmityltriphosphate
• Step A Palmitol (1.0 g, 4.12 mmol) and DMAP (0.73 g, 5.9 mmol) in DCM (20 mL) was cooled to -20°C under an argon atmosphere. / r>-Nitrobenzenesulfonyl chloride (1.09 g, 4.9 mmol) was added and the mixture was stirred at 5°C under an argon atmosphere for 18 h. The reaction mixture was poured into cold sodium bicarbonate solution (saturated, 20 mL) and the organic layer was washed with citric acid (10%), brine, and dried over MgSO 4 .
• Step B The bis(tetrabutylammonium) salts of 3 '-azido-3' -deoxythymidine 5'- ⁇ -P- borano- ⁇ ,7-(difluoromethylene)triphosphate (183 mg, 0.146 mmol) was coevaporated with anhydrous DMF (3 X 5 mL). For the third coevaporation, approximately half the volume of DMF was removed and the mixture cooled to 0°C under an argon atmosphere. Palmityl 4- nitrobenzenesulfonate (123 mg, 0.29 mmol) was added.
• Step A A slurry of sodium hydride (320 mg, 8.0 mmol) in anhydrous DMF (10 mL) was treated with phenol (0.9 g, 10.0 mmol) dissolved in anhydrous DMF (2 mL) under an argon atmosphere. After stirring for 1 h at room temperature gas evolution had ceased. 15-Crown-5 (1.59 mL, 8.0 mmol) was added and stirring continued for 1 h. This mixture was used immediately in Step 2.
• Step B The triethylammonium salts of 3 '-azido-3 '-deoxythymidine 5'- ⁇ -P-borano- ⁇ , ⁇ -(difluoromethylene)triphosphate (0.21 mmol) was coevaporated with anhydrous DMF (3 X 5 mL). For the third coevaporation, approximately half the volume of DMF was removed and anhydrous methanol (0.3 mL) was added, followed by N,N'-dicyclohexylcarbodiimide (200 mg). After stirring at room temperature under an argon atmosphere for 3 h, the mixture was concentrated in vacuo and the above procedure was repeated once more.
• Example 28 3 ' - Azido-3 ' -deoxythymidine 5 ' - ⁇ -P-b orano- 3 ,Y-diluoromethylene- ⁇ - O-(4- nitrophenvDtriphosphate (43)
• Step A The preparation of l-(3-O-acetyl-2,5-dideoxy-5 ⁇ iodo- ⁇ 8-D-tAreo- pentofuranosypthymine (49)
• Step B The preparation of l-[3-O-acetyl-2,5-dideoxy-5-(di-O-ethylphosphono)-ff-D- t/zreo-pentofuranosyl]thvmine (50)
• Step C The preparation of l-[2,5-dideoxy-5-(di-O-ethylphosphono)-3-O-mesyl- 3-D- t/2reo-pentofuranosyl]thvmine (51)
• Step D The preparation of l-r3-azido-3, 5-dideoxy-5-(di-O-ethylphosphono)- ⁇ -D- ervt/zro-pentofuranosyllthymine (52)
• Step E The preparation of 3'-azido-3',5'-dideoxythymidine-5'-phosphonic acid (53)
• Step F The preparation of 3'-azido-3',5'-dideoxythymidine 5'-( 3, ⁇ -difluoromethylene)- triphosphate (55)
• reaction mixture was stirred at -15 to -5°C for 45 min, then poured into ice-cold aq. NaHC ⁇ 3 (1%, 1 L) and vigorously shaken. The layers were separated and aqueous layer extracted with CH 2 C1 2 (2 x 75 mL). Combined extract was dried (Na 2 SO ), concentrated and rapidly purified on silica gel column using hexanes and hexanes/Et 2 O as eluents. Product-containing fractions were concentrated and used immediately for the next step.
• Step B l-r6-Diethylphosphono-6,6-difluoro-2,3,5,6-tetradeoxy- ⁇ / ⁇ -D- allofuranosyl]thymine (60)
• Step C l- 6,6-Difluoro-6-phosphono-2.3,5.6-tetradeoxy- ⁇ / ⁇ -D-allofuranosyllthvmine (61)
• Step D l-r6,6-Difluoro-6-( ⁇ , ⁇ -difluoromethylenetriphosphono)-2,3,5,6-tetradeoxy- ⁇ / ⁇ - D-allofuranosyl]thymine (62)
• Step A The preparation of iodomethyl pivalate
• Step B 3 '-Azido-3 '-deoxythymidine 5'- ⁇ , ⁇ , ⁇ -O-(tripivaloyloxymethyl- ⁇ , ⁇ - imidodiphosphate (68)
• a mixture of 3 '-azido-3 '-deoxythymidine 5'-imidodiphosphate (5'-O- imidodiphosphate of AZT was prepared according to the literature. (Ma et al, J. Med. Chem. 1992, 35, 1938-1941) (H+ form, 57 mg, 0.13 mmol) and tributylstannyl methoxide (125 mg, 0.39 mmol) in methanol (3 mL) was stirred at 25°C for 30 min. Methanol was removed by evaporation. To thoroughly remove methanol, the residue was coevaporated with acetonitrile three times.
• Step C 3 '-Azido-3 '-deoxythymidine 5'- ⁇ -P-borano- ⁇ -O-(S-pivaloyl-2-thioethyl)- ⁇ , ⁇ - (difluoromethylene triphosphate (70)
• Step A r(2,3-O-Di ⁇ almitoylglyeroxy)carbonyl-3-propionoxy]-2-ethanol
• Step C 3 '-Azido-3 '-deoxythymidine 5'- ⁇ -P-borano- ⁇ -O-r(2,3-O- dipalmitoylglyceroxy)carbonyl-3-propionoxy1-2-ethyl)- ⁇ , ⁇ - (difluoromethylene)triphosphate (79)
• the reaction mixture was stirred for 24 hours, extracted with 50% water/acetonitrile (3 x 6 mL), filtered, and concentrated to remove partial acetonitrile.
• the aqueous mixture was purified by reverse-phase HPLC to give 20 mg of the titled compound 79.
• HIV Reverse Transcriptase HIV Reverse Transcriptase
• This assay was used to measure the ability of the nucleotide mimics of the present invention to inhibit the enzymatic synthesis of complementary strand DNA from a DNA-primed template of homopolymeric RNA.
• This assay is a modification of a published procedure (Seville, et al, "Fluorometric Assay for DNA Polymerases and Reverse Transcriptase", BioTechniques 1996, 21, 664-72).
• DNA polymerase activity was measured in a reaction buffer containing primed- RNA template and dTTP diluted to appropriate concentrations in assay buffer.
• Nucleotide mimics of the present invention were diluted in buffer and pipetted into the wells of a 96-well plate. The reaction was initiated by addition of enzyme and allowed to proceed at 37°C for 10 minutes. The reaction was quenched by addition of 5 ⁇ L 0.2 M EDTA, pH 8.0.
• Blank reactions were prepared in parallel with the test reactions in which either enzyme or dTTP was omitted from the reactions, substituted by an appropriate volume of enzyme diluent or assay buffer, respectively.
• Inhibition [1-(RFU in test reaction - RFU in blank) / (RFU in control reaction - RFU in blank)] x 100.
• Inhibition constants (K) were determined for representative compounds that exhibited > 50% inhibition at 10 ⁇ M when tested in the HIV RT homopolymer polymerization assay. Each inhibitor was titrated over an appropriate range of concentrations, and inhibition constants were determined using the following equations:
• Representative compounds of the present invention tested in the HIV RT-catalyzed DNA polymerization assay exhibited inhibition constants less than 100 ⁇ M.
• Certain compounds of the present invention were also tested to determine their ability to inhibit HIV RT-catalyzed DNA polymerization on a heteropolymeric primed-RNA template in the presence of all four natural deoxynucleotide triphosphate (dNTP) substrates.
• This assay is a modification of a published procedure (Parker et al, "Mechanism of Inhibition of Human Immunodeficiency Virus Type 1 Reverse Transcriptase and Human DNA Polymerases ⁇ , ⁇ , and ⁇ by the 5'-Triphosphates of Carbovir,3'-Azido-3'-deoxythymidine,2'-3'- Dideoxyguanosine,and3 '-Deoxythymidine", J. Biol. Chem. 1991, 266 (3), 1754-62).
• dNTPs 50 ⁇ M dNTPs (dATP, TTP, dGTP, dCTP)
• HIV Reverse Transcriptase Purified, Type B, 66 kDa subunit
• HIV RT DNA polymerase activity was measured in a reaction buffer containing primed-ribosomal RNA template and dNTPs diluted to appropriate concentrations in assay buffer and pipetted into 1.5 mL microcentrifuge tubes. Nucleotide mimics of the present invention were diluted in buffer and tested at various concentrations up to 5 ⁇ M final concentration. The reaction was initiated by addition of enzyme and allowed to proceed at 42°C for 60 to 90 minutes. The reaction was quenched by addition of 12 ⁇ L 0.2 M ⁇ DTA, pH 8.0.
• RNR Ribonucleotide Reductase
• This assay was used to measure the ability of the nucleotide mimics of the present invention to inhibit the enzymatic reaction catalyzed by RNR enzyme.
• CDP cytidine 5 '-diphosphate
• dCDP 2'-deoxycytidine 5 '-diphosphate
• oxidized thioredoxin is formed.
• This protein is reduced by NADPH in a reaction catalyzed by thioredoxin reductase.
• RNR activity was measured by spectrophotometric determination of NADPH oxidation at 340 nm using a UV/VIS spectrophotometer.
• the compounds were tested at various concentrations up to 500 ⁇ M final concentration.
• the nucleotide mimics of the present invention were added to irreversible inactivation mixtures at the desired concentrations.
• An identical control was run in which the inhibitor was replaced with H 2 O. After a 3-minute incubation, 20 ⁇ L was removed from the inactivation mixtures and assayed for activity.
• the standard RNR assay is performed in a 96-well plate (Corning). Aliquots from the inactivation mixtures were added to an appropriate volume of assay buffer pipetted into the plate wells. The reactions were initiated by the addition of the substrate CDP. The reactions were allowed to proceed for 5 minutes at 25°C. The oxidation of NADPH was monitored at 340 nm on a microplate spectrophotometer (Molecular Devices Corp, Sunnyvale, CA). Initial velocity data (n A min "1 ) was collected and fit to the equations below.
• % Inhibition [l-(mA min "1 in test reaction - mA min “1 in blank) / (mA min "1 in control reaction - mA min "1 in blank)] x 100.
• Representative compounds of the present invention tested in the RNR inhibition assay exhibited inhibition constants less than 250 ⁇ M.
• This assay was used to measure the ability of the nucleotide mimics of the present invention to inhibit the polymerization of tubulin subunits and subsequent assembly of microtubule protein.
• This assay is a modification of a published procedure (Xu et al, "Interaction of Tubulin with Guanosine 5'-O-(l-Thiotriphosphate) Diastereoisomers: Specificity of the ⁇ -Phosphate Binding Region", Biochemistry 1994, 33, 11884-90).
• % Inhibition [l-(mA min "1 in test reaction - mA min “1 in blank) / (mA min "1 in control reaction - mA min "1 in blank)] x 100.
• Inhibition constants (Kj) were determined for representative compounds that exhibited > 50% inhibition at 250 ⁇ M when tested in the tubulin polymerization assay. Each inhibitor was titrated over an appropriate range of concentrations, and inhibition constants were determined using the following equations:
• This assay was used to measure the ability of the nucleoside derivatives of the present invention to interfere with normal microtubule protein dynamics, including the rate of polymerization, the rate of depolymerization, and stabilization of microtubule protein.
• This assay is a modification of the method described in O. Monasterio and S.N. Timasheff, " Inhibition of Tubulin Self- Assembly and Tubulin-Colchicine GTPase Activity by Guanosine 5'- ( ⁇ -Fluorotriphosphate)", Biochemistry 26: 6091-99 (1987).
• the compounds were tested at various concentrations up to 500 ⁇ M final concentration.
• Polymerization assays were performed in 96-well microplates (Costar), in assay buffer containing tubulin.
• the nucleotide mimics of the present invention were diluted in buffer and pipetted into the plate wells. Samples were kept on ice until the reactions were initiated by moving the plates into a 37°C incubator. The polymerization reaction was allowed to proceed at 37°C for 30 minutes, monitored at 340 nm using a microplate spectrophotometer with a temperature-controlled incubator (Molecular Devices, Sunnyvale, CA).
• This assay was used to measure the ability of the nucleotide mimics of the present invention to inhibit hydrolysis of GTP by tubulin.
• the enzymatic conversion of GTP to GDP + inorganic phosphate (Pj) is monitored in a coupled-spectrophotometric assay using the EnzChek Phosphate Assay Kit (Molecular Probes, Inc, Eugene, OR).
• This assay is a modification of a published procedure (Vandecandelaere et al., "Phosphate Release during Microtubule Assembly: What Stabilizes Growing Microtubules?", Biochemistry 1999, 38, 8179-88).
• GTP hydrolysis assays were performed in 96-well microplates (Costar), in assay buffer containing tubulin, GTP, MESG, and PNP.
• the nucleotide mimics of the present invention were diluted in buffer and pipetted into the plate wells. Samples were kept on ice until the reactions were initiated by moving the plates into a 37°C incubator.
• the hydrolysis reaction was initiated by addition of GTP, and allowed to proceed at 37°C for 10 minutes. The reaction was monitored at 360 nm using a microplate spectrophotometer with a temperature-controlled incubator (Molecular Devices, Sunnyvale, CA). Initial velocity data (mA min "1 ) was collected and fit to the equations described above.
• CCRF-CEM Human CCRF-CEM cells were obtained from American Tissue Culture Collection (ATCC) and grown according to ATCC specifications. Briefly, CCRF-CEM, a lymphoblastoid cell line, was grown and maintained as a suspension culture in RPMI 1640 medium containing 2 mm L-glutamine, 10 mM HEPES, 1 mM sodium pyruvate, 4.5 g/L glucose, 1.5 g/L sodium bicarbonate and supplemented with 10% (v/v) dialyzed and heat-inactivated fetal bovine serum. CEM cells were grown at 37°C in a 95% humidified environment and 5% CO 2 atmosphere.
• ATCC American Tissue Culture Collection
• Cytotoxicity Assays MTT Assay.
• MTT 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide
• CCRF-CEM cells (3 x 10 4 ) were plated in 96-well plates in RPMI media. The next day, cells were incubated with different concentrations (0-200 ⁇ M) of the nucleotide mimics of the present invention for 72 hr. Following treatment, MTT (2mg/ml in PBS) dye was added to each well so that the final concentration was 0.5 mg/ml and then incubated for 4 hr at 37°C Media and MTT dye were removed without disturbing the cells and 100% DMSO was added to dissolve the precipitate. After a 10 minute incubation at room temperature, the optical density values were measured at 540 nm, using the Spectra Max Plus plate reader. Survival was expressed as the percentage of viable cells in treated samples relative to non-treated control cells.
• Bacterial cells (2 x 10 4 ) growing in exponential phase were plated in 96-well plates and treated with different concentrations (0-200 ⁇ g/ml) of the nucleotide mimics of the present invention. The plates were incubated overnight at 37°C and then examined spectrophotometrically at 600 nm to determine the minimum concentration of each compound that inhibited replication of bacteria as determined by no increase in absorbance at 600 nm.
• nucleotide mimic 50% (v/v) fetal calf senim
• reaction mixtures were made up and incubated at 37°C. At appropriate times aliquots of 25 ⁇ l were removed and added to 65 ⁇ l ice-cold methanol. These solutions were incubated for at least one hour at -20°C and typically overnight. After incubation samples were centrifuged for at least 20 minutes at high speed in a microcentrifuge. The supernatant was transferred to a clean tube and the extract was dried under vacuum in a LabConco Centrivap Concentrator. The dried extracts were resuspended in dH 2 O and filtered to remove particulate before analysis on reverse phase HPLC.
• the reverse phase HPLC columns used for the analysis were either a Phenomenex C18 Aqua column (2 X 100 mm) or the Phenomenex C18 Aqua column (3 X 150 mm) used with the appropriate guard column.
• the HPLC was run at 0.2 ml/min (for the 2 X 100 mm column) or at 0.5 ml/min (for the 3 X 150 mm column) with the following buffer system: 5 mM tetrabutylammonium acetate, 50 mM ammonium phosphate, and an acetonitrile gradient running from 5% up to as high as 60%. The amount of remaining parent compound at each time point was used to determine the half-life of the compound.
• the lysis buffer was composed of the following: 50 mM Tris-HCl, pH 7.4 (100 ⁇ l/ml 10X stock), 20% glycerol (200 ⁇ l/ml), and 0.5% Triton X-100 (5 ⁇ l/ml). 100 ⁇ l of LB was added to each microfuge tube containing 10 7 frozen CEM cells. After the cells were lysed the extracts were centrifuged at high speed in a microcentrifuge for 5 minutes and the clarified cell extract transferred to a new tube.
• the cell stability reaction mixtures contained concentrations of buffer, magnesium, nucleotide, and cell extract as shown below:
• the reverse phase HPLC columns used for the analysis were either a Phenomenex C18 Aqua column (2 X 100 mm) or the Phenomenex C18 Aqua column (3 X 150 mm) used with the appropriate guard column.
• the HPLC flow rate was either 0.2 ml/min (for the 2 X 100 mm column) or 0.5 ml/min (for the 3 X 150 mm column) with the following buffer system: 5 mM tetrabutylammonium acetate, 50 mM ammonium phosphate, and an acetonitrile gradient running from 5% up to as high as 60%. The amount of remaining parent compound at each time point was used to determine the half-life of the compound.
• CEM cells were resuspended in RPMI at 1 X 10 7 cells/mL. The appropriate amount of each compound was added to well and then 0.5 ml of the cells was aliquoted into the appropriate number of wells of a 24-well plate and incubated the cells at 37°C. Cells were harvested after 24 hours of incubation. To harvest cells they were transferred to a microfuge tube and spun for one minute at high speed. The samples were washed in PBS containing 1 mg/ml BSA and re-centrifuged. The PBS was then aspirated and 200 ⁇ L of ice-cold 60% methanol was added to extract the nucleotides. The extracts were incubated ovemight at -20°C.
• the limit of detection was estimated to be 5 pmol/10 cells.
• Cell Preparation - CEM-SS cells were passaged in T-75 flasks prior to use in the antiviral assay. On the day preceding the assay, the cells were split 1 :2 to assure they were in an exponential growth phase at the time of infection. Total cell and viability quantification was performed using a hemacytometer and trypan blue exclusion. Cell viability was greater than 95% for the cells to be utilized in the assay. The cells were resuspended at 5 x 10 4 cells/ml in tissue culture medium and added to the dmg-containing micro titer plates in a volume of 50 ⁇ l.
• HIV-IRF HIV-IRF. This vims was obtained from the NIH AIDS Research and Reference Reagent.
• the assay plates were stained with the soluble tetrazolium- based dye MTS (CellTiter Reagent Promega) to determine cell viability and quantify compound toxicity.
• MTS is metabolized by the mitochondrial enzymes of metabolically active cells to yield a soluble formazan product, allowing the rapid quantitative analysis of cell viability and compound cytotoxicity.
• the MTS is a stable solution that does not require preparation before use.
• 20 ⁇ L of MTS reagent was added per well.
• the microtiter plates were then incubated 4-6 hrs at 37°C for the HIV cytoprotection assay; the incubation intervals were chosen based on empirically determined times for optimal dye reduction.
• Adhesive plate sealers were used in place of the lids, the sealed plate was inverted several times to mix the soluble formazan product and the plate was read spectrophotometrically at 490 nm with a Molecular Devices Vmax plate reader. Table 13. Anti-HIV Screening

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