AU2007203174B2 - 2'-Fluoronucleosides - Google Patents

Description

1. AUSTRALIA Patent Act 1990 (Clh) Complete Specification (Divisional) University of Georgia Research Foundation and Emory University Blake Dawson Waldron Patent Services Level 39. 101 ColLins Street Melbourne VIC 3000 Telephone: + 61 3 9679 3065 Ref: WJP KMC 03 1419 7131 Fax: + 61 3 96793111 2'-FLUORONUCLEOSIDES The invention described herein was made with Government support under grant number A132351 awarded by the National Institutes of Health. The United States 5 Government has certain rights to this invention. This invention is in the area of pharmaceutical chemistry, and in particular, includes 2'-fluoronucleosides and methods for their preparation and use. BACKGROUND OF THE INVENTION 10 Synthetic nucleosides such as 5-iodo-2'-deoxyuridine and S-flnoro-2'-deoxyuridine have been used for the treatment of cancer and herpes viruses for a number of years. Since the 1980's, synthetic nucleosides have also been a focus of interest for the treatment of HIV, hepatitis, and Epstein-Ban viruses. In 1981, acquired immune deficiency syndrome (AIDS) was identified as a disease 15 that severely compromises the human immune system, and that almost without exception leads to death. In 1983, the etiological cause of AIDS was determined to be the human immunodeficiency virus (HV). Ia 1985, it was reported that the synthetic nucleoside 3' azido-3'-dcoxythymidine (AZT) inhibits the replication of human immunodeficiency virus. Since then, a number of other synthetic nucleosides, including 2',3'-dideoxyinosine (DDI), 20 2',3'-dideoxycytidine (DDC), and 2',3'-dideoxy-2',3'-idehydrothymidine (D4T). have been proven to be effective against HIV. After cellular phosphorylation to the 5'-triphosphate by cellular kinases, these synthetic nucleosides are incorporated into a growing strand of viral DNA, causing chain termination due to the absence of the 3'-hydroxyl group. They can also inhibit the viral enzyme reverse transcriptase. 25 The success of various synthetic nucleosides in inhibiting the replication of HIV in vivo or in vitro has led a number of researchers to design and test nucleosides that substitute a heteroatom for the carbon atom at the Y-position of the nucleoside. European Patent Application Publication No. 0 337 713 and U.S. Patent No. 5,041,449, assigned to BioChem Pharmna, Inc., disclose racemic 2-substiuted-4-substituted- 1,3-dioxolanes that exhibit 30 antiviral activity. U.S. Patent No. 5,047,407 and European Patent Application No. 0 382 526, also assigned to BioChern Pharma, Inc., disclose that a number of racemic 2-substituted-5substituted-1.3-oxathiolane nucleosides have antiviral activity, and specifically report that the racemic mixture of 2-hydroxymethyl-5-(cytosin-1-yl)-1,3-oxathiolane (referred to below as BCHI-1 89) has approximately the same activity against lV as AZT, with little toxicity. The (-)-enantiomer of the racemate BCH- 189, known as 3TC, which is covered by U.S. Patent 5 No. 5,539,116 to Liotta et al., is currently sold for the treatment of HIV in combination with AZT in humans in the U.S. It has also been disclosed that cis-2-hydroxymethyl-5-(5-fluorocytosin-1-yl)-1,3 oxathiolane ("FTC") has potent 1HIV activity. Schinazi, e aL, "Selective Inhibition of Human Immunodeficiency viruses by Racemates and Enantiomers of cis-5-Fluoro-l-f2 10 (Hydroxymethyl)-1,3-Oxathiolane-5-yl]Cytosinc" Antimicrobial Agents and Chemotherapy, November 1992, pp. 2423-2431. See also U.S. Patent No. 5210,085; WO 91/11186, and WO 92/14743. Another virus that causes a serious human health problem is the hepatitis B vims (referred to below as "HBV). HBV is second only to tobacco as a cause of human cancer. 15 The mechanism by which HBV induces cancer is unknown. It is postulated that it may directly trigger tumor development, or indirectly trigger tumor development through chronic inflammation, cirrhosis, and cell regeneration associated with the infection. After a two to six month incubation period in which the host is unaware of the infection, HBV infection can lead to acute hepatitis and liver damage, that causes abdominal 20 pain, jaundice, and elevated blood levels of certain enzymes. HBV can cause flminant hepatitis, a rapidly progressive, often fatal form of the disease in which massive sections of the liver are destroyed. Patients typically recover from acute hepatitis. In some patients, however, high levels of viral antigen persist in the blood for an extended, or indefinite, period, causing a chronic 25 infection. Chronic infections can lead to chronic persistent hepatitis. Patients infected with chronic persistent HBV are most common in developing countries. By mid-I 991, there were approximately 225 million chronic carriers of HBV in Asia alone, and worldwide, almost 300 million carriers. Chronic persistent hepatitis can cause fatigue, cirrhosis of the liver, and hepatocellular carcinoma, a primary liver cancer. 30 In western industrialized countries, high risk groups for HBV infection include those in contact with HBV carriers or their blood samples. The epidemiology of HBV is very 2 similar to that of acquired immune deficiency syndrome, which accounts for why HBV infection is common among patients infected with HIV or AIDS. However, HBV is more contagious than Vl. Both FTC and 3TC exhibit activity against HBV. Furman, et al., "The Anti-Hepatitis 5 B Virus Activities, Cytotoxicities, and Anabolic Profiles of the (-) and (+) Enantiomers of cis-5-Fluoro-l-[2-(Hydroxymethyl)-1,3-oxathiolane-5-yl]-Cytosine" Antimicrobial Agents and Chemotherapy, December 1992, pp. 2686-2692; and Cheng, et al., Journal ofBiological Chemisay, Volume 267(20), pp.13938-13942 (1992). A human serum-derived vaccine has been developed to immunize patients against 10 HBV. While it has been found effective, production of the vaccine is troublesome because the supply of human serum from chronic carriers is limited, and the purification procedure is long and expensive. Further, each batch of vaccine prepared from different serum must be tested in chimpanzees to ensure safety. Vaccines have also been produced through genetic engineering. Daily treatments with a- interferon, a genetically engineered protein, has also 15 shown promise. Hepatitis C virus ("HCV") is the major causative agent for post-transfusion and for sporadic non A, non B hepatitis (Alter, H. I (1990) J. Gastro. HJpatol. 1:78-94; Dienstag, J. L. (1983) Gastro 85-439462), Despite improved screening, HCV still accounts for at least 25% of the acute viral hepatitis in many countries (Alter, H. 3. (1990) supra; Dienstag, I L. 20 (1983) supra; Alter M. J. et al.(1990a) J.AMA. 264:2231-2235; Alter M.J. er al (1992) N. Engl. J. Med. 327:1899-1905; Alter, M.. er al. (1990b) N Engl. J. Med 32:1494-1500). Infection by HCV is insidious in a high proportion of chronically infected (and infectious) carriers who may not experience clinical symptoms for many years. The high rate of progression of acute infection to chronic infection (70-100%) and liver disease (>50%), its 25 world-wide distribution and lack of a vaccine make HCV a significant cause of morbidity and mortality. A tumor is an unregulated, disorganized proliferation of cell growth. A tumor is malignant, or cancerous, if it has the properties of invasiveness and metastasis. Invasiveness refers to the tendency of a tumor to enter surrounding tissue, breaking through the basal 30 laminas that define the boundaries of the tissues, thereby often entering the body's circulatory 3 system. Metastasis refers to the tendency of a tumor to migrate to other areas of the body and establish areas of proliferation away from the site of initial appearance. Cancer is now the second leading cause of death in the United States. Over 8,000,000 persons in the United States have been diagnosed with cancer, with 1,208,000 new diagnoses 5 expected in 1994. Over 500,000 people die annually from the disease in this country. Cancer is not fully understood on the molecular level. It is known that exposure of a cell to a carcinogen such as certain viruses, certain chemicals, or radiation, leads to DNA alteration that inactivates a "suppressive" gene or activates an "oncogene". Suppressive genes are growth regulatory genes, which upon mutation, can no longer control cell growth. 10 Oncogenes are initially normal genes (called prooncongenes) that by mutation or altered context of expression become transforming genes. The products of transforming genes cause inappropriate cell growth. More than twenty different normal cellular genes can become oncogenes by genetic alteration. Transformed cells differ from normal cells in many ways, including cell morphology, cell-to-cell interactions, membrane content, cytoskeletal structure, 15 protein secretion, gene expression and mortality (transformed cells can grow indefinitely). All of the various cell types of the body can be transformed into benign or malignant tumor cells. The most frequent tumor site is lung, followed by colorectal, breast, prostate, bladder, pancreas, and then ovary. Other prevalent types of cancer include leukemia, central nervous system cancers, including brain cancer, melanoma, lymphorna, erythroleukemia, 20 uterine cancer, and head and neck cancer. Cancer is now primarily treated with one or a combination of three years oftherapies: surgery, radiation, and chemotherapy. Surgery involves the bulk removal of diseased tissue. While surgery is sometimes effective in removing tumors located at certain sites, for example, in the breast, colon, and skin, it cannot be used in the treatment of tumors located in 25 other areas, such as the backbone, nor in the treatment of disseminated neoplastic conditions such as leukemia. Chemotherapy involves the disruption of cell replication or cell metabolism. It is used most often in the treatment of leukemia, as well as breast, lung, and testicular cancer. There are five major classes of chemotherapeutic agents currently in use for the 30 treatment of cancer: natural products and their derivatives; anthacyclines; alkylating agents; 4 antiproliferatives (also called animctabolites); and hormonal agents. Chemotherapeutic agents are often referred to as antineoplastic agents. The alkylating agents are believed to act by alkylating and cross-linking guanine and possibly other bases in DNA, arresting cell division. Typical alkylating agents include 5 nitrogen mustards, ethyleneimine compounds, alkyl sulfates, cisplatin, and various nitrosoureas. A disadvantage with these compounds is that they not only attach malignant cells, but also other cells which are naturally dividing, such as those of bone marrow, skin, gastro-intestinal mucosa, and fetal tissue. Antimetabolites are typically reversible or irreversible enzyme inhibitors, or 10 compounds that otherwise interfere with the replication, translation or transcription of nucleic acids. Several synthetic nucleosides have been identified that exhibit anticancer activity. A well known nucleoside derivative with strong anticancer activity is 5-fluorouracil. 5 Fluorouracil has been used clinically in the treatment of malignant tumors, including, for 15 example, carcinomas, sarcomas, skin cancer, cancer of the digestive organs, and breast cancer. 5-Fluorouracil, however, causes serious adverse reactions such as nausea, alopecia, diarrhea, stomatitis, leukocytic thrombocytopenia, anorexia, pigmentation, and edema. Derivatives of 5-fluorouracil with anti-cancer activity have been described in U.S. Patent No 4,336,381, and in Japanese patent publication Nos. 50-50383, 50-50384, 50-64281, 51 20 146482, and 53-84981. U.S. Patent No. 4,000,137 discloses that the peroxidate oxidation product of inosine, adenosine, or cytidine with methanol or ethanol has activity against lymphocytic leukemia. Cytosine arabinoside (also referred to as Cytarabin, araC, and Cytosar) is a nucleoside analog of deoxycytidine that was first synthesized in 1950 and introduced into clinical 25 medicine in 1963. It is currently an important drug in the treatment of acute myeloid leukemia. It is also active against acute lymphocytic leukemia, and to a lesser extent, is useful in chronic myclocytic leukemia and non-Hodgkin's lymphoma. The primary action of araC is inhibition of nuclear DNA synthesis. Handschumacher, R. and Cheng, Y., "Purine and Pyrimidine Antimetabolites", Cancer Medicine, Chapter XV-1, 3rd Edition, Edited by J 30 Holland, et al., Lea and Febigol, publishers. 5 5-Azacytidine is a cytidine analog that is primarily used in the treatment of acute myelocytic leukemia and myelodysplastic syndrome. 2-Fluoroadenosine-5'-phosphate (Fludara, also referred to as FaraA)) is one of the most active agents in the treatment of chronic lymphocytic leukemia. The compound acts by 5 inhibiting DNA synthesis. Treatment of cells with F-araA is associated with the accumulation of cells at the G1/S phase boundary and in S phase; thus, it is a cell cycle S phase-specific drug. Incorporation of the active metabolite, F-araATP, retards DNA chain elongation. F-araA is also a potent inhibitor of ribonucleotide reductase, the key enzyme responsible for the formation of dATP. 10 2-Chlorodeoxyadenosine is useful in the treatment of low grade B-cell neoplasms such as chronic lymphocytic leukemia, non-Hodgkins' lymphoma, and hairy-cell leukemia. In designing new biologically active nucleosides, there have been a number of anempts to incorporate a fluoro substituent into the carbohydrate ring ofthe nucleoside. Fluorine has been suggested as a substituent because it might serve as an isopolar and 15 isosteric mimic of a hydroxyl group as the C-F bond length (1.35 A) is so similar to the C-O bond length (1.43 A) and because fluorine is a hydrogen bond acceptor. Fluorine is capable of producing significant electronic changes in a molecule with minimal steric perturbation. The substitution of fluorine for another group in a molecule can cause changes in substrate metabolism because of the high strength of the C-F bond (116 kcal/mol vs. C-H - 100 20 kcal/mol). A number of references have reported the synthesis and use of 2'-arabinofluoro nucleosides (i.e., nucleosides in which a 2-fluoro group is in the "up"-configuration). There have been several reports of 2-fluoro-f-D-arabinofiranosyl nucleosides that exhibit activity against hepatitis B and herpes. See, for example, U.S. Patent No. 4,666,892 to Fox, et al.; 25 U.S. Patent No. 4,211,773 to Lopez, et al; Su, el al., Nucleosides. 136, "Synthesis and Antiviral Effects of Several 1-(2-Deoxy-2-fluoro-f-D-arabinofuranosyl)-5-alkyluracilsf" "Some Structure-Activity Relationships," J. Med. Chem., 1986, 29, 151-154; Borthwick, et al., "Synthesis and Enzymatic Resolution of Carbocyclic 2'-Ara-fluoro-Guanosine: A Potent New Anti-Herpetic Agent," J. Chem. Soc., Chem. Common, 1988; Wantanabe, er al., 30 "Synthesis and Anti-HV Activity of 2'-"Up"-Fluoro Analogues of Active Anti-Aids Nucleosides 3'-Azido-3-deoxythymidinc (AZT) and 2',Y-didcoxycytidine (DDC),". Med 6 Chem. 1990. 33,2145-2150; Manin, et lt "Synthesis and Antiviral Activity of Monofluoro and Difluoro Analogues of Pyrimidine Deoxyribonucleosides against Human immunodeficiency Virus (HIV-1)," . Med, Chem. 1990, 33,2137-2145; Sterzycki, et aL, 'Synthesis and Anti-HIV Activity of Several 2'-Fluoro-Containing Pyrimidine Nucleosides," 5 J. Med Chem. 1990, as well as EPA 0 316 017 also filed by Sterzycki, et al.; and Montgomery, et al, "9- (2-Deoxy-2-fluoro-0-D-arabinofuranosyl)guanine: A Metabolically Stable Cytotoxic Analogue of 2-Deoxyguanosine." U.S. Patent No. 5,246,924 discloses a method for treating a hepatitis infection that includes the administration of 1-(2-deoxy-2' fluoro-g-D-arabinofuranosyl)-3-ethyluracil), also referred to as "FEAU." U.S. Patent No. 10 5,034,518 discloses 2-luoro-9-(2-deoxy-2-fluoro-p-D-arabino-furanosyl)adenine nucleosides which exhibit anticancer activity by altering the metabolism of adenine nucleosides by reducing the ability of the compound to serve as a substrate for adenosine. EPA 0 292023 discloses that certain p-D-2'-fluoroarabinonucleosides are active against viral infections. U.S. Patent No- 5,128,458 discloses p-D-2',3'-dideoxy-4-thioribonucleosides as 15 antiviral agents. U.S. Patent No. 5,446,029 discloses that 2,3',-dideoxy-3'-fluoronucleosides have antihepatitis activity. European Patent Application No. 0 409 227 A2 discloses certain 3'-substituted p-D pyrimidine and purine nucleosides for the treatment of hepatitis B. It has also been disclosed that L-FMAU (2-fluoto-5-methyl 20 P-L-arabinofuranosyluracil) is a potent anti--BV and anti-EBV agent. See Chu, et al, "Use of 2'-Fluoro-5-methyl-p-L-arabinofuranosyluraci as a Novel Antiviral Agent for Hepatitis B Virus and Epstein-Bar Virus" Antimicrobial Agents and Chemotherapy, April 1995 pages. 979-981; Balakrishna, et al., "Inhibition of Hepatitis B Virus by a Novel L-Nucleoside, 2' Fluoro-5-Methyl--L-arabinofuranosyl Uracil," Antimicrobial Agents and Chemotherapy 25 Feb 1996, pages 380-356; U.S. Patent Nos. 5,587,362; 5,567,688; and 5,565,438. U.S. Patent Nos. 5,426,183 and 5,424,416 disclose processes for preparing 2'-deoxy 2',2'-difluoronucleosides and 2'-deoxy-2T-fluoro nucleosides. See also "Kinetic Studies of 2',2'-difluorodeoxycytidine (Gemitabine) with Purified Human Deoxycytidine Kinase and Cytidine Deaminase," BioChemical Pharmacology, Vol. 45 (No. 9) pages 4857-1861, 1993. 30 U.S. Patent No. 5,446,029 to Eriksson, et .t, discloses that certain 2',3'-didcoxy-3 fluoronuciensides have hepatitis B activity. U.S. Patent No. 5,128,458 discloses certain 2',3'. 7 dideoxy-4'-thioribonucleosides wherein the 3'-substituent is I1 azide or fluoro. WO 94/14831 discloses certain 3'-fluoro-dihydropyrimidine nucleosides. WO 92/08727 discloses 0-L-2'-deoxy-3'-fluoro-5-substituted uridine nucleosides for the treatment of herpes simplex I and 2. 5 EPA Publication No. 0 352 248 discloses a broad genus of L-ribofuranosyl purine nucleosides for the treatment of HIV, herpes, and hepatitis. While certain 2'-fluorinated purine nucleosides fall within the broad genus, there is no information given in the specification on how to make these compounds in the specification, and they are not among specifically disclosed or the preferred list of nucleosides in the specification. The 10 specification does disclose how to make 3'-ribofuranosyl fluorinated nucleosides. A similar specification is found in WO 88/09001, filed by Aktiebolaget Astra. European Patent Application 0 357 571 discloses a broad group of P-D and a-D pyrimidine nucleosides for the treatment of AIDS which among the broad class generically includes nucleosides that can be substituted in the 2' or 3'-position with a fluorine group. 15 Among this broad class, however, there is no specific disclosure of 2'-fluorinated nucleosides or a method for their production. EPA 0 463 470 discloses a process for the preparation of (5S)-3-fluoro-tetrahydro-5 [(hydroxy)methyl)-2-(3H)-ftranone, a known intermediate in the manufacture of 2'-fluoro 2',3'-dideoxynucleosides such as 2'-fluoro-2',3'-dideoxycytidine. 20 U.S.S.N. 07/556,713 discloses P-D-2'-fluoroarabinofuranosyl nucleosides, and a method for their production, which are intermediates in the synthesis of 2',3'-dideoxy-2' fluoroarabinosyl nucleosides. U.S. Patent No. 4,625,020 discloses a method of producing 1-halo-2-deoxy-2 fluoroarabinofuranosyl derivatives bearing protective ester groups from 1,3,5-tri-O-acyl 25 ribofumnose There appears to be a lack of disclosure of p-L-2'-fluoro-ribofuranosyl nucleosides for medicinal uses, including for HIV, hepatitis (B or C), or proliferative conditions. At least with respect to 2'-ribofuiranosyl nucleosides. this may be because of the prior perceived difficulty in placing a fluoro group in the 2'-ribofuranosyl configuration. With respect to L 30 2'-fluoro-2',Y-unsaturated purine nucleosides, it may be because the purine nucleosides are unstable in acidic media, resulting in glycosyl bond cleavage. 8 In light of the fact that HIV acquired immune deficiency syndrome, AIDS-related complex, and hepatitis B and C viruses have reached epidemic levels worldwide, and have tragic effects on the infected patient, there remains a strong need to provide new effective pharmaceutical agents to treat these diseases that have low toxicity to the host. Further, there 5 is a need to provide new antiproliferative agents Therefore, it is an object of the present invention to provide a method and composition for the treatment of human patients infected with hepatitis B or C. It is another object of the present invention to provide a method and composition for the treatment of human patients infected with HIV. 10 It is a further object of the present invention to provide new antiprolife-ative agents. It is still another object of the present invention to provide a new process for the preparation of 2'-fluoro-ribofuranosyl nucleosides. It is yet another object of the present invention to provide a new process for the preparation of 2',3ideoxy-2',3'-didehydro-2 -fluoro-L-glycero-pent-2-eno-furanosyI 15 nucleosides. SUMMARY OF THE INVENTION In one cmbodiment of the invention, a 2-a-fluoro-nucloside is provided of the structure: 20 0 25 R1 wherein Base is a purine or pyrimidine base as defined further herein; R' is OhA, OR 3 , N, CN, halogen, including F, or CF,, lower alkyl, amino. 30 lowcralkyiaminu, di(lower)alkylamino, or alkoxy, and base refers to a purine or pyrimidine base; 9 R is H, phosphate, including monophosphatc. diphosphate, triphosphate, or a stabilized phosphate prodrug; acyl, or other pharmaceutically acceptable leaving group which when administered in vivo, is capable of providing a compound wherein R2 is H or phosphate; sulfonate ester including alkyl or arylaikyl sulfonyl including methanesulfony, benzyl, S wherein the phenyl group is optionally substituted with one or more substituents as described in the definition of aryl given above, a lipid, including a phospholipid, an amino acid, peptide, or cholesterol; and

R

3 is acyl, alkyl, phosphate, or other pharmaceutically acceptable leaving group which when administered in vivo, is capable of being cleaved to the parent compound. 10 In a second embodiment, a 2'-fluoronucleoside is provided of the formula: V Y=4 S ,6 CHF wherein the substituents are as defined above. In a third embodiment, a 2'-fluoronucleoside is provided of the formula; 20 Base x RF X= S, C14 25 wherein the substituents are as defined above. In a fourth embodiment, a 2'-flooronucleoside is provided of the structure: 30 10 Sase

OR'

2 F R' 5 X-s, CH2 wherein the substituents are as defined above. These 2'-fluoronucleosides can be either in the p-L or f-D configuration. The p-L configuration is'preferred. The 2'-fluoronueleosides are biologically active molecules which are useful in the 10 treatment of hepatitis B, hepatitis C or HV. The compounds are also useful for the treatment of abnormal cellular proliferation, including tumors and cancer. One can easily determine the spectmrn of activity by evaluating the compound in the assays described herein or with another conflnmatory assay. In another embodiment, for the treatment of hepatitis or HIV, the active compound or 15 its derivative or salt can be administered in combination or alternation with another antiviral agent, such as an anti-HIV agent or anti-hepatitis agent, including those of the formula above, In general, in combination therapy, an effective dosage of two or more agents are administered together, whereas during alternation therapy, an effective dosage of each agent is administered serially. The dosages will depend on absorption, inactivation, and excretion 20 rates of the drug as well as other factors known to those of skill in the art. It is to be noted that dosage values will also vary with the severity of the condition to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens and schedules should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions. 25 Nonlimiting examples of antiviral agents that can be used in combination with the compounds disclosed herein include 2-hydroxymethyl-$-(5-fluorocytosin- -yl)- 1,3 oxathiolane (FTC); the (-)-enantiomer of 2-hydroxymethyl-5(cytosin-1-yi)-1,3-oxathiolane (3TC); carbovir, acyclovir, interferon, famciclovir, pencicluvir, AZT, DDI, DDC, D4T, abacavir, L-(-)-FMAU, L-DDA phosphate prodrugs, and P-D-dioxolane nucleosidces such as 30 P-D-dioxolanyl-guanine (DG), P-D-dioxolanyl-2,6-diaminopurine (DAPD), and @-D dioxolanyl-6-chloropurine (ACP), non-nucleoside RT inhibitors such as nevirapine, MKC 11 442. DMP-266 (sustiva) and also protease inhibitors such as indinavir, saquinavir. AZT, DMP-450 and others. The compounds can also be used to treat equine infectious anemia virus (EIAV), feline immunodeficiency virus, and simian immunodeficiency virus. (Wang, S., Montelaro. 5 R., Schinazi, R.F., Jagerski, B., and Mellors, J.W.: "Activity of nucleoside and non nucleoside reverse transcriptase inhibitors (NNRTI) against equine infectious anemia virus (EIAV)." First National Conference on Human Retro viruses and Related Infections, Washington, DC, Dec. 12-16, 1993; Sellon D.C., "Equine Infectious Anemia," Vet. Clin. North Am. Equine Pract. United States, 9: 321-336, 1993; Philpott, M.S., Ebner, J.P., 10 Hoover, E.A., "Evaluation of 9-(2-phosphonylmethoxyethyl) adenine therapy for feline immunodeficiency virus using a quantitative polymerase chain reaction," Vet. Immunol. Immunopathol. 35:155166, 1992.) A new and completely diastereoselective method for the introduction of fluorine into a non-carbohydrate sugar ring precursor is also provided. The method includes reacting a 15 chiral, non-carbohydrate sugar ring precursor (4S)-5-(protected oxy)-pentan-4-olide, which can be prepared from L-glutamic acid, with an electrophilic source of fluorine, including but not limited to N-fluoro-(bis)benzenesulfonimide, to yield key intermediate fluorolactone 6. The fluorolactone is reduced to the lactol and acetylated to give the anomeric acetate and then used for the synthesis of a number of novel p-L-t-2'-fluoronucleosides. The corresponding 20 D-enantiomer can also be synthesized using D-glutanic acid as a starting material. In an alternative embodiment, a fluorinated glycal is prepared which is dehydrogenated and then converted to a 2',3'-dideoxy-2',3'-didehydro-2'-fluoronucleoside or a p-L or J-D..rabinosyl-2'-fluoronucleoside, as discussed further below. A method for the facile preparation of 2',3'-dideoxy-2',3'-didchydro-2' 25 fluoronucleosides is also presented that includes the direct condensation of silylated 6 chloropurine with key immediate, which is prepared from L-2,3-0-isopropylidene glyceraldenhyde. DETAILED DESCRIPTION OF THE INVENTION 30 The invention as disclosed herein is a compound, method and composition for the treatment of HIV, hepatitis (B or C), or abnormal cellular proliferation, in humans or other 12 host animals, that includes administering an effective amount of a 2'-fluoro-nucleoside, a pharmaceutically acceptable derivative, including a compound which has been alkylated or acylated at the 5'-position or on the purine or pyrimidine, or a pharmaceutically acceptable salt thereof, optionally in a pharmaceutically acceptable carrier. The compounds of this 5 invention either possess amtiviral (i.e., anti-IV-1, anti-HIV-2, or anti-hepatitis ( B or C)) activity, or antiproliferative activity, or are metabolized to a compound that exhibits such activity. In summary, the present invention includes the following features: (a) p-L and p-D-2-fluoronucleosides, as described herein, and pharmaceutically 10 acceptable derivatives and salts thereof; (b) p-L and p-D-2T-fluoronucleosides as described herein, and pharmaceutically acceptable derivatives and salts thereof for use-in medical therapy, for example for the treatment or prophylaxis of an HIV or hepatitis (B or C) infection or for the treatment of abnormal cellular proliferation; 15 (c) 2 ',3'-Dideoxy-2',3-didehydro-2'-fluoro-L-glycero-pen-2-eno-furanosy nucleosides, and pharmaceutically acceptable derivatives and salts thereof for use in medical therapy, for example for the treatment or prophylaxis of an HV or hepatitis (B or C) infection or for the treatment of abnormal cellular proliferation (d) use of these 2'-fluoronucleosides, and pharmaceutically acceptable derivatives 20 and salts thereof in the manufacture of a medicament for treatment of an HIV or hepatitis infection or for the treatment of abnormal cellular proliferation; (e) pharmaceutical formulations comprising the 2'-fluoronucleosides or a pharmaceutically acceptable derivative or salt thereof together with a pharmaceutically acceptable carrier or diluent; 25 (1) processes for the preparation of p-L and P-D-2'-a-fluoronucleosides, as described in more detail below, and (g) processes for the preparation of 2',3'-dideoxy-2',3'-didehydro-2'-fluoro-L glycero-pent-2-eno-furanosyl nucleosides. 1. Active Compound, and Physiologically Acceptable Derivatives and Salts Thereof 30 A 2T-a-fluoro-nucleoside is provided of the structure: 13 Base 0 R2 5 F wherein R' is H, OH, OR-, N), CN, halogen, including F, or CF 3 , lower alkyl, amino, loweralkylamino, di(lower)alkylamino, or alkoxy, and base refers to a purine or pyrimidine 10 base. R2 is 1H, phosphate, including monophosphate, diphosphatc, triphosphatc, or a stabilized phosphate prodrug; acyl, or other pharmaceutically acceptable leaving group which when administered in vivo, is capable of providing a compound wherein R' is H or phosphate, sulfonate ester including alkyl or arylalkyl sulfonyl including methanesulfonyl, benzyl, wherein the phenyl group is optionally substituted with one or more substituents as described 15 in the definition of aryl given above, a lipid, an amino acid, peptide, or cholesterol; and RI is acyl, alkyl, phosphate, or other pharmaceutically acceptable leaving group which when administered in vivo, is capable of being cleaved to the parent compound. In a second embodiment, a 2-fluoronucleoside is provided of the formula: 20 Base Y

R

2 0 RI F Y=O, S, CH 2 , CHF 25 In a third embodiment, a 2-fluoronucleoside is provided of the formula: Base ore x 30 R'F X= S. CH 2 14 In a fourth embodiment, a 2-fluoronucleoside is provided of the structure: Base 5 F R' X- S, CH2 The term alkyl, as used herein, unless otherwise specified, refers to a saturated 10 straight, branched, or cyclic, primary, secondary, or tertiary hydrocarbon of C 1 to CIO, and specifically includes methyl, ethyl, propyl, isopropyl, cyclopropyl, butyl, isobutyl, t-butyl, pentyl, cyclopentyl, isopentyl, neopentyl, hexyl, isobexyl, cyclohexyl, cyclohexylmethyl, 3 methylpentyl,2,2-dimethylbutyl, and 2,3-dimethylbutyl. The alkyl group can be optionally substituted with one or more moieties selected from the group consisting of hydroxyl, amino, 15 alkylamino, arylamino, alkoxy, asyloxy, nitro, cyano, sulfonic acid, sulfate, phosphonic acid, phosphate, or phosphonate, either unprotected, or protected as necessary, as known to those skilled in the art, for example, as taught in Greene, e at, Protective Groups in Organic Synthesis, John Wiley and Sons, Second Edition, 1991, hereby incorporated by reference. The term lower alkyl, as used herein, and unless otherwise specified, refers to a C, to 20 C, saturated straight, branched, or if appropriate, a cyclic (for example, cyclopropyl) alkyl group. The term alkylamino or arylamino refers to an amino group that has one or two alkyl or aryl substituents, respectively. The term "protected" as used herein and unless otherwise defined refers to a group 25 that is added to an oxygen, nitrogen, or phosphorus atom to prevent its further reaction or for other purposes. A wide variety of oxygen and nitrogen protecting groups are known to those skilled in the an of organic synthesis. The tenn aryl, as used herein, and unless otherwise specified, refers to phenyl, biphenyl, or naphthyl, and preferably phenyL The aryl group can be optionally substituted with one or more moieties selected from the group consisting of 30 hydroxyl, amino, alkylamino, arylamino, alkoxy, aryloxy, nitro, cyano, sulfonic acid, sulfate, phosphonic acid, phosphate, or phosphonate, either unprotected, or protected as necessary, as 15 known to those skilled in the art, for example, as taught in Greene, et al, Protective G in Oreanic Synthesis. John Wiley and Sons. Second Edition, 1991. The term alkaryl or alkylaryl refers to an alkyl group with an aryl substituent. The term aralkyl or arylalkyl refers to an aryl group with an alkyl substituent. 5 The tern halo! as used herein, includes chloro, bromo, iodo, and fluoro. The term purine or pyrimidine base includes, but is not limited to, adenine, N alkylpurines, NI-acylpurincs (wherein acyl is C(O)(alkyl, aryl, alkylayl, or arylalkyl), N' benzylpurine, N-halopurine, N-vinylpurine, W-acetylenic purine, N'-acyl purine, N'-hydroxyalkyl purine, NthioaIkyl purine, N-alkylpurines, W-Akyl-6-thiopurines, 10 thymine, cytosine, 5-fluorocytosine, 5-methylcytosine, 6-azapyrinidine, including 6-azacytosine, 2- and/or 4-mercaptopyrnidine, uracil, 5-halouracil, including 5-fluorouracil, C'-alkylpyrimidines. C'-benzylpyrimidines,

C

3 -halopyrimidines, Ckvinylpyrimidine.

CI

acetylenic pyrimidine, C'-acyl pyrimidine, C 5 -hydroxyalkyl purine, C-amidopyrimidine, C cyanopyrimidine, C-nitropyrimidine, C-aminopyrimidine, N-alkylpurines, N7-alkyl-6 15 thiopurines, 5-azacytidinyl, 5-azauracilyl, triazolopyridinyl, imidazolopyridinyl, pyrrolopyrimidinyl, and pyrazolopyrimidinyl. Purine bases include, but are not limited to, guanine, adenine, hypoxanthine, 2

,

6 -diaminopurine, and 6-ehioropurine. Functional oxygen and nitrogen groups on the base can be protected as necessary or desired. Suitable protecting groups are well known to those skilled in the art, and include trimethylsilyl, 20 dimethylhexylsilyl, t-butyldimethylsilyl, and t-butyldiphenylsilyi, trityl, alkyl groups, acyl groups such as acetyl and propionyl, methanesulfonyl, and p-toluenesulfonyl. The active compound can be administered as any derivative that upon administration to the recipient, is capable of providing directly or indirectly, The parent compound, or that exhibits activity itself Nonlimiting examples are the pharmaceutically acceptable salts 25 (alternatively referred to as "physiologically acceptable salts"), and a compound which has been alkylated or acylated at the 5'-position or on the purine or pyrimidine base (alternatively referred to as "pharmaceutically acceptable derivatives"). Further, the modifications can affect the biological activity of the compound, in some cases increasing the activity over the parent compound. This can easily be assessed by preparing the derivative and testing its 30 antiviral activity according to the methods described herein, or other method known to those skilled in the arL 16 The term acyl refers to a carboxylic acid ester in which the non-carbonyl moiety of the ester group is selected from straight, branched, or cyclic alkyl or lower alkyl, alkoxyalkyl including metboxymethyl, aralkyl including benzyl. aryloxyalkyl such as phenoxymethyl. aryl including phenyl optionally substituted with halogen, C, to C, alkyl or C, to C, alkoxy, 5 sulfonate esters such as alkyl or aralkyl sulphonyl including methanesulfonyl. the mono, di or triphosphate ester, trityl or monomethoxytrityl, substituted benzyl, trialkylsilyl (e.g. dimethyl t-butylsilyl) or diphenylmethylsilyl. Aryl groups in the esters optimally comprise a phenyl group. As used herein, the term "substantially free of' or "substantially in the absence of" 10 refers to a nucleoside composition that includes at least 95% to 98%, or more preferably, 99% to 100%, of the designated enantiomer of that nucleoside. Nucleotid! Prodrug Formulations Any of the nucleosides described herein can be administrated as a nucleotide prodrug to increase the activity, bioavailability, stability or otherwise alter the properties of the 15 nucleoside. A number of nucleotide prodrug ligands are known. In general, alkylation, acylation or other lipophilic modification of the mono, di or triphosphate of the nucleoside will increase the stability of the nucleotide. Examples of substituent groups that can replace one or more hydrogen on the phosphate moiety are alkyl, aryl, steroids, carbohydrates, including sugars, 1,2-diacylglycerol and alcohols. Many are described in R. Jones and N. 20 Bischofberger, Antiviral Research, 27 (1995) 1-17. Any of these can be used in combination with the disclosed nucleosides to achieve a desired effect. The active nucleoside can also be provided as a 5'-phosphoether lipid or a 5'-ether lipid, as disclosed in the following references, which are incorporated by reference herein; Kucera, L.S., N. Iyer, E. Leake, A. Raben, Modest E.K., D.L.W., and C. Piantadosi. 1990. 25 "Novel membrane-interactive ether lipid analogs that inhibit infectious HIV-1 production and induce de fective virus formation." AIDS Res. Hum. Retro Viruses. 6:491-50 1; Piantadosi, C. I Marasco C.J., S.L. Morris-Natschke, K.L. Meyer, F. Gumus, J.R. Surles, K.S. Ishaq, L.S. Kucera, N. Iyer, C.A. Wallen, S. Piantadosi, and E.J. Modest. 1991. "Synthesis and evaluation of novel ether lipid nucleoside conjugates for anti-HIV activity." J. Med. Chem. 30 34:1408.1414; Hosteller, K.Y., D.D. Richman, D.A. Carson, L.M. Stuhmiller, G.M. T. van Wijk. and 8, van den Bosch. 1992. "Greatly enhanced inhibition of human 17 immunodeficiency virus type I replication in CEM and 11T4-6C cells by 3'-deoxythymidinc diphosphate dimyristoylglycerol. a lipid prodrug of 3,-deoxythymidine." Animicrob. Agents Chemother. 36:2025.2029; Hosetler, K.Y., L.M. Stuhuniller, H.B. Lenting, H. van den Bosch, and D.D. Richman, 1990. "Synthesis and antiretroviral activity of phospholipid 5 analogs of azidothymidine and other antiviral.nucleosides." J. Biol. Chem. 265:61127. Nonlimiting examples of U.S. patents that disclose suitable lipophilic substituents that can be covalently incorporated into the nucleoside, preferably at the 5'-OH position of the nucleoside or lipophilic preparations, include U.S. Patent Nos. 5.149,794 (Sep. 22, 1992, Yatvin et al); 5,194,654 (Mar. 16, 1993, Hostetler et al., 5,223,263 (June 29, 1993, Hostetler 10 et al.); 5,256,641 (Oct 26, 1993, Yatvin et al); 5,411,947 (May 2, 1995, Hostetler et al.); 5,463,092 (Oct. 31, 1995, Hostetler et al.); 5,543,389 (Aug. 6, 1996, Yatvin et al.); 5,543,390 (Aug. 6,1996, Yatvin et al.); 5,543,391 (Aug. 6, 1996, Yarvin et al.); and 5,554,728 (Sep. 10, 1996; Basava et al.), all of which are incorporated herein by reference. Foreign patent applications that disclose lipophilic substituens that can be attached to the nucleosides of the 15 present invention, or lipophilic preparations, include WO 89/02733, WO 90/00555, WO 91/16920, WO 91/18914, WO 93/0091d, WO 94/26273, WO 96/15132, EP 0 350 287, EP 93917054.4, and WO 91/19721. Nonlimiting examples of nucleotide prodrugs are described in the following references: Ho, D.H.W. (1973) "Distribution of Kinase and dearninase of 1f-D 20 arabinofuranosylcytosine in tissues of man and muse." Cancer Res. 33, 2816-2820; Holy, A. (1993) Isopolar phosphorous-modified nucleotide analogues," In: De Clercq (Ed.), Advasa in Antiviral Drug Design Vol. I, JAI Press, pp. 179-231; Hong, C.., Nechaev, A., and West, CR. (1979a) "Synthesis and antitunmor activity of 1 -P-D-arabino-furanosylcytosine conjugates of cortisol and cortisone." Bicohem. Biophys. Ar. Commun. 88, 1223-1229; Hong, 25 C.I., Nechaev, A., Kirisits, AJ. Buchheit, D.J. and West, C.R. (1980) "Nucleoside conjugates as potential antitumor agents. 3. Synthesis and antitunor activity of I-(0-D arabinofuranosyl) cytosine conjugates of corticosteriods and selected lipophilic alcohols." J Med Chem. 28, 171-177; Hlosteller, K.Y., Stuhmiller, L.M., Lenting, H.B.M. van den Bosch, Hi. and Richman J Biol. Chem. 265, 6112-6117; Hosteller, K.Y., Carson, D.A. and Richman, 30 D.D. (1991); "Phosphatidylazidothymidine: mechanism of antiretroviral action in CEM cells." I Blt Chem. 266, 11714-11717; Hosteller, K.Y.. Korba, 13. Sridhar, C., Gardener, M. 18 (1994a) "Antiviral activity of phosphaidyl-dideoxycytidine in hepatitis B-infected cells and enhanced hepatic uptake in mice." Antiviral Res. 24, 59-67; Hosteller, K.Y., Richman. D.D., Sridhar. C.N. Feigner, P.L. Feigner, J., Ricci, J., Gardener, M.F. Selleseth, D.W. and Ellis, M.N. (1994b) "Phosphatidylazidothymidine and phosphatidyl-ddC: Assessment of uptake in 5 mouse lymphoid tissues and antiviral activities in human inmunodeficiency virus-infected cells and in rauscher leukemia virus-infected mice." Animicrobial Agents Chemother. 38. 2792-2797; Hunston, R.N., Jones, A.A. McGuigan, C., Walker, R.T., Balzarini, J., and DeClercq, E. (1984) "Synthesis and biological properties of some cyclic phosphotriesters derived from 2'-deoxy-5-flourouridine." . Med. Chem. 27, 440-444; Ji, Y.H., Moog, C., 10 Schmitt, G., Bischoff, P. and Luu, B. (1990); "Monophosphoric acid esters of 7-fp hydroxycholesterol and of pyrimidine nucleoside as potential antitumor agents: synthesis and preliminary evaluation of antitumor activity." J Med. Chem. 33 2264-2270; Jones, A.S., McGuigan, C., Walker, R.T., Balzarini, J. and DeClercq, E. (1984) "Synthesis, properties, and biological activity of some nucleoside cyclic phosphoramnidates." J CheM Soc Perikin 15 Trans. 1, 1471-1474; Juodka, BA. and Smrt, J. (1974) "Synthesis of diribonucleoside phosph (P-N) amino acid derivatives." Coll Czech. Chem. Comm. 39, 363-968; Kataoka, S., Imai, J., Yamaji, N., Kato, M., Saito, M., Kawada, T. and Imai, S. (1989) "Alkylated cAMP derivatives; selective synthesis and biological activities." Nucleic Acids Res. Sym. Ser. 21, 1 2; Kataoka, S., Uchida, "(cAMP) benzyl and methyl triesters." Heterocycles 32, 1351-1356; 20 Kinchington, D., Harvey, J.J., O'Connor, T.J., Jones, B.C.N.M., Devine, K.G., Taylor Robinson D., Jeffries, D.J. and McGuigan, C. (1992) "Comparison of antiviral effects of zidovudine phosphoramidate an dphosphorodiamidate derivates against HIV and ULV in vitro." Antiviral Chem. Chemother. 3, 107-112; Kodama, K., Morozumi, M., Saithoh, K.I., Kuninaka, H., Yosino, H. and Saneyoshi, M. 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(1960) "The metabolism of exogenously supplied nucleotides by Escherichia coli.," J Biot Chem. 235, 457-465; Lucthy, J., Von Daeniken, A., Friederich, J. Manthey, B., Zwcifel, J., Schlatter, C. 5 and Bean, M.H. (1981) "Synthesis and toxicological properties of three naturally occurring cyanoepithioalkancs". Mitt. Geg. Lebensmittelunters. Hyg. 72, 131-133 (Chem. Abstr. 95. 127093); McGigan, C. Tollerfield, S.M. and Riley, P.a. (1989) "Synthesis and biological evaluation of some phosphate triester derivatives of the anti-viral drug Ara." Nucleic Acids Res. 17, 6065-6075; McGuigan, C., Devine, KG., O'Connor, T.J., Galpin, S.A, Jeffries, D.J. 10 and Kinchington, D. (1990a) "Synthesis and evaluation of some novel phosphoramidate derivatives of 3'-aido-3'-deoxythynidine (ALT) as anti-IV compounds." Antiviral Chem. Chemother. 1 107-113; McGuigan, C., O'Connor, T.J., Nicholls, S.R. Nickson. C. and Kinchington, D. (1990b) "Synthesis and anti-1HIV activity of some novel substituted dialkyl phosphate derivatives of AZT and ddCyd." Antiviral Chem Chemother. 1, 355-360; 15 McGuigan, C., Nicholls, S.R, O'Connor, T., and Kinchington, D. (1990c) "Synthesis of some novel dialkyl phosphate derivative of 3'-modificd nucleosides as potential anti-AIDS drugs." Antiviral Chem. Chemother. 1, 25-33; McGuigan, C., Devin, K.G., O'Connor, T.J., and Kinchington, D. (1991) "Synthesis and anti-IV activity of some haloalkyl phospboramidate derivatives of 3-azido-3'-deoxythylmidine (AZT); potent activity of the 20 trichloroethyl methoxyalaninyl compound." Antiviral Res. 15,255-263; McGuigan, C., Pathirana, R-N., Balzarini, J. and DeClercq, E. (1993b) "Intracellular delivery of bioactive AZT nucleotides by aryl phosphate derivatives of AZT." J, Med. Chem. 36, 1048-1052. Alkyl hydrogen phosphate derivatives of the anti-HIV agent AZT may be less toxic than the parent nucleoside analogue. Antiviral Chemn. Chemother. 5, 271-277; Meyer, R. B., 25 Jr., Shuman, DA. and Robins, R.K. (1973) "Synthesis of purine nucleoside 3', 5'-cyclic phosphoranidates." Tetrahedron Lett. 269-272; Nagyvary, J. Gohil, R.N., Kirchner, C.R. and Stevens, J.D. (1973) "Studies on neutral esters of cyclic AMP," Biochem. Biophys. Res. Commun. 55, 1072-1077; Namane, A. Gouyette, C., Fillion, MY., Fillion, G. and Huynh Dinh, T. (1992) "Improved brain delivery of AZT using a glycosyl phosphotriester prodrug." 30 J. Med. Chem. 35, 3039-3044; Nargeot, J. Nerbonne, J.M. Engels, J. and Leser, H.A. (1983) Natt. Acad Sci. U.S.A. 80, 2395-2399; Nelson, K.A., Bentrude, W.G. Stser, W.N. and 20 Hutchinson, J.P. (1987) "The question of chair-twist equilibria for the phosphate rings of nucleoside cyclic 3', 5' monophosphates. 'HNMR and x-ray crystallographic study of the diastereomers of thymidine phenyl cyclic 3, 5'-monophosphate." J. Am. Chem- Soc. 109, 40584064; Nerbonne, J.M., Richard, S., Nargeot, J. and Lester, H.A. (1984) "New 5 photoactivatable cyclic nucleotides produce intracellular jumps in cyclic AMP and cyclic GMP concentrations." Nature 301, 74-76; Neumann, J.M., Herv6, M., Debouzy, J.C., Guerra, F.1., Gouyette, C., Dupraz, B. and Huyny-Dinh, T. (1989) "Synthesis and transmembrane transport studies by NMR of a glucosyl phospholipid of thymidine." J. Am. Chem. Soc. I11, 4270.4277; Ohno, R., Tatsumi, N., Hirano, M., Imai, K. Mizoguchi, H., 10 Nakamura, T., Kosaka, M., Takatuski, K., Yamaya, T., Toyama K, Yoshida, T., Masaoka, T., Hashimoto, S., Ohshima, T., Kimura, L, Yamada, K. and Kimura, _ (1991) "Treatment of myclodysplastic syndromes with orally administered I -P-D-arabinouranosylcytosine -5' stearylphosphate." Oncology 48,451-455. Palomino, E., Kessle, D. and Horwitz, 1.P. (1989) "A dihydropyridine carrier system for sustained delivery of 2', 3' dideoxynucleosides to the 15 brain." . Med Chem. 32,22-625; Perkins, RM., Barney, S. Wittrock, R., Clark, P.H., Levin, R. Lambert, D.M., Petteway, S.R., Serafinowska, H.T., Bailey, S.M., Jackson, S., Harriden, M.R. Ashton, R, Sutton, D., Harvey, IJ. and Brown, A.G. (1993) "Activity of BRL47923 and its oral prodrug, SB203657A against a rauscher murine leukemia virus infection in mice." Antiviral Res. 20 (Suppl. 1). 84; Piantadosi, C., Marasco, CJ., Jr., Norris 20 Natschke, S.L., Meyer, K.L., Gumus, F., Sales, J.R., Ishaq, K.S., Kucera, L.S. lyer, N., Wallen, C.A., Piantadosi, S. and Modest, E.J. (1991) "Synthesis and evaluation of novel ether lipid nucleoside conjugates for anti-HIV-1 activity." J Med Chem. 34, 1408-1414; Pompon, A., Lefebvre, L, Imbach, .L., Kahn, S. and Farquhar, D. (1994). "Decomposition pathways of the mono- and bis(pivaloyloxymethyl) esters of azidothymidine-5'-monophosphate in cell 25 extract and in tissue culture medium; an application of the 'on-line ISRP-cleaning HPLC technique." Antiviral Chem Chemother. 5, 91-98; Postemark, T. (1974) "Cyclic AMP and cyclic GMP." Annu. Rev. Pharmacol. 14,23-33; Prisbe, E.J., Martin, J.C.M., McGhee, D.P.C., Barker, M.F., Smee, D.F. Duke, A.E., Matthews, T.R. and Verheyden, J.PJ. (1986) "Synthesis and antiherpes virus activity of phosphate an phosphonate derivatives of 9-[(1, 3 30 dihydroxy-2-propoxy)methyll guanine." J Med Chem. 29, 671-675; Pucch, F., Gosselin, G. Lefebvrc, I, Pompon, a., Aubertin, A.M. Dirn, and Imbach, .L. (1993) "Intracellular delivery 21 of nucleoside monophosphate through a reductase-mediated activation process." Anfivral Res. 22, 155-174; Pugaeva. V.P., Klochkeva, S.I., Mashbits, F.D. and Eizengad, R.S. (1969). "Toxicological assessment and health standard ratings for ethylene sulfide in the industrial atmosphere." Gig. Trf Prof Zabol. 14,47-48 (Chem. Abstr. 72,212); Robins, R.K. (1984) 5 "The potential ofnucleotide analogs as inhibitors of Retro viruses and tumors." Pharm. Res. 11-18; Rosowsky, A., Kim. S.H., Ross and J. Wick, M.M. (1982) "Lipophilic 5' (alkyl phosphate) esters of I -p-D-arabinofuranosyleytosine and its N 4 -acyl and 2.2'-anhydro 3'0-acyl derivatives as potential prodrugs." J Med. Chem. 25, 171-178; Ross, W. (1961) "Increased sensitivity of the walker turnout towards aromatic nitrogen mustards carrying 10 basic side chains following glucose pretreatment" Biochem. Pharm. 8, 235-240; Ryu, EK., Ross, R.J. Matsushita, T., MacCoss, M., Hong, C.I. and West, C.R. (1982). "Phospholipid nucleoside conjugates. 3. Synthesis and preliminary biological evaluation of I-p-. arabinofuranosyleytosine 5' diphosphate [-], 2-diacylglycrols." J. Med. Chem. 25, 1322 1329; Saffhill, R. and flume, W.J. (1986) "The degradation of 5-iododeoxyuridine and 5 15 brotnoetboxyuridine by serum from different sources and its consequences for the use of these compounds for incorporation into DNA." Chem. ioL. Interact. 57, 347-355; Saneyoshi, M., Morozumi, M., Kodama, K., Machida, J., Kuninaka, A. and Yoshino, H. 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An example of a useful phosphate prodrug group is the S-acyl-2-thioethyl 30 group, also referred to as "SATE". 22 11. Combination and Allernation Therapy it has been recognized that drug-resistani variants of HIV and BIV can emerge after prolonged treatment with an antiviral agent. Drug resistance most typically occurs by mutation of a gene that encodes for an enzyme used in viral replication, and most typically in 5 the case of HIV, reverse transcriptase, protease, or DNA polymerase, and in the case of HBV, DNA polymerase. Recently, it has been deronsti-ated that the efficacy of a drug against HIV infection can be prolonged, augmented, or restored by administering the compound in combination or alternation with a second, and perhaps third, antiviral compound that induces a different mutation from that caused by the principle drug. Alternatively, the 10 pharmacokinetics, biodistribution, or other parameter of the drug can be altered by such combination or alternation therapy. In general, combination therapy is typically preferred over alternation therapy because it induces multiple simultaneous stresses on the virus. The second antiviral agent for the treatment of HIV, in one embodiment, can be a reverse transcriptase inhibitor (a "RTr'), which can be either a synthetic nucleoside (a 15 "NRTI") or a non-nucleoside compound (a "NNRTI"). In an alternative embodiment, in the case of HIV, the second (or third) antiviral agent can be a protease inhibitor. In other embodiments, the second (or third) compound can be a pyrophosphate analog, or a fusion binding inhibitor A list compiling resistance data collected in vitro and in vivo for a number of antiviral compounds is found in Schinazi, et al, Mutations in retroviral genes associated 20 with drug resistance, International Antiviral News, 1997. Preferred compounds for combination or alternation therapy for the treatment of HBV include 3TC, FTC, L-FMAU, interferon, P-D-dioxolanyl-guanine (DXG), J-D-dioxolanyl 2,6-diaminopurine (DAPD), and P-D-dioxolanyl-6-chloropurine (ACP), faniciclovir, penciclovir, BMS-200475, bis porn PMEA (adefovir, dipivoxil); lobucavir, ganciclovir, and 25 ribavarin. Preferred examples of antiviral agents that can be used in combination or alternation with the compounds disclosed herein for HIV therapy include cis-2-hydroxymethyl-5-(5 tluorocytosin-1-yl)-1,3-oxathiolane (FTC); the (-)-enantiomer of 2-hydroxymethyl-5 (cytosin- I-yl)- 1,3-oxathiolane (3TC); carbovir, acyclovir, foscarnet, interferon, AZT, DDI, 30 DDC, D4T, CS-87 (3'-azido-2',3'-dideoxy-uridine), and P-D-dioxolane nucleosides such as 23 P-D-dioxolanyl-guanine (DXG), P-D-dioxolanvl-2,6-diaminopurine (DAPD), and S-D dioxolanyl-6-chloropurine (ACP), MKC-442 (6-benzyl-I.(ethoxymethyl)-5-isopropyl uracil. Preferred protease inhibitors include crixivan (Merck), nelfinavir (Agouron), ritonavir (Abbott), saquinavir (Roche), DMP-266 (Sustiva) and DMP-450 (DuPont Merck). 5 A more comprehensive list of compounds that can be administered in combination or alternation with any of the disclosed nucleosides include (I S,4R)-4-[2-amino-6-cyclopropyl mino)- 9 H-purin-9-yl]-2-cyclopentene-1 -methanol succinate ("1592", a carbovir analog; GlaxoWellcome); 3TC: (-)-B-L-2,3'-dideoxy-3'-thiacytidine (GlaxoWellcome); a-APA R18893: a-nitro-anilino-phenylacetamide; A-77003; C2 symmetry-based protease inhibitor 10 (Abbott); A-75925: C2 symmetry-based protease inhibitor (Abbott); AAP-BHAP: bisheteroarylpiperazine analog (Upjohn); ABT-538: C2 symmetry-based protease inhibitor (Abbott); AzddU:3'-azido-2',3'-dideoxyuridine; AZT: 3'-azido-3'-deoxythymidine (GlaxoWelicome); AZT-p-ddl: 3'-azido-3'-deoxythymidilyl-(5',5')-2',3'-dideoxyinosinic acid (lvax); BHAP: bisheteroarylpiperazine; BILA 1906: N-{ 1S-[[[3-[2S-{(1 ,1 15 dimethylethyl)amino]carbonyl)-4R-]3-pyridinylmethyl)thio]-1 -piperidinyll-2R-hydroxy-1S (phenylmethyl)propyljamino]carbonyl-2-methylpropyl }-2-quinolinecarboxamide (Bio Mega/Boehringer-Ingelheim); BILA 2185; N-(1,i -dimethylethyl)-l -[28-[[2-2,6 dimethyphenoxy)-1-oxoethyljamino]-2R-hydroxy-4-phenylbutyl]4R-pyridinylthio)-2 piperidinecarboxamide (BioMega/Boehringer-Ingelheim); BM+5 1.036: thiazolo 20 isoindolinone derivative; BMS 186,318: aninodiol derivative HIV-1 protease inhibitor (Bristol-Myers-Squibb); d4API: 9-[2,5-dihydro-5-(phosphonomethoxy)-2-furaneladenine (Gilead); d4C: 2',3'-didehydro-2',3'-dideoxycytidine; d4T; 2',3'-didehydro-3'-dcoxythymidine (Bristol-Myers-Squibb); ddC; 2',3'-dideoxycytidine (Roche); ddl: 2',3'-dideoxyinosine (Bristol-Myers-Squibb); DMP-266: a 1,4-dihydro-2H-3, I -benzoxazin-2-one; DMP-450: 25 {[4R-(4-a,5-a,6-b,7-b)]-hexahydro-5,6-bis(hydroxy)-1,3-bis(3-anino)phenyl]methyl)-4,7 bis(phenylmethyl)-2H-1,3-diazepin-2-one)-bistmesylate (Avid); DXG:(-)--D-dioxolane guanosine (Triangle); EBU-dM:5-ethyl-l-ethoxynethyl-6-(3,5-dimethylbenzyl)uracil;

E

EBU: 5-ethyl-1-etboxymethyl-6-benzyluracil; DS: dextran sulfate; E-EPSeU:1 (ethoxymethyl)-(6-phenylselenyl)-5-ethyluracil; E-EPU: -(ethoxymethyl)-(6-pheny-thio)-5 30 ethyluracil; FTC:-2',3'-dideoxy-5-fluoro-3'-thiacytidine (Triangle); HBY097:S-4 isopropoxycarbonyl-6-methoxy-3-(methylthio-methyl)-3,4-dihydroquinoxalin-2(1H)-thione; 24 HEPT: I-[(2-hydroxyethoxy)methyll-6-(phenylthio)thymine; HIV-l:human immunodeficiency virus type 1; 1M2763: 1,1'-(1,3-propanediyl)-bis-1,4,8, 1I etraazacyclotetradecane (Johnson Matthey); JM3100:1,1'-[1,4-phenylenebis-(methylene)] bis-1,4,8,11-tetraazacyclotetradecane(Johnson Matthey); KNI-272: (2S.3S)-3-amino-2. S hydroxy-4-phenylbutyric acid-containing tripeptide; L-697,593;5-ethyl-6-methyl-3-(2 phthalimido-ethyl)pyridin-2(IH)-one; L-735,524:hydroxy-aninopernane aide HIV-.1 protease inhibitor (Merck); L-697,661: 3-([(-4,7-dichloro-1,3-benzoxazol-2 yl)mcthyl)amino)-5-ethyl-6-methylpyridin -2(1 H)-one; L-FDDC:(-)-B-L-5-fluoro-2,3' dideoxycytidine; L-FDOC:(-)-B-L-5-fluoro-dioxoane cytosine; MKC442:6-benzyl-1 10 ethoxymethyl-5-isopropyluracil (I-EBU; Triangle/Mitsubishi); Nevirapine:11-cyclopropyl 5,11-dihydro-4-methyl-61-T-dipyridol[3,2-b:2',3'-ediaepin-6-one (Boehringer-Ingelhein); NSC648400:1-benzyloxymethyl-5-ethyl-6-(alpha-pyridylthio)uracil (E-BPTU); P9941: [2 pyridylacetyl-IlePbeAla-y(CHOH)]2 (Dupont Merck); PFA: phosphonoformatc (foscamet; Astra); PMEA: 9-(2-phosphonylxmethoxyethyl)adenine (Gilead); PMPA: (R)-9-(2 15 phosphonylmetboxypropyl)adenine (Gilead); Ro 31-8959: hydroxyethylanine derivative HIV- protease inhibitor (Roche); RPI-312: peptidyl protease inhibitor, 1-[(3s)-3-(n-alpha benzyloxycarbonyl)-1-asparginyl)-amino-2-hydroxy-4-phenylbutyryl}-n-tet-butyl--proline amide; 2720: 6-chloro-3,3-dimcthyl-4-(isopropenyoxycarbonyl)-3,4-dihydro-quinoxalin 2(IH)thione; SC-52151: hydroxyethylurea isostere protease inhibitor (Searle); SC-55389A: 20 hydroxyethyl-urea isostere protease inhibitor (Seaile); TIBO R82150: (+)-(5S)-4,5,6,7 tetrahydro-5-methyl-6-(3-methyl-2-butenyl)imidazo(4,5,1-jk][1,4]-benzodiazepin-2(11l) thione (Janssen); TIBO 82913: (+)-(5S)-4,5,6,7,-tetrahydro-9-chloro-5-methyl-6-(3-methyl 2-butenyl)imidazo[4,5,ljk]-[1,4]benzo-diazepin-2(1H)-thione (Janssen); TSAO-m3T:[2',5' bis-O-(tert-butyldimethylsilyl)-3'-spiro-5'-(4'-amino-1l',2'-oxathiole-2',2'-dioxide)]-b-D 25 pentofLiranosyl-N3-methylthymine; U90152:1-[3-[(I-methylethyl)-amino]-2-pyridinyl-4-[[5 [(nethylsulphonyl)-amino]-IH -indol-2y1]carbonyl]piperazine; UC: thiocarboxanilide derivatives (Uniroyal); UC-781 =N-[4-chloro-3-(3-methyl-2-butenyloxy)phenyll-2-methyl-3 furancarbothioamide; UC -82 =N-[4-chloro-3-(3-methyl-2-butenyloxy)phenylj-2-methyl-3 thiophenecarbothioamide; VB 11,328: hydroxyethyl-sulpbonamide protcase inhibitor 30 (Vertex); VX-478:hydroxyethylsulphonamide protease inhibitor (Vertex); XM 323: cyclic urea protease inhibitor (Dupont Merck). 25 Combination Therapy for the Treatment of Proliferative Conditions In another embodiment the compounds, when used as an antiproliferative, can be administered in combination with another compound that increases the effectiveness of the therapy, including but not limited to an antifolate, a 5-fluoropyrimidine (including 5 5 fluorouracil), a cytidine analogue such as j-L-1,3-dioxolanyl cytidine or I-L-1,3-dioxolanyl 5-fluorocytidine, antimetabolites (including purine antimetabolites, cytarabine, fudarabine, floxuridinc, 6-mercaptopurine, methotrexatc, and 6-thioguanine), hydroxyurea, mitotic inhibitors (including CPT-1 1, Etoposide (VP-21), taxol, and vinca alkaloids such as vincristine and vinblastine, an alkylating agent (including but not limited to busulfan, 10 chlorambucil, cyclophosphamide, ifofamide, mechlorethamine, melphalan, and thiotepa), nonclassical alkylating agents, platinum containing compounds, bleomycin, an anti-tumor antibiotic, an anthracycline such as doxorubicin and dannomycin, an anthracenedione, topoisomerase 11 inhibitors, hormonal agents (including but not limited to corticosteroids (dexamethasone, prednisone, and methylprednisone), androgens such as fluoxynesterone and 15 methyltestosterone, estrogens such as diethylstilbesterol, antiestrogens such as tamoxifen, LHRH analogues such as leuprolide, antiandrogens such as flutarnide, aminoglutethimide, megestrol acetate, and medroxyprogesterone), asparaginase, carmustinc, lomustine, hexamethyl-melamine, dacarbazine, miotane. streptozocin, cisplatin, carboplatin, levamasole, and leucovorirt The compounds of the present invention can also be used in 20 combination with enzyme therapy agents and immune system modulators such as an interferon, interleukin, tumor necrosis factor, macrophage colony-stimulating factor and colony stimulating factor. HI. Process for the Preparation of Active Compounds In one embodiment of the invention, a diastereoselective reaction for effecting the 25 introduction of fluorine into the sugar portion of novel nucleoside analogs is provided. This synthesis can be used to make both purine and pyrimidine derivatives. The key step in the synthetic route is the fluorination of a chiral, non-carbohydrate sugar ring precursor (4S)-5-(protected-oxy)-pentan-4-olide, for example, (4S)-5-(t-butyldiphenylsiloxy) pentan-4-olide 4 using an electrophilic fluorine source, including, but not limited to, 30 N-fluoro-(bis)benzenesulfonimide 5. This relatively new class ofN-fluorosulfonimide reagents was originally developed by B-arnette in 1984 and since then has seen much 26 refinement and use as a convenient and highly reactive source of electrophilic fluorine (Barnette, W. - J. Am. Chem. Soc. 1984, 106,452.; Davis, F. A.; Han; W., Murphy, C. K. J. Org. Chem. 1995,60,4730; Snieckus, V.; Beaulieu, F.; Mohri, K.; Han, W.; Murphy, C. K.; Davis, F. A. Tetrahedron Lett. 1994, 35(21), 3465). Most often, these reagents are used to 5 deliver fluorine to nucleophiles such as enolates and metalated aromatics (Davis, F. A.; Han; W., Murphy, C. K. J. Org. Chem. 1995, 60,4730). Specifically, N-fluoro-(bis)benzenesulfonimide (NFSi) is an air stable, easily handled solid with sufficient steric bulk to stereoselectively fluorinate the enolate of silyl-protected lactone 4. As a nonliniting example of this process, the synthesis of fluorolactone 6 and its use as a common 10 intermediate in the synthesis of a number of novel a-2'-fiuoro nucleosides is described in detail below. Given this description, one of ordinary skill can routinely modify the process as desired to accomplish a desired objective and to prepare a compound of interest. Any source of electrophilic fluorine can be used that fluorinates the precursor (43)-5-(protected-oxy)-pentan-4-olide, for example, (45)-5-(t-butyl 15 diphenylsiloxy)-pentan-4-olide. Alternative sources of electrophilic fluorine include N fluorosulfams (Differding, et al, Tet. Lett. Vol. 29, No. 47 pp 6087-6090 (1988); Chemical Reviews, 1992, Vol 92, No. 4 (517)), N-fluoro-O-benzenedisulfonimide (Tel. Let. Vol. 35, pages 3456-3468 (1994), Tet. Lett. Vol 35. No. 20, pages 3263-3266 (1994)); J. Org. Chem. 1995, 60,4730-4737), 1-fluoroethene and synthetic equivalents (Matthews, Tet. Lett. Vol. 35, 20 No. 7, pages 1027-1030 (1994); Accufluor fluorinating agents sold by Allied Signal, Inc., Buffalo Research Laboratory, Buffalo, New York (NFTh (1-fluoro-4-hydroxy-1,4-diazoa bicyclo[2.2.2]octane bis(tetrafluoroborate)), NFPy (N-fluaropyridinium pyridine heptafluorodiborate), and NFSi (N-fluorobenzenesulfonimide); clectrophilic fluorinating reagents sold by Aldrich Chemical Company, Inc., including N-fluoropyridinium salts ((1 25 fluoro-2,4,6-trimethylpyridinium triflate, 3,5-dichloro-1 -fluoropyridiniumn triflate, 1 fluoropyridiniun triflate, 1-fluoropyridinium tetrafluoroborate, and I -fluoropyridinium pyridine heptafluorodiborate) see also J. Am. Chem. Soc., Vol 112, No. 23 1990); N fluorosulfonimides and-arnides (N-fluoro-N-methyl-p-toluenesulfonarnide, N-fluoro-N propyl-p-toluenesulfonamide, and N-fluorobenzenesulfonimide) ; N-fluoro-quinuclidinium 30 fluoride (1 Chem. Soc. Perkin Trans (1988, 2805-2811); perfluoro-2,3,4,5 tetrahydropyridine and perfluoro-( 1 -methylpyrrolidine), Banks, Cheng, and Haszeldine, 27 Heerocyclic Polyfluoro-Compoun Part I (1964); 1-fluoro-2-pyridone. J. Org. Chem., 1983 48, 761-762; quaternary stereogenic centers possessing a fluorine atom (J. Chen. Soc. Perkin Trans. pages 221-227 (1992)); N-fluoro-2,4,6-pyridinium triflate, Shimizu, Tetrahedron Vol 50(2), pages 487-495 (1994); N-fluoropyridinium pyridine heptafluorodiboratc, J. Org. 5 Chem. 1991, 56, 5962-5964; Umernoto, et al., Bull. Chem. Soc. Jpn., 64 1081-1092 (1991); N-fluoroperfluoroalkylsulfonimides, J. Am. Chem. Soc., 1987, 109, 7194-7196; Purrington, ct al., Lewis Acid Mediated Fluorinations of Aromatic Substrates, J Org. Chem. 1991, 56, 142-145. A significant advantage of this methodology is the ability to access separately either 10 the "natural" (ia) D or the "unnatural" (1b) L enantiomer of the nucleosides by appropriate choice of L - or D - glutamic acid starting material, respectively. R21 R, N R,1 .O i5 . O HO N O RI = H, CFa, F

R

2 = OH. NH 2 , NHAc FF 15 lb Lactone 4 was synthesized by the route shown in Scheme I from L-glutamic 20 acid as described by Ravid et al. (Tetrahedron 1978, 34, 1449) and Taniguchi et al. (Tetrahedron 1974, 30,3547). Scheme 1 H- COOH HOOC BDPSO 25 H 0 < XNH NaNO2 / HCI O 50-70% 2 3 4 The enolate of lactone 4, prepared at -78 *C with LiHMDS in THF, is known to be stable. Several syntheses using this enolate have been performed, including addition of 30 electrophiles such as diphenyldiselenide, diphenyldisulfide, and alkyl halides in high yield (Iiotta, D. C-; Wilson, L. J. Tetrahedron Lett. 1990, 3/(13), 1815; Chu, C. K.; Babu, I. R. 28 Beach. J. W.;Ahn, S. K., Huang, H.; Jeong, L. S.; Lee. S. J. J. Org. Chem., 1990, 55 1418; Kawakami, H.; Ebata, T.; Koseki. K.; Matsushita, H.; Naoi, Y.; Ioh, K. Chem. Le. 1990, 1459). However, addition of a THF solution of 5 to the enolate of 4 gave poor yields of the desired monofluorinaled product 6. Numerous by-products were formed including what was 5 surmised to be a difluorinated lactone that is inseparable from other impurities. For this reason, the order of addition of the reagents was altered such that lactone 4 and NFSi 5 were dissolved together in THF and cooled to -78D C. Slow addition of LiHMDS resulted in a reaction yielding 6 as the only product in addition to a small amount of unreacted starting material (eq 1). 10 Equation I F Ol, ,N, . O T B D P S O -OD S 0 ci TEPS-y 15 45(') 2. LIMMDS Fluorolactone 6 could be obtained in 50 - 70 % yield after silica gel column chromatography and crystallization. This reaction yielded a single diastereomer of 6, presumably due to the interaction of the sterically bulky TBDPS group and the bulky 20 fluorinating reagent S. Identification of fluorolactone 6 as the a or "down" fluoro isomer was accomplished by comparison to previously published NMR data and by X-ray crystal structure determination of its enantiomer 20. Lactone 6 was transformed into the anoineric acetate 8 as shown in Scheme 2. It is of interest to note that lactol 7 exists exclusively as the P anomer and that acetate 8 shows no 25 detectable a anomer by NMR, as reported by Niihata et al. (Bull. Chem. Soc. Jpn. 1995; 68, 1509). 29 Scheme 2 6 DIBALH TBDPSO-yOH Ac 2 O, DMAP TBDPSO OVAC F F 7 10 Coupling of 8 with silylated pyrimidine bases was performed by standard Vorbmggen methodology (Tetrahedron Lett. 1978, 15, 1339) using TMS triflate as the Lewis acid. Alternatively, any other Lewis acid known to be useful to condense a base with a carbohydrate to form a nucleoside can be used, including tin chloride, titanium chloride, and other tin or titanium compounds. A number of bases were successfully coupled in high yields 15 ranging frotm 72% - 100 % after column chromatography (eq 2, Table 1). Equation 2 F2TMS F, R, TBDPSOn/AC N OTMS TBDPS (2) 20 TMS ifte F 72-l00% B fra- 2:1 9.10,11,12,13 Table 1. Glycosylation of Substituted Pyrimidines with 8 Cmpd. R, R yield 25 9 F OH 87% 10 F NH2 99% 11 H NHAc 91% 12 14 NH, 72% 13 CH., O 89% 30 30 Proton NMR indicated that the ratio of P to a nucleoside anomers was approximately 2:1 in all cases. The silyl protected nucleosides could not be resolved by column chromatography into the separate anomers. However, after deprotection of the 5'oxygcn with N1 4 F in methanol (eq 3), the a and P anomers could be readily separated and the results 5 arc summarized in Table 2. Equation 3

F

2 0 4b T8D0$ N~ Nl- 4 F,/M.OH HObjJ 50-70% F F :a - 2:1 14a, 15a, 16a, 17a, 1Ba 14b, 1Sb, 1cb,17b, Ib 9, 10, 11, 12. 13 Table 2. Deprotection of Nucleosides 15 Ra R 2 yield b yield F OH 14a 19% 14b 48% F NH 2 15a 27% I5b 51% H NIIAc 16a 17% 16b 31% H NH 2 17a - 17b 20 CH, OH 18a 12% 18b 33% The classification ofthe free nucleosides as a or P was based on the chemical shift of the annmeric proton (Table 3) and on the polarity of the nucleosides as observed by thin layer chromatography. A trend for all of the c / pairs of free nucleosides was observed in that the 25 less polar compound of the two had an anomeric proton chemical shift that was notably upfield from that of the more polar compound. 31 Table 3. Anomeric Proton Chemical Shift (ppm) Cmpd. a p 14 a,b 6.11 5.89 15 a,b 6.08 5.92 5 16 a,b 6.09 5.90 17 a,b 6.05 5.92 18 a,b 6.11 5.93 The correlation between anomeric proton chemical shift and absolute structure was 10 verified by comparison of I8a (Niihata, S.; Ebata, T.; Kawakami, H.; Matsushida, H. Bull. Chen. Soc. Jpn. 1995, 68, 1509) and 18h (Aerschot, A. V.; Herdewijn, P.; Balzarini, J.; Pauwels, R; De Clercq, E. J. Med Chem. 1989, 32, 1743) with previously published spectmal data and through X-ray crystal structure determination of 14b and 15b. This finding is the opposite of the usual trend for nucleosides in which the a anomer is normally the less polar of 15 the two. Presumably, in the "down" 2' - fluorinated nucleosides, the strong dipole of the C-F bond opposes the C-N anomeric bond dipole in the P isomer and reduces the overall molecular dipole. Conversely, the a anomer has a geometry that allows reinforcement of the molecular dipole through the addition of the C-F and C-N bond dipoles. Thus, the a anomer is more polar than the P anomer in the case of a-2'-fluoro nucleosides. 20 The a and P anomers 172 and 17b could not be separated by column chromatography because the free amino group caused the nuclcosides to streak on silica gel. Therefore, it was necessary to use M-acctylcytosine to prepare II and then resolve 16a and 16b. The acetyl group was removed quantitatively with a saturated solution of ammonia in methanol in order to obtain separated 17a and 17b. When 5-fluorocytosine was used as the base 25 (compound 10), the anomers 15a and 15b were easily separated and no streaking on silica gel was observed. Of the ten nucleosides listed in Table 2, it appears that only 17b (Martin, J. A; Bushnell, D. J.; Duncan, I. B.; Dunsdon, S. J.; Hall, M. J.; Machin, P. J.; Merrett, J. H.; Parks, K E. B.; Roberts, N. A.; Thomas, G. J.; Galpin. S. A.; Kinchington, D. J Med. 30 Chem- 1990, 33(8), 2137; Zenchoff, G. B.; Sun, R.; Okabe, M. J Org. Chem. 1991, 56, 32 4392). Ia (Niihata. S.; EbataT.; Kawakami, H.; Marsushida, H. Bul. Chem. Soc.Jpn. 1995, 68, 1509). and l8b (Aerschot, A. V.; Herdewijn, P.; Balzarini, .; Pauwels, R.; De Clercq, E. . Med Chem. 1989. 32, 1743) have been synthesized previously. They, like the numerous known 2'- P or "up" fluoro nucleoside analogs" have been synthesized front 5 natural precursors (i.e, they are in the P-D configuration), It appears that no P-L-2'-fluoro ribofuranosyl nucleosides have been identified in the literature prior to this invention. Fluorine is usually introduced into these molecules through nucleophilic attack on an anhydro-nucleoside (Mengel, R.; Guschlbauer, W. Angew Chem., lIni Ed Engl. 1978, 17, 525) or through replacement and inversion of a stereochemically fixed hydroxyl group with 10 diethylaminosulfur trifluoride (DAST) (Herdewijn, P.; Aerschot, A. V.; Kerremans, L. Nucleosides Nucleotides 1989, 8(1), 65). One advantage of the present methodology is that no hydroxyl group is needed for fluorine introduction. Thus, the process is not limited to natural nucleosides or sugars as starting materials, and provides an easy to access the unnatural enantiomers of the 2'-fluoro nucloosides. 15 Accordingly, several unnatural nucleosides were synthesized using this synthetic route with D-glutamic acid 19 as the starting material (Scheme 3). The sugar ring precursor 20 was fluorinated in the manner described above and coupled with various silylated bases (Table 4). 33 Scheme 3 TEDPSO DLBALJ-{ iEDPSO D-Glutamic acid $ F to 212

AC

2 O TEDPSO .1t g I 2 A c2O A C sily laied base DMAP 0 TM$tiflate TDPSO F tMJr~ n 22 O 23,24,25 1 0 RNF fl F 26a, 27a. 283 25b, 27b, 22b 15 Table 4. Yields of Unnatural Nucleoside Analogs cmpd. yield Ra yield b yield (23-25) 20 23 87% CH OH 26a 24% 26b 61% 24 85% F OH 27a 35% 27b 51% 25 99% F NH 2 28a 34% 28b 52% 34 Scheme 4 -EDPSO OH 1. Methanesulfonyl chloride TBDPSO 2. triethyl amine 5 F F 7 29 Successful synthesis of 29, as shown in Scheme 4, allows access to two categories of 10 nucleosides. The first is the class of compounds known as 2*,3*-dideoxy-2',3'-didehydro-2-2' fluoro-nucleosides, 30, and the second is the "up"-fluoro or arabino analogs, 31, of the nucleosides described in Scheme 5 below. Sebeme 5 15

R

2 HO R HNR K HR, = H, CH 3 , F, CI, etc. O F R 2 = OH, NH 2 20 F 30 31 25 Compounds 30 and 31 may be synthesized from a common intermediate 32, which may be accessed through selenylation of fluorogycal 29. 35 Scheme 6 1. PhSeCj M TBDPS 3 2 .siylaea base H R 5 F ,---- F 0 29 SePh 32 Raney Ni (l (exidaOn) I7D 10 R2 HO HO N H F 31 30 15 Selenylated compound 32 may be transformed into the "up" fluoro analog 31 through reduction with Raney nickel. Alternatively, oxidation of the selenide 32 with NaTO 4 or hydrogen peroxide followed by thermal elimination of the selenoxide intermediate lead to 30. Both of these transformations on the unfluorinated systems are well documented and have been reported (Wurster, J. A-; Ph.D. Thesis, Emory University, 1995; Wilson, L. J.; Ph.D. 20 Thesis, Emory University, 1992). In addition, the synthesis of the enantiomers of nucleosides 30 and 31 is also possible since they arise flom the enantiomer of 29. An alternative route for the preparation of compounds of the type represented by 30, the 2 ',3'-dideoxy-2',3'-didhydro-2'-flouro-nucleosides, is shown in Scheme 7. This route 25 provides simple, direct access to this class of compounds utilizing a wide range of silylated bases and has been successfully completed. 36 Scheme 7 Ro- I. TmS.ci RO 2. LOA RDVO"%a 1. DIRAL--. CH,01 RO-

V

7 O 3. Phi~eBr _02. A-20, DMAP V>O',AC R FTBDPS THF. .75C 71% 6 F 88% Soft SePh at7 66 66% silytated base, TMSOTI 10 N 0 I N H F "M' O H F % S&Ph 30 3B Formation of silyl ketene acetal from 6 allows for the stereosclective addition of phenyl Is selenium bromide to generate compound 36 as a single isomer. Reduction and acetylation of this compound proceeds smoothly and in high yield over the two steps to 37. The a orientation of the phenyl selenyl group allows for stereoselection in the subsequent glycosylation step, and synthesis of the P isomer of the nucleoside 38 is accomplished in good yield. Compound 38 may be oxidized with hydrogen peroxide in dichloromethane to 20 yield the elimination product 39, but in our experience, it was merely necessary to adsorb 38 onto silica gel and allow to stand for several hours, after which time 39 could be eluted from a plug column in nearly quantitative yield. Removal of the protected group from 39 to obtain the final compound 30 was performed as before and resulted in a good yield (81%) of product nucleoside. 25 Scheme 8 N RIN TMPSO THDPSO F H N O 30 OH HO He F 30 -'i 21 33 34 35 37 The same series of chemical transformations that were used for the synthesis of 30 and 31 may also be used for the synthesis of 34 and 35. Experimental Section 5 General Procedures: N - Fluoro - (bis)benzenesulfonimide 5 was obtained from Allied Signal, and was used without further purification. All other reagents were obtained from Aldrich Chemical Company and were used without further purification. Melting points were determined on a Thomas Hoover capillary melting point apparatus and are uncorrected. IR spectra were 10 obtained on a Nicolet Impact 400 FT-IR spectrometer. 'H NMR and "C NMR spectra were rmorded on either NT - 360 or Varian 400 MHz spectrometer. TLC plates were silica gel 60 F1 (0.25 nun thickness) purchased from EM Science. Flash chromatography was carried out with silica gel 60 (230-400 mesh ASTM) from EM Science. All reactions were performed in flame-dried glassware under an atmosphere of dry argon. Solvents were removed by rotary 15 evaporation. Elemental analyses were performed by Atlantic Microlab, Inc, Atlanta, GA, (2S,4R) -5 - (r -butyldiphenylsiloxy) - 2 - fluoropentan - 4 - olide (20). To a flask was added (4R) - 5 - (I -butyldiphenylsiloxy) - pentan - 4 - olide (20.0 g, 0.0564 mol, 1.0 eq.) and N - fluoro - (bis)benzenesulfonimide (NFSi) 5 (17.80 g, 0.0564 mol, 1.0 eq.) in 250 mL of anhydrous THF The solution was cooled to -78 *C and 68.0 mL (0.0680 mol, 1.2 eq.) of a 20 1.0 M solution of LiHMDS in THF was added dropwise over a period of I hr. This was allowed to stir at -78 "C for an Additional 2 his. and was then warmed to room temperature to stir for one hour. After completion, the reaction was quenched with 10 mL of saturated

NH

4 Cl solution, The mixture was diluted with three volumes of diethyl ether and was poured onto an equal volume of saturated NaHCO 3 . The organic layer was washed a second time 25 with saturated NaHCO 3 and once with saturated NaCl. The organic layer was dried over MgSO, filtered, and concentrated to a light yellow oil. The oil was purified by silica gel column chromatograpy using a 30 % diethyl ether /70 % hexanes solvent system. The resultant white solid was then crystallized from hot hexanes to yield 13.04 g (62 % yield) of a transparent crystalline solid: Rr (30 % diethyl cther / 70 % hexanes)= 0.26; rmp 115-116 *C. 30 'H NMR (360 MHz, CDCI 3 ) d 7.63 - 7.60 (mo, 4H), 7.45 - 7.35 (in, 6H), 5.49 (dt, J 52.9 and 7 9 Hz, IH), 4,69 (d, J = 9.36 Hz, 111), 3.91 (d, J - 11.5 Hz, 111), 3.60 (d, I = 11.5 Hz, 1N), 38 2.72 - 2.40 (m, 2-), 1.05 (s. 911); 'C NMR (100 MHz, CDCl) d 172.1 (d, J = 20.5 Hz), 135.5. 135.4, 132-3, 131.7, 130.1. 128.0, 127.9, 85.6 (d, J - 186.6 Hz), 77.3 (d, j = 5.3 Hz), 65.0, 31.8 (d, I= 20.5 Hz), 26.7, 19.1; IR (thin film) 2958, 1796, 1252, 1192, liii, 1016 cm; HRMS calculated for [M + Li] C-H 25 OFSiLi : 379.1717. Found: 379.1713. Anal. Calc. 5 CHAFFS : C, 67.71; H, 6.76. Found: C, 67.72; H, 6.78. 5 - 0 - (f -butyldiphenylsilyl) - 2,3 - dideoxy -2 - fluoro - (L) - erythron - pentofuranose (21). To a flask was added lactone 20 (12.12 g, 0.0325 mol, 1.0 eq.) and 240 mL of anhydrous THF. The solution was cooled to - 78 "C and 65 mL (0.065 moi, 2-0 eq.) of a 1.0 M solution of DIBALH in hexanes was added dropwise over a period of 30 mi. This was 10 allowed to stir at - 78 "C for 3 his., after which time the reaction was quenched by the slow addition of 2.93 mL (0.163 mol, 5.0 eq.) of water. The reaction was allowed to warm to room temperature and stir for I hr., after which time a clear gelatinous solid formed throughout the entire flask. The reaction mixture was diluted with two volumes of diethyl ether and was poured onto an 15 equal volume of saturated aqueous sodium potassium tartrate solution in an Erlenneyer flask. This was stirred for 20 min. until the emulsion had broken. The organic layer was separated and the aqueous layer was extracted three times with 250 mL of diethyl ether. The combined organic layers were dried over MgSO,, filtered, and concentrated to a light yellow oil. The product was purified by silica gel column chromatography using a 6:1 hexanes / ethyl acetate 20 solvent system. The resulting clear oil was crystallized from boiling hexanes to give 11.98 g (98 % yield) of a white crystalline solid: R, (30 % diethyl ether /70 % hexanes) =0.33; mp 66-67 *C. 'H NMR (360 MHz, CDCIl) d 7.68 - 7.66 (m, 4H), 7.55 - 7.38 (m, 6H), 5.39 (t, J 7.6 Hz, 11-1), 4.99 (dd, J= 52.2 and 4.3 Hz, IH), 4.52 (m, 1IH), 3.88 (dd. J= 10.8 and 2.5 Hz, 11), 3.65 (d, J = 7.9 Hz, IH), 3.49 (dd. J: 7.9 and 1.8 Hz, 1H), 2.44 - 2.07 (in, 2H), 1.07 25 (s, 9H); "C NMR (100 MHz, CDCl,) d 135.7, 135.5, 132.2, 132.1. 130.2, 130.0, 129.8, 127.9, 127.7, 99.8 (d, J = 31.1 Hz), 96.6 (d, J= 178.3 Hz), 79.4, 64.8, 29.9 (d, J= 212 Hz), 26.8, 19.2; IR (thin film) 3423, 2932, 1474, 1362, 1113 cma; HRMS calculated for [M + Li] C H 27

O

2 FSiLi : 381.1874. Found: 381.1877. Anal. Calc. C2,H1OFSi : C, 67.35; H, 7.27. Found: C, 67.42; H, 7.31. 30 1 -O - Acetyl - 5 - 0 - (f -butyldiphenylsilyl) - 2,3 - dideoxy - 2 - fluoro - (L) - erythron pentofuranose (22). To a flask was added lactol 21 (8.50 g, 0.0227 mol, 1.0 eq.) and 170 39 mL of anhydrous CH 3 Cl,. Then, DMAP (0.277 g, 0.00277 mol, 0.1 eq.) and acetic anhydride (13.5 mL, 0.143 mot, 63 eq.) were added and stirred at room temperature overnight. Upon completion, the reaction was poured onto saturated NaHCO, solution. The organic layer was separated, and the aqueous layer was extracted three times with chloroform. The combined 5 organic layers were dried over MgSO 4 , filtered, and the solvent removed to yield a light yellow oil. The oil was purified by silica gel column chromatography using an 8:1 hexanes / ethyl acetate solvent system to give 9.85 g (99 % yield) of a clear colorless oih R (30 % diethyl ether / 70 % hexanes) = 0.44; 'H NMR (360 MHz, CDCI) d 7.69 - 7.67 (m, 4H), 7A3 - 7.38 (m, 6H), 6.30 (d, I 1A0 Hz, 1H), 5.06 (d, J =54.9 Hz, 18), 4.53 (m, 11), 3.81 10 (dd, J= 10.8 and 4.3 Hz, lH), 3.72 (dd, J 10.8 and 4.3 Hz, 1H), 2.38 - 2.12 (m, 2H), 1.89(s, 3H), 1.07 (s, 911); "C NMR (100 MHz, CDC,) d 169.4, 135.6, 135.5, 133.2, 133.1, 129.8, 129.7, 127-8, 127.7, 99.3 (d, J -34. liz), 95.5(d, 3= 178.2 Hz), 81.4, 65.3, 31.6 (d,1=20.5 Hz),26.8, 21.1, 19.3; IR (thin film) 3074,2860, 1750, 1589, 1229,1113 cr'; HRMS calculated for [M - OCOCH,} C 21

H

4 0 2 FSi: 357.1686. Found: 357.1695. Anal. Cale.. 15 C)H2,0 4 FSi : C, 66.32; H, 7.02. Found: C, 66.30; H, 7.04. Representative procedure for the coupling of a silylated base with 22: (L) - 5' - 0 (I -butyldiphenylsilyl)-2',3-dideoxy-2'-fluoro-5-fluorocytidine (25). To a flask equipped with a short-path distillation head was added 5 - fluorocytosine (2.01 g, 15.6 mmol, 5.0 eq), 35 mL of 1,1,1,3,3,3 - hexamethyldisilazane, and a catalytic amount (- mg) of (NH) 2

SO

4 . 20 The white suspension was heated to boiling for 1 hr. until the base was silylated and reaction was a clear solution. The excess HMDS was distilled off and the oily residue that remained was placed under vacuum for I hr. to remove the last traces of HMDS. A white solid resulted which was dissolved, under argon, in 5 mL of anhydrous 1,2 - dichloroethane. To this clear solution was added a solution of acetate 22 (1.30 g, 3.12 mmol, 1.0 eq.) in 5 mL of anhydrous 25 1,2 - dichloroethane. To this was added, at room temperature, trimethylsilyl trifluoromethanesulfonate (3.32 mL, 17.2 mmol, 5.5 eq.). The reaction was monitored by TLC (10 % methanol /90 % CHZCZ) and was observed to be complete in 4 hrs. The reaction mixture was poured onto saturated NaHCO,. The organic layer was then separated, and the aqueous layer was extracted three times with chloroform. The combined organic layers were 30 dried over MgSO 4 , filtered, and the solvent removed to yield a white foam. The compound was purified by silica gel column chromatography using a gradient solvent system from 100 40 % CHI,Cl, to 10 % methanol in CH.Cl.- The compound was isolated as 1.51 g (99 % yield) of a white foam: mixture of anomers Rr (100% EtOAc) = 0.36; mp 74-80 *C. 'H NMR (400 MHz, CDC 3 ) d 8.84 (bs, 1H), 8.04 (d, J =6.4 Hz, 0.67H), 7.67 - 7-63 (m. 411), 7.51 - 7.39 (m, 6.33H), 6.11 (d, 3= 20 Hz, 0.33H), 5.98 (d, J -16.4 Hz, 0.67H), 5.88 (bs. 1H), 5.41 (d, J 5 = 52.4 Hz, 0.33H), 5.23 (dd, J = 50.4 and 4 Hz, 0.67H), 4.56 (m, 0.3311), 4.45 (m, 0.67H), 4.23 (dd, J = 12.0 and 1.6 Hz. 0.67H), 3.89 (dd, J = 11.2 and 3.2 Hz, 0.33 H), 3.74 - 3.66 (m, 1H), 2.45 - 1.96 (m. 2H), 1.09 (s, 611), 1.06 (s, 3H); "C NMR (100 MHz, CDC1 3 ) d 158.6 (d, J= 14.4 Hz), 158.4 (d, I= 14.4 Hz), 153.9, 153.8, 136.6 (d, J= 240.5 Hz), 136.3 (d, J= 239.7 Hz), 135.6. 135.56, 135.5, 135-4, 133.1, 132.9, 132.5, 132.4, 130.1, 130.0, 129.9, 127.9, 10 127.8, 125.8 (d, Ja 33.4 Hz), 124.6 (d, J - 32.6 Hz), 96.5 (d, 3 182.0 Hz), 91.7 (d, J= 185.1), 90.7 (d, J=35.6 Hz), 87.7 (d, 3 = 15.2 Hz), 81.5, 79.5, 64.9, 63.0, 33.5 (d, J= 20.5 Hz), 30.6 (d, 3 20.4 Hz), 26.9, 26.8. 19.22, 19.18; IR (thin film) 3300, 2960, 1682, 1608, 1513, 1109 cm '; HRMS calculated for [M + Li] C 25 HiN,0 3 SiFzLi: 492.2106. Found:492.2085. Anal, Calc. C,,H2N 3 0 3 SiF 2 - 1/2 H20: C, 60.71; H, 6.11; N. 8.50. Found: 15 C, 60.67; H, 6.03 ; N, 8.44. Representative Procedure for the deprotection of silyl-protected nucleosides: a- and p (L) - 2',3' - dideoxy -2' - fluoro -5 - fluoro cytidine (2Sa and 2Mb): Nucleoside 25(1.098 g, 2.26 nnol, 1.0 eq.) was dissolved in 15 mL of methanol to which was added ammonium fluoride (0.838 g, 22.6 mmol, 10.0 eq.). This was stirred vigorously for 24 hrs., after which 20 time TLC (15 % ethanol / 85 % ethyl acetate) revealed that the reaction was complete. The reaction mixture was diluted with three volumes of ethyl acetate and was filtered through a small (1 cm) silica gel plug. The plug was rinsed with 200 mL of 15 % ethanol /85 % ethyl acetate solution and the solvent was removed to yield a white foam. The compound was purified by silica gel column chromatography using a 15 % ethanol /85 % ethyl acetate 25 solvent system which also effected the separation of the a and t anomers. The yield of a as a white foam was 0.190 g (0.768 mmol, 34 % yield) and the yield of P as a white foam was 0.290 g (L17 nunol, 52 % yield): (28a) Rr (15 % ltOH , 85 % ETOAc)=0.22; mp 199-203 "C (dec.), 'H NMR (400 MHz, CD2OD) d 7.78 (d, J 6.8 H4z, 1H), 6.07 (d, J = 19.2 Hz, I H), 5.37 (d, I . 54.0 Hz, 1H), 4.60 (m, 111), 3.80 (dd, J = 12.0 and 3.2 Hz, I H), 3.56 (dd, J = 30 12.4 and 4.4 Hz, I H), 2.40-2.00 (in, 214); "C NMR (100 MHz,DMSO - d6) d 157.7 (d, J= 13.6 Hz), 153.2, 135.9 (d, J= 239.0 Hz), 126.2 (d, 3= 31.1 Hz), 92.4 (d, J = 183.6 Hz), 86.7 41 (d, J 15.2 Hz), 79.6, 62.7, 33.3 (d, J= 20.5 Hz); IR (KBr) 3343, 3100, 1683, 1517, 1104 cm''; HRMS calculated for [M +Li) C,H 1 NO,F.Li : 254.0929. Found: 254.0919. Anal. Calc.. C,H 1 1 N,0 3

F

2 1/2 H20: C, 42.19; H, 4.72; N, 16.40. Found: C, 42.44; 1-1, 4.56; N, 16.56. (28b) Rr (15 % EtOH ,85 % EtOAc)= 037; mp 182-186'C (dec.). 'H NMR (400 5 MHz, DMSO- d) d 8.32 (d, J= 7.6 Hz, I1H), 7.79 (bs, 11), 7.53 (bs, 181), 5.81 (d, J= 16.8 Hz, 1H), 5.37 (t, J= 4.8 Hz), 5.18 (dd, J = 51.6 and 3.2 Hz, IH), 4.32 (m, 111), 3.88 (dd, J= 12.0 and 2.8 Hz, IH), 3.59 (dd, ] = 12.4 and 2.4 Hz, IH), 2.20-1.99 (m, 2H); '"C NMR (100 MHz, DMSO - d6) d 157.7 (d, ]= 13.7 Hz), 153.2, 136.1 (d, J = 237.4 Hz), 125.3 (d, J M 33A Hz), 97.3 (d, J = 176.8 Hz), 89.9 (d, J - 35.7 Hz), 81.6, 60.2,30.3 (d,);-- 19.7 Hz); IR (KBr) 10 3487,2948, 1678, 1509, 1122 cm4; HRMS calculated for [M + Li] C,H 1

N

3 0 3

F

2 Li : 254.0929. Found: 254.0935. Anal. Calc.. C,H 1 N0,F 2 : C, 43.73; H, 4.49; N, 17.00. Found: C, 43.69; H, 4.53; N, 16.92. (D) - 5' - 0 - ( -butyldiphenylsilyl) - 2',3'-dideoxy-2'-fluoro-5-fluorouridine (9). mixture of anomers R 1 (1:1 hexanes / EtOAc)= 0.48; mp 65 - 70 "C. 'H NMR (400 MHz, CDCI 3 ) d 15 10.0 (bm, 1H), 7.99 (d, J = 5.6 Hz, 0.631H), 7.65 (in, 4H), 7.42 (m, 6.371), 6.12 (dd, J = 18.0 and 1.6 Hz, 0.37H), 6.00 (d, Jw 16 Hz, 0.63H), 5.37 (dd, J 54.6 and 2.4 Hz, 0.37H), 5.22 (dd, J -50.4 and 4 Hz, 0.63H), 4.57 (m, 0.37H), 4.44 (m, 0.631-1), 4.22 (dd, J - 12.2 and 2.0 Hz, 0.63H), 3.92 (dd, J = 11.2 and 3.2 Hz, 0.37 H), 3.70 (m, IH), 2.22 (m, 2H), 1.09 (s, 5.67H), 1.074 (s, 3.33H); C NMR (100 MHz, CDCI) d 157.2(d, J= 31.7 Hz), 157.1 (d, J= 20 25.8 Hz), 149.1, 148.8, 140.4 (d, ] = 236.6 Hz), 140.1 (d, J 235.2 Hz), 135.6, 135.5, 135.4, 132.9, 132.7, 132.4, 132.3, 130.1, 130.0, 129.9, 127.9, 127.8, 125.1 (d, J = 34.9 Hz), 123.6 (d, J 34.1 Hz). 96.4 (d, J = 182.0 Hz), 92.0 (d. J= 185.9 Hz), 90.2 (d, J= 37.2 Hz), 87.0 (d, J = 15.2 Hz), 81.7,79.8,64.8, 63.0, 33.3 (d, J = 21.2 Hz), 31.0 (d, J 21.2 Hz), 26.9, 26.8, 19.2; IR (thin film) 3185, 1722, 1117 cm -'; HRMS calculated for [M + 1) C 25

HN

2 0 4 SiF 2 : 25 487.1866. Found: 487.1853. Anal. Calc. C 2 5 HsN 2 0 4 SiF, : C, 61.71; H, 5.80; N, 5.76. Found; C, 61.72; H, 5.86; N, 5.72 . (D))- 5' - 0 - (I -butyldiphenylsilyl) - 2',3' - dideoxy - 2' - fluoro - 5 - fluorocytidine (10). mixture of anomers Rl (100% EtOAc)=0.36; np 75 - 81 'C. 'H NMR (400 MHz, CDC1) d 8.50 (bm, IH), 8.05 (d, J = 6.0 Hz, 0.67H), 7.67 - 7.63 (m, 4H), 7.51 - 7.39 (m, 6.33R), 6.10 30 (d,J= 20 Hz, 0.33H), 5.98 (d, J = 16.4 Hz, 0.67H), 5.62 (bm, 1H), 5.41 (d, J = 52.4 Hz, 0.33H), 5.23 (dd, J = 51.6 and 4 Hz, 0.67H), 4.57 (m, 0.33H), 4.48 (m, 0.67H), 4.24 (dd, J= 42 12.4 and 2.0 Hz. 0.67H), 3.89 (dd, 3= 11.2 and 3.2 Hz, 033 Hi), 3.74 -3.66 (m, 1H), 2.39 1.95 (m, 2-), 1.09 (s, 6H), 1.06 (s, 3H); "C NMR (100 MHz, CDCI 3 ) d 158.4 (d, J= 14.4 liz), 158.3 (d, J = 15.2 Hz), 153.8, 153.7, 136.5 (d, J= 240.5 lz), 136.2 (d, J 241.8 iz), 135.59, 135.56, 135.4, 133.0, 132.9, 132.5, 132.4, 130.1, 130.0, 129.9, 127.9, 127.8, 124.8 5 (d, J= 31.9 Hz), 96.5 (d, 1 181-3 Hz), 91.8 (d, J = 175.2 Hz), 90.7 (d, J = 24.9 Hz), 87.8 (d, J 212 Hz), 81,6, 79.6,64.9,63.0, 33.5 (d, J = 19.7 Hz), 30.6 (d, J = 21.3 Hz), 26.9, 26.8, 19.2, 14.2; IR (thin film) 3304, 2959, 1680, 1621, 1508, 1105 cm -'; HRMS calculated for [M + Li] C2sHN,O 3 SFXi: 492.2106. Found:492.21 10. Anal. Calc. C2, 2 N,OSiF 2 : C, 61.84; 11, 6-02; N, 8.65. Found: C, 61.86; H, 6.09; N, 8.55. 10 (D) - N-acetyl-5'-O-Q -butyldiphenylsilyl)-2',3'-dideoiy-2'-lnoro-cytidine (11). mixture of anomers R,(15 % EtOH , 85 % EtOAc) 0.75; mp 81-86 *C . 'H NMR (400 MHz, CDCb) d 10.58 (bs, 1H), 8.40 (d, J 7.2 Hz, 0.61H), 7.86 (d, J= 7.6 Hz, 0.38H), 7.67 - 7.65 (m. 4H), 7.51 -7.41 (m, 611), 7.27 (d, J= 8.4 Hz, 1H), 6.12 (t, J 15.8 Hz, IH), 5.51 (d, J - 52.6 Hz, 0.38H), 5.21 (dd, J = 50.8 and 2.9 Hz, 0.61H), 4.62 (m, 0.38H), 4.54 (m, 0.61H), 4.28 (d, J 15 11.5 Hz, 0.6111). 3.95 (dd, J= 11.9 and 3.2 Hz, 0.381), 3.79 - 3.70 (m, 11), 2.46 - 2.04 (m, 5H), 1.12 (s, 5,49H), 1.07 (s, 3.42H); 3C NMR (100 MHz, CDCl,) d 171.5,171.3,163.4, 154.9, 144.9, 144.1, 135.5, 135.4, 133.0, 132.8, 132.5, 132.2, 130.2, 130.1, 129.9, 128.0, 127.8, 96.8 (d, 3= 91.1 Hz), 96.2 (d, J= 147.9 Hz), 92.3,91.2 (d,]= 35.7 Hz), 90.5, 88.5 (d, J - 15.9 Hz), 81.9, 80.1, 64.7, 62.9, 33.5 (d, I= 20.5 Hz), 30.5 (d, I = 20.5 Hz), 26.9, 26.8, 20 24.9,24.8, 19.3,19.2; IR (thin film) 3237,2932, 1722,1671,1559,1493,1107 cmnv; HRMS calculated for [M+Li) C 21

H

3

NO

4 FSiLi : 516.2306. Found: 516.2310. Anal. Calc.. C2H 32

N

3 0 4 FSi : C, 63.63; H, 6.33; N, 8.24. Found; C, 63.45; H, 6.42; N, 8.09. (D) - 5' - 0 - ( -butyidiphenylsilyl)-2',3'-dideoxy-2'-fluoro-cytidine (12). mixture of anomers R,(15 % EtOH, 85 % EIOAc)=0.50; mp 98-104 'C. 'H NMR (360 MHz, CDC 3 ) 25 d 7.97 (d, J= 7.2 Hz, 0.64H, H-6), 7.65 (m, 4H), 7.47 - 7.38 (m, 6.36H), 6.15 (d, J =20.5 Hz, 0.36H), 6.05 (d, J = 16.6 Hz, 0.64H), 5.83 (d, J 7.9 Hz, 0.36H), 5.46 (d, J =7.2 Hz, 0.64H), 5.30 - 5.10 (mn 1H), 4.55 (m, 0.36M), 4.44 (m, 0.64H), 4.22 (d, 3 = 9.7 Hz, 0.64H), 3.88 3.63 (m, 1.36H), 2.38 - 1.95 (m, 2H), 1.09 (s, 5.76H), 1.06 (s, 3.24H); 'C NMR (100 MHz,

CDCI

3 ) d 166.1, 155.8, 141.5, 140.5,135.6, 135.4, 133.1, 132.9, 132.8, 132.4,130.1,130.0, 30 129.8, 128.0, 127.9, 127.8, 96.7 (d, J 181.3 Hz), 93.4 (d, J = 140.3 Hz), 94.5, 90.8 (d, J = 35.6 Hz), 90.8, 87.8 (d, J = 15.9 Hz), 81.2, 79.4, 65.0,63.2, 33.7 (d, J = 21.2 Hz), 30.8 (d, J= 43 20.4 Hz), 26.9. 26.8, 19.3, 19.2: IR (thin film) 3470. 3339. 1644. 1487, 1113 cm-'; HRMS calculated for fM + Li] C, 5

H

3

N

3

O

3 FSiLi : 474.2201. Found: 474.2198. Anal. Calc..

C

25 HXN,0 3 FSi : C, 64.21; H, 6.47 ; N, 8.99. Found: C, 64.04 ; H, 6.58 ; N, 8.76. a - (D) - 2',3' - Dideoxy -2' - fluoro -5- fluorouridine (14a), R (100 % EtOAc)= 0.38; 5 mp 153-155 'C. 'H NMR (360 MHz, CDOD) d 7.80 (d, J = 6.8 Hz, I H), 6.11 (d, = 18.7 Hz, 1H), 5.35 (d, I = 52.9, 1H), 4.59 (m, I H), 3.81 (d, J= 11.9 Hz, IH), 3.57 (dd, J= 12.6 and 3-6 Hz, 1N), 2.36-2.15 (m, 2H); "C NMR (100 MHz, CD 3 OD) d 159.6 (d, J = 25.8 Hz), 150.7, 141.5 (d, J = 230.6 Hz), 127.0 (d, J= 34.9 Hz), 93.9 (d, J 185.1 Hz), 88.5 (d, J " 15.1 Hz), 81.8, 64.3,34.3 (d, J= 20.5 Hz); IR (KBr) 3421, 3081, 1685, 1478, l1 cm"; HRMS 10 calculated for (M + Li] C,HN 2 0 4

F

2 Li; 255.0769. Found: 255.0778. Anal. Cale..

C,HIN

2 0 4

F

2 : C, 43.56; H, 4.06; N, 1129 - Found: C, 43.59; H, 4.11; N, 11.17. p- (D)- 2',3' - Dideoxy -2' - fluoro -5- fluorouridine (14b). Rr (100% EtOAc)= 0.54; mp 152-154( C. 'H NMR (360 MHz, CD 3 OD) d 8.41 (d, J= 7.2 Hz, IH), 5.89 (d, 1= 16.6 Hz, 1H), 5.21 (ddJ, 3 51.5 and 3.6 Hz, 1H), 4.41 (m, 1H), 4.00 (d, J 12.6Hz. IH), 3.67 (d, 15 J = 12.2 Hz, I H), 2.25-2.09 (m, 2H); '3C NMR (100 MHz, CD 3 OD) d 159.7 (d, J= 25.8 Hz), 150.7, 141.8 (d, J = 229.8 Hz), 126.3 (d, J = 36.4 Hz), 98.3 (d, I - 179 Hz), 91.9 (d, I 37.1 Hz), 83.6, 61.9, 31.9 (d, J=20.5 Hz); IR (KBr) 3417, 3056, 1684, 1474, 1105 cm''; HRMS calculated for [M + Li] CHN 2

O

4

F

2 Li : 255.0769. Found: 255.0764. Anal. Calc.. C,1N 2 0F 2 : C, 43.56; H, 4.06; N, 11.29. Found: C, 43.37; H, 3.98; N, 11.22. 20 m - (D) - 2',3' - Dideoxy - 2' - fluoro - 5 - fluorocytidine (15*). Rr (l5 % EtOH , 85 % EtOAc)= 0.22; mp 198-202 *C (dec.). 'H NMR (400 MHz. CD 3 OD) d 7.78 (d, J = 6.8 Hz, 1H), 6.07 (d, J = 18.8 Hz, IH), 5.37 (d, I = 54.0 Hz, IH), 4.59 (m, 1H), 3.80 (dd, = 12.0 and 3.2 H3z, 1), 3.57 (dd, J . 12.4 and 4.4 Hz. IH), 2.38-2.14 (n, 2H); "C NMR (100 MHz, CDOD) d 159.9 (d, I = 13.6 Hz), 156.5, 138.3 (d, J 240.4 Hz), 127.5 (d, J= 33.4 Hz), 93,6 25 (d, J= 184.3 1Hz), 89.5 (d, J - 15.9 Hz), 81.8, 64.4, 34.5 (d, I= 20.5 Hz); IR (KBr) 3486, 3098, 1681, 1519, 1108 cm'; HRMS calculated for [M + Li] C,H,,N,0,F 2 Li : 254.0929. Found: 254.0929. Anal. Cale.. CHN,OF 2 - 1/2 1420: C, 42.19; H, 4.72; N, 16.40. Found: C, 41.86; H, 4.75; N, 16.36. p- (D)- 2',3' - Dideoxy -2'- fluoro -5- fluorocytidibe (15b). R,(15 % EtOH,85% 30 EtOAc) = 0.37; mp 181-183 *C (dec.). 'H NMR (400 MI-z, CDOD) d 8.45 (d, 3 =7.2 Hz, 11H), 5.92 (dd, J - 16.2 and 1.2 Hz, I H), 5.18 (dd, J 50.8 and 4.0 Hz, 1H), 4.46 (m, 111), 44 4.05 (dd, J = 12.4 and 2.4 Hz. 11), 3.72 (dd. J = 12.8 and 2.4 Hz. 1H), 2.27-2.05 (m, 2H); "C NMR (100 MHz, CDOD) d 159.9 (d, ) = 13.6 Hz), 156.5, 138.5 (d, I = 240.5 H4z), 126.9 (d, J= 33.4 Hz), 98.4(d, J = 179.0 Hz), 92.5 (d. J = 36.4 liz), 83.6, 61.9, 31.6 (d, 3 20.5 Hz); IR(KBr) 3494, 2944, 1689, 1522, 1106 cm-'; HRMS calculated for [M + Li] C,H1N 3 0 3 F,U 5 254.0929. Found; 254.0936. Anal. Calc.. C8,HN,0OF 2 : C, 43.73; H, 4.49; N, 17.00. Found: C, 43.84; H, 447; N, 17.05. a - (D) - N- acetyl - 2',3' - dideoxy - 2' - fluoro - cytidine (162). Rr (15 % EtOH , 85 % EtOAc)- 0.40; mp 208-212 "C. 'H NMR (360 MHz, DMSO - d 6 ) d (10.91, bs, 1H), 8.05 (d, 3=7.2 Hz, I H), 7.25 (d, J =7.2 Hz, 1 H), 6.08 (dd, J = 19.1 and 2.9 Hz, IH), 5.42 (d, J= 10 52.2 Hz, 1-), 4.97 (bs, 1H), 4.54 (m. IH), 3.63 (d, J= 13.0 Hz, 1H), 3.47 (d, J = 13.3 Hz, 1H), 2.35-2.15 (m, 2R), 2.11 (s, 3H); "C NMR (100 MHz, DMSO - d6) d 171.0, 162.6, 154.3, 145.7, 94.9, 92.0 (d, J = 183.6 Hz), 87.5 (d, J = 15.9 Hz), 80.2, 62.6, 33.3 (d, I= 19.7 Hz), 24.4; IR (KBr) 3436,3227, 1702, 1661, 1442, 1102 cur'; HRMS calculated for [M + Li} C,,HN,O 4 FLi : 278.1128. Found: 278.1136. Anal. Cale.. C 1 H 4

N

3 0F : C, 48.71; H, 15 5.20; N, 15.49. Found: C, 48.73; H, 5.23; N, 15.52. p - (D) - N- acutyl - 2',3' - dideoxy - 2' - fluoro - cytidine (16b). R (15 % EtOH, 85 % EtOAc) -0.50; mp 174-178 "C. 'H NMR (360 MHz, DMSO -d d (10.90, bs, IH), 8.46 (d, J= 7.2 Hz, IH), 7.18 (d, 3=7.2 Hz, 1H), 5.90 (d, J - 16.9 Hz, IH), 5.27 (d, J = 52 .9 Hz, 1H), 5.27 (bs, IfH), 4.39 (m, 114), 3.88 (d, J= 13.0 Hz, 1M), 3.61 (d, J= 13.0 Hz, IH), 2.09 (s, 20 3H), 2.20-1.85 (m, 2H); "C NMR (100 MHz, DMSO - di) d 171.0, 162.6, 154.4, 144.7, 97.0 (d, J 177.5 Hz), 95.0, 90.7 (d, J = 36.6 Hz), 82.2, 60.3, 30.3 (d, J = 19.7 Hz), 24.3; IR (KBr) 3447, 3245, 1703, 1656, 1497, 1122 cm; HRMS calculated for [M + Li)

C,

1 H,4N 3 0 4 FLi: 278.1128. Found: 278.1133. Anal. Calc.. CIH, 4 N,0 4 F: C, 48.71; 11, 5.20; N, 15.49 . Found: C, 48.65; H, 5.22; N, 15.46. 25 a - (D) - 2',3' - Dideoxy - 2' - fluoro - cytidine (17a). R, (15 % EtOH, 85 % EtOAc) = 0.08; mp 234-237 C (dcc.). 'H NMR (400 MHz, DMSO - ) d 7.52 (d, J 7.6 Hz, I H), 7.21 (bm, 2H), 6.05 (dd, J- 20.4 and 3.2 Hz, I H), 5.73 (d, J = 7.2 Hz, I H), 5.28 (d, J - 52.4 Hz, 111), 4.93 (t, J = 5.6 Hz, IH), 4.45 (m, I H), 3.58 (i, 1H), 3.43 (m, 1H), 226-2.13 (m, 2H); 3 C NMR (100 MHz, DMSO - d,) d 165.8, 155.0, 141.6, 93.3, 92.2 (d, J - 182.8 Hz), 86.6 (d, 30 J= 15.1 Hz), 79.4, 62.8, 33.3 (d, J= 19.7 Hz); IR (KBr) 3366, 3199, 1659, 1399,1122 cm-; 45 HRMS calculated for tM + Lij C,HN,,FLi : 236.1023. Found: 236,1014. Anal. Calc.. C,H 12

N

3 0 3 F : C. 47.16; H, 5.28; N, 18.33. Found: C, 47.40; H, 5.34; N, 18.51. P3- (D) - 2',3' - Dideoxy - 2' -fluaro - cytidine (17b). Nucleoside 25 (0.160 g, 0.59 mmol) was dissolved in 10 mL of saturated methanolic ammonia. After stirring for 5 min., the 5 reaction was complete. The methanolic ammonia was removed and the resultant white solid was placed under vacuum and heated gently in a 60 "C water bath for 2 hrs. to remove the acetamide by-product through sublimation. The white solid was crystallized from 5 % methanol / 95 % methylene chloride to give a quantitative yield of a white crystalline solid. R(15 % EtOH, 85 % EtOAc)= 0.18;imp 191-195 *C (dec.). 'H NMR (360 MHz, CDOD) 10 d8.10(d, J-7.2Hz, IH), 5.92(d,J= 17.3 Hz, 1H), 5.82 (dJ-7.6Hz, IH),5.13 (d,J= 50.0 Hz, IH), 4.39 (i, 1H-1), 3.97 (d, J= 12.2 Hz, IH), 3.68 (dd, J = 13.0 and 2.5 Hz, IlH), 2.21-2.00 (m, 211); C NMR (100 MHz, CDOD) d 165.9, 155.0, 140.8. 97.3 (d, i= 176.8 Hz), 93.6,90.3 (d, J 35.6 Hz), 81.3, 60-7, 31.0 (d, J =20.5 Hz); IR (KBr) 3397,3112, 1680, 1400, 1178, 1070 cm*; HRMS calculated for [M + Li] C,H, 2

N

3 03Fli : 236.1024. 15 Found: 236.1028. Anal. Calc.. C,H, 2

N

3 0,F : C, 47.16; H, 5.28; N, 18.33 . Found: C, 47.01; H, 5.21; N, 18.29. (L) - 5' - O - ( -butyldiphenylsilyl) - 2',3' - dideoxy -2'. flurs - thymidine (23). mixture of anomers Rr(10 % MeOH / 90 % CH2Cl2) 036; mp 61-65 *C. IH NMR (360, MHz, CDCl) d 9.48 (bs, 111), 7.67 (m, 4H), 7.45 - 7.37 (in, 7H), 6.15 (dd, J W 20.2 and 3-2 Hz, 20 0.36H), 5.99 (d. 1 18.4 Hz, 0.64H), 5.34 (d, J= 51.8 Hz, 0.36H), 5.24 (dd, J = 52.2 and 4.3 Hz, 0.64H), 4.59 (m, 0.3611), 4.45 (tn, 0.64H), 4.17 (dd, I = 12.2 and 2.5 H1z, 0.64H), 3.91 (dd, J 11.9 and 2.9 Hz, 0.36H), 3.21 (dd, J 11.5 and 2.9 Hz, 0.641H), 3.68 (dd, J= 10.8 and 3.6 Hz, 0.36H), 2.40 - 2.12 (m, 2H), 1.94 (s, 1.0811), 1.61 (s, 1.9211). 1.10 (s, 5.76H), 1.07 (s, 3.24H); "C NMR (100 MHz, CDCI 3 ) d 164.1, 164.0, 150.4, 150.2, 136.4, 135.6, 135.5, 25 135.4, 135.3, 135.2,133.0, 132.8, 132.6, 130.1, 130.0, 129.9, 127.94, 127.90,127.8, 110.8, 109.8, 96.4 (d, J = 181.3 Hz), 92.1 (d, J = 185.8 Hz), 90.7 (d, S = 36.4 Hz), 86.6 (d, J 15.2 Hz), 80.9, 79.4, 64.9,63.6,33.4 (d, 1 =20.5 Hz), 32.0 (d, J=21.2 Hz), 27.0, 26.8, 19.4, 19.2, 12.6, 12.2; IR (thin film) 3183, 3050, 1696, 1506, 1188 cm "; HRMS calculated for JM + Li] CNHN2O 4 SiF; 489.2197. Found: 489.2175. Anal. CaIc. CJ1,HN 2

O

4 SiF : C, 64.71; H, 647; 30 N, 5.80. Found: C, 64.88; U, 6.56; N, 5.76. 46 (L) - 5'- 0 - (t-butyidiphenylsilyl) - 2',3' - dideoxy - 2' fluoro - 5 - fluorouridine (24). mixture of anomers Rr (1:1 hexanes / EtOAc) = 0-48; mp 65 - 71 *C. 'H NMR (400 MHz, CDCl,) d 9.08 (bs, 0.44), 9,00 (bs, 0.6H) 8.01 (d, I = 5.4 Hz, 0.6H). 7.65 (m, 4H), 7.42 (m. 6.4H), 6.10 (dd, J = 20.2 and 1.4 Hz, 0.4H), 6.00 (d, J = 16.0 Hz, 0.6H), 5.35 (dd, J = 52.4 5 and 1.6 Hz, 0.4H), (5.22, dd, J = 51.2 and 4 Hz, 0.6H), 4.57 (m, OAH), 4.44 (m, 0.611), 4.22 (dd, J= 12.4 and 2.0 Hz, 0.6H), 3.91 (dd, J - 11.2 and 2.9 Hz, 0.4H), 3.70 (m, 1H), 2.45 2.00 (m, 2H), 1.09 (s, 5.4H), 1.07 (s, 3.6H); 1C NMR (100 MHz, CDCI,) d 156.9 (d, J= 26.5 Hz), 148.8, 148.6, 140.3 (d, J = 236.7 Hz), 140.1 (d, I = 235.1 Hz), 135.6, 135.5, 135.4, 132.9, 132.7, 132.4, 132.3, 130.2, 130.1, 129.9, 127.9, 127.8, 125.1 (d, J = 34.9 Hz), 123.6 (d, 10 J = 34.2 Hz), 96.4 (d, J = 182.9 Hz), 92.0 (d, I= 186.6 Hz), 90.2 (d, J= 36.0 Hz), 86.9 (d, J= 15.1 Hz), 8 L7, 79.8, 64.8, 63.0, 33.2(d, J= 20.5 Hz), 30.9 (d, 3 20A Hz), 26.9, 26.8, 19.2; IR (thin film) 3191, 1719, 1113 cm ; HRMS calculated for [M + Li) CsHN,O 4 SiFLi: 493.1946. Found: 493.1952. Anal. CaIc. C 2

SH

2

IN

2

O

4 SiF2 : C, 61.71; H, 5.80; N, 5.76. Found: C, 61.73; H, 5.83; N, 5.77. 15 i - (L) - 2',3' - Dideoxy - 2' - fluoro - thymidine (26a). Rf (100 % EtOAc)= 0.25; mp 147-149'C. 'H NMR (360 MHz, CDOD) d 7A5 (s, IH), 6.11 (dd, 319.4 and 2.9 Hz, IH), 5.30 (d, J - 53.6 Hz, 1H), 4.58 (m, IH), 3.79 (dd, J= 12.2 and 2-2 Hz, 1H), 3.55 (dd, J 122 and 3.6 1z, 1H), 2.40 - 2.15 (m, 2H), 1.87 (s, 3H); 'C NMR (100 MHz, CD 3 OD) d 166.6, 152.3, 138.6, 110.5, 93.9 (d, J = 185.1 Hz), 88.3 (d, I = 15.1 Hz), 81.7. 64.4,34.5 (d, J 20 =20.5 Hz), 12.6; IR (KBr) 3436, 3166, 1727, 1667, 1362, 1186 cm-'; HRMS calculated for [M + Li] C, 1 JN2O 4 FLi :251.1019. Found: 251.1014. Anal. Calc.. C, 0 H8,N 2 0 4 F : C,49.18; H, 5.37; N, 11.47. Found: C, 49.32; H, 5.40; N, 11.29. p - (L) - 2',3' - dideoxy - 2' - fluoro - thymidine (26b). R (100 % EtOAc) -0.38; np 186-188C . 'H NMR (360 MHz, CD 3 OD) d7.94 (s, 1H), 5.93 (d, J 17.6 Hz, 11), 5.20 (d, 25 J -51.8 Hz, IH),4.40 (m, IH), 3.98 (d, J = 11.9Hz 1H), 3.68(d, J13.0 Hz, 1H), 2.37 2.10 (m, 2H), 1.83 (s, 3H); 'C NMR (100 MHz, CD 3 OD) d 166.7, 152.3, 138.2, 111.0, 98.4 (d, J = 178.3 Hz), 92.1 (d, Jm 36.4 Hz), 83.1, 62.4, 32.5 (d, J1 20.5 Hz), 12.6; IR (KBr) 3478, 3052, 1684, 1363, 1192, 1005 cm-; Anal. Calc.. C, 0 1,N,0 4 F: C, 49.18; 11,5.37; N, 11.47. Found: C, 49.29; H, 5.44; N, 11.36. 30 a - (L) - 2',3' - dideoxy - 2' - fluoro - 5 - fluorouridine (27a). Rr (100 % EtOAc) = 0.38; mp 155-157 *C). 'H NMR (400 MHz, CD 3 OD) d 7.80 (d, J = 6.8 Hz, I H), 6.13 (d, J= 20.0 Hz, 47 lI h), 5.35 (d. J = 54.4 Hz, 1 H), 4.63 (m, I H), 3.81 (dd, J = 11.9 and 3.2 Hz, 1H). 358 (dd, J= 12.4 and 2.0 Hz, 1IH), 2.41-2.15 (m, 2H); "C NMR (100 MHz, CD 3 OD) d 159.6 (d. =25.8 Hz), 150.7, 141.5 (d,J= 230.6 Hz), 127.0 (d, J = 34.9 Hz), 93.9 (d, J= 184.3 11z), 88.5 (d. I= 15.1 Hz), 81.9, 64.3, 34.3 (d, J = 20.5 Hz); IR (RBr) 3401, 3098, 1661, 1458, 1018 cm-'; 5 HRMS calculated for [M + Li] CHON04F 2 Li : 255.0769. Found: 255.0771. Anal. Calc..

CH

1

N

2 0F2 : C, 43.56; H. 4.06; N, 11.29. Found: C, 43.70; H, 4.17; N, 11 .15. - (L) - 2',3' - dideoxy -2'- fluoro - 5 - fluorouridine (27b). R 1 (100 % EtOAc) = 0.54; mp 153-156"C, 'H NMR (400 MHz, CDOD) d 8.46 (d, J=6.8 Hz, IH), 5.94 (d, J= 16.4 Hz, 1H), 5.25 (dd, J= 51.6 and 4.0 Hz, 1H), 4.41 (m, IM), 4.05 (dd, J= 12.8 and 2.4 Hz, II), 10 3.72 (dd, J - 12.4 and 2.4 Hz, 1H), 2.34-2.09 (m, 2H); "C NMR (100 MHz, CDJOD) d 159.7 (d, J 25.8 Hz), 150.7, 141.8 (d. J 230.6 Hz). 126.3 (d, J -357 liz), 98.3 (d, J= 184.6 Hz), 91.9 (d, J= 36.4 Hz), 83.6, 61.9, 31.9 (d, - 20.5 Hz); IR (KBr)3482, 3037, 1702, 1654, 1402, 1103 cr'; HRMS calculated for [M + Li] C,H, 0 N,0 4

F

2 Li : 255.0769. Found: 255.0764. Anal. Calc.. C,HN 2 0 4

F

2 : C, 43.56; H, 4.06; N, 11.29. Found: C, 43.59; H, is 4.06; N, 11.17. PREPARATION OF L-2'-FLUORO-2',3'-UNSATURATED NUCLEOSIDES A second facile synthesis of unsaturated 2'-fluoronucleosides has also now been acccomplished and is described below. The synthesis involves reacting a protected 20 pyrimidine or purine base with key intermediate 309 in the presence of a Lewis acid, as described generally in Scheme 9 below. Representative compounds made according to this synthesis are described in Tables 5-6. 48 Scheme 9 OH L.GICa~*ttrjacwflf 301 302 E 0 Wi F a IF F 307---0E *306 30& ADS 305 309 FIesrr:t) -mnthd&Plypmfen. DMF. pTsOH Iii) Nso. H,0 (n11) (E0P(C)CMFCC 2 EL NmHDS, THF. ,-7 *C iv) c.MCL EtCH M 7TSMCL inmMte, CH2C, (vi) DIAL44. CHg,0, -75 60i) AMgO, pyddhne. CH 2 C1. 49 Scheme 9 309 Yx R N F 100. 312-X.M' F x 31 0 CO 3 33 - XNHB Y H 3 13 NH Y H -a 324.OltY 66, Y. 325 cXb* H2. * 3 2 XMY- 4~XuC.. egnt;(0 dlOte utall. TMSOTI. DCE (II) tbybwtd thiwe. ThMuO~T. DCE (HU) Elytsted N-B34v in., TMa0T, CHaCN fry 5-F-cytcIne, TM'BOTI. CHCN (v) TBhF. Q4CN 1v4 WMM 50 Scheme 9 OF; 309 9V N N j2i 327 0 32 h 129300 x Y N N NN 33 x.191H 3 - ;X.a-,,Y-F 340:X-N y.p NVDME (v) NHrM1 gON PAM DCE ITAP CJ CN (IV) $1 P- N -. -- . a U - 2a N 3 Us 4 ,- - -, . ± r vi .j 0.. . & o m - -me n - - '9a a a * - - u e N. a 6 v - e - e 77 - . - t 1* 0 7 52. , r u-- -a-- m * S- -r - -- ---- ma p . a - - S a N - -- g a 'C a OP P a ad .: -am-- - P0 aSe md n z * * * Ca O * a i 5I a e a S .a .- a e 5 ripi53 £ C 2. vi I. In 9 U * a z .2. - U o - -, ~ a 2. * j U u$ ~- ? a. St 2 . ~ i - K , a U- 3 U, It . .4 U~ a, A 1 5 Intl a - ~ a #1 £4 a a - -t - -, ~ vi w 5~.. a *0t ~ * - N ~" 2 ~ a , * a : ~ : g a- U cv -. .;-2 w N - ft C - 6 S 6 o a a a a W a ~ ~ ~ - et n 2 C U W - w * Q 54 Table 5 no. mp 'C(solvr jaID! dcn faruam,

STPC,

3 1-IJN,5i CH-I N 11 symp CI FN,0,si C. H- N 5 fl 144-146(A) -20A47 (t 036, CHCI,) Cz)1 3 FNO,Si C, H. N 13 1I341 (A) -57.68 (e-031. C1 3 CH,,NPoSi C.N.N, 14 SYp C, H, N is SYTup C,HI, N 16 161-162 (C) -13A 12 (c 0.20, MeON) CHF ,0.311o C. ,KN I D 17 13&-137 (E) +l38.55(C0.14,Meofj) CI,O021 2 C,H.N i8 149151 (D) -30.44 (cO0.20, MeWu C,JIJN,0, C, H, N 19 116.119(E) + 132.42 (c 025,.McOH) C,.$l%0Q, C, H.N 20 200202-ec IC) -54.89 (c 039. C102J) CjId~FNO, C, Hi, N 21 170. 172 (C) +136.38 (c 0.45, CHCI 3 ) Cf,~, 1 .J~ C.H, N 15 22 198-200 d=c (B) -21-3 1 (c 025, Mcli) CH 1 j*NP,4r1 C. , .N 23 120-121 (E) 4-159.15 (C021. McOH) CHJN,O, C, HN 25 )1 C. H., N 25 syny C. Ii. N 26 sN" Cm 1 11J0Ni C, H. N 20 27 fom +9W9 (c 0.20. CC!) CWsTCH~i C, H. N 28 SYWP -tr13967 (c 0.1B, CHcQ~ CMHJZCINOm C, H.N 29 310 foam C. N 31 180.182(A) +13.33(c 054, ClC!,) CsI~NIY4ASiOUaswnp CH.N IS 32 129-130 (A) -'9022 (e 0-23. CHICII) C,JbFCD4,OA; C.NN 33 134-286 (A) + 116.53 (c 0. 13. did 1 3) C,.HJNAio-arn C., N 3d 123-130$A) +$9.87 (c 0,13, CI1 3

C,H

21

FCIN

12 Si C. H, N 35 183.139(C) -54.01 (c 0.17, MoON CHJ,,Oao2 C, H. N 36 1694171 (C) +160.62 (c 0. 19, MoON) C,J% 5 0.3MeO$j C. H, N 30 37 123.130 (E) -5021 (C 020' MeOH C, 0 HYNO00.2, C.NHN 38 52go dec (C) + 1 69.60 (c O.20, M014 C,,HFN 4 O,0uI1,o C,N 39 1355I18t r= (B) -56.15 (CO0.1-6, MCOH) CH. N 40 IHOdam(B3) +178.22 (c 0.10, McOH)CHN 41 155.IS6,dec(D) +10.6.4(cO0.17, McON) C. H, N 35 42 15D-152 (B) +147-49 (cO .17, MeON) C, H, N 43 >20D0dec () +24A42(cU.10, DW C,H1%N 44 '220 dc(B) +51.6: (C 0.10, DMF C, H, Soi1ventz; A; EIOAC-hexmcs, D; 0CC!-McOH, C; CHC3~rMcOM D. TY 40 cyclabeXar, E; Iyophilyzed 235 Previously. the synthesis of 2',3' -unsaturated D-nucleosides has been accomplished via eleimination reaction starting from readily available nucleoside analog, which involved a lengthy modification for individual nucleosides. Several groups reported D-2'-fluoro-2',3' unsaturated pyrimidine nucleosides by the elimination of suitable 2-fluorinated nucleoside 5 analogs(Martin, J. A.. er at.. J Med. Chem. 1990, 33, 2137-2145; Stezycki, it Z., et al., J, Med Chem. 1990, 33, 2150-2157). This strategy for the synthesis of L-Fd4N, however, is accompanied by additional difficulties in the preparation of L-nucleosides as the starting material. There are few examples of the synthesis of 2',3'-unsaturated purine nucleosides by direct condensation due to the lability of the 2,3-unsaturated sugar moiety under the coupling 10 conditions in the presence of Lewis acid, except one case of the pyrimidine analog using a thiophenyl intermediate (Abdel-Medied, A. W.-S., er al., Synthesis 1991, 313-317; Sujino, K., et al., Tetrahedron Len. 1996, 37, 6133-6136). In contrast to the 2,3-unsaturated sugar moiety, the 2-fluoro-2,3-unsaturated sugar, which bears enhanced stability of glycosyl bond during the condensation with a heterocycle, was expected to become more suitable for the 15 direct coupling reaction. Thus, (R)-2-fluorobutenolide 506, as a precusor for the key intermediate 508, was chosen, which was prepared from L-glyceraldehyde acetonide 501. Starting from L-glyceraldehyde acetonide., a mixture of (E)-502/(Z)-2 (9:1 by 'H NMR) was obtained via the Horner-Emmons reaction in the presence of triethy I a fluorophosphonoacetate and sodium bis (trimethylsily1) aide in THF (Thenappan, A., et al, 20 J. Org. Chem., 1990, 55, 4639-4642; Morikawa, T., et al., Chem. Pharm. Bull. 1992, 40, 3189-3193; Patrick, T. B., et al, J Org. Chem. 1994, 59, 1210-1212). Due to the difficulties in separating (E)-5021(Z)-502 isomers, the mixtures were used in the following cyclization reaction under acidic condition to give the desired lactone 503 and uncyclized dio 504. The resulting mixture was converted to the silyl lactone 506 was subjected to reduction with 25 DIBAL-H in C14 2 C1 2 at 78* C to give the Jactol 507. The lactol 507 was treated with acetic anhydride to yield key intermediate 508, which was condensed with silylated 6-chloropurine under Vorburggen conditions to afford anomeric mixtures 509. Treatment of 509 with TBAF in THF gave free nucleosides 510 and 511, which was readily separated by silica gel column chromatography. Adenine analogs 512 and 513 were obtained by the treatement of 30 compound 510 and 511 with mercaptoethanol and NaOMe a steel bomb at 90*C, respectively. Treatment of compounds 510 and 511 with mercaptoetharnol and NaOMe afforded the inosine analogs 514 and 515, respectively. The sterochemical assignment of 56 these compounds was based on the NOESY spectroscopy (cross peak between H-T and H 4' in B-isomer 512). Any discussion of documents, acts, materials, devices, articles or the like which has been 5 included in the present specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed in Australia before the priority date of each claim of the application. 10 The terms "comprise", "comprises" and "comprising" as used throughout the specification are intended to refer to the inclusion of a stated component or feature or group of components with or without the inclusion of a further component or feature or group of components or features. 57 58. Scheme 10. Synthesis of L-2'-Fluoro.d4 -Adenine and -Hypoxanthine by Direct Condensation C- 4 0 COFt 4 F0iF OH OHF OH o, F +CO2t +t 501o 0502 (z- 502 503 504 A.0- o-..OR N F (6% 2 ' (1% ) ' 508 507 106 505 (702% frum 1) 3(63) N vii ern i N OH F F 511 (4S%) N 513 iX.Samf(7t VMJ MetzN ^ 75% kntu()0 (EM )PO)CFCOE. (COhSlbNNa. TMW, .78 "C (i) HCOEf0l (4H) TBDMS imiols. CH 2 02 (hv) 3 M DBAL-H in C1202. CH2.-78 t (') AczO. pyr. C0lA4dwziytIud &-CIpuri, TMSO. DCE *I' -IAF, CH3CN (viU) NH/MeL 90 C ) S(CH020M MWe MeOHsfl Table 7 -Median Effective (-C, 0 2nd 1jnhibitorv (1C50) Conicentration of L,2'. Fluoro-d4lAdenine ann Hvpoxntine against H-IV-1 in PBM Compound No- EC~a (Pkf) EC-0, (PM cyroiaxicily (PBM cells) (PBM _______________ PMC Vcr- Cells CEM cells cell$ 512 1-5 15.1 >100 >100 >100 S13 47,6 332 >100 >100 >100 514 >1]00 >100 >100 >300 >100 15 515 >100 >100 >100 >100 >100 316(0) >100 >100 >100 >100 >100 317 (a) >100 > 100 >i00 >-100 >100 3184) >)00 >100 >100 >100 >100 31910) >100 >100 >)00 5100 >100 20 3220p) 0.51 4.3 >)Go >)00 >100 323 (a) >100 >100 >100 >100 >100 335(0) 1.5 13.1 >300 >100 >100 336 (a) 47.6 332 >100 >100 >100 337 (A) >100 >100 >100 >100 >100 25 338 (M) >100 >100 >100 >100 AZT 0.004 0.04 >100 29.0 14.3 59 Experimental section. Melting points were determined on a Mel-temp 11 laboratory device and are uncorrected. Nuclear magnetic resonance spectra were recorded on a Bruker 250 and AMX400 400 MHz spectrometers with tetramethylsilane as the internal reference; chemical 5 shifts (6) are reported in parts per million (ppm), and the signals are described as S (singlet), d (doublet), t (triplet), q (quartet), br s (broad singlet), dd (doublet of doublet), and m (multiplet). UV spectra were obtained on a beckman DU 650 spectrophotometer. Optical rotations were measured on a Jasco DIP-370 Digital Polarimeter. Mass spectra were measured on a Micromass Inc. Autospec High Resolution double focussing sector (EBE) MS 10 spectrometers. Infrared spectra were recorded on a Nicolet 510 FT-R spectrometer. Elemental analyses were performed by Atlantic Microlab, Inc.. Norcross, GA. All reactions were monitored using thin layer chromatography on Analtech, 200 mm silica gel GF plates. Dry 1,2-dichloroethane, dichloromethane, and acetonitrile were obtained by distillation from CaH 2 prior to use. Dry THF was obtained by distillation from Na and benzophenone when 15 the solution became purple. 1r(S)-Glyceraldehyde acetonide (302). A solution of L-gulonic-y-lactonc (175 g, 0.98 mol) in DMF (I L) was cooled to 0 *C and p-toluenesulfonic acid (L.1 g, 5.65 mmol) was added portionwise with stirring. To the resulting solution, 2-methoxypropene (87.7 g, 0.92 mol) was added dropwise through a dropping funnel at 0 C. The reaction mixture was warmed up 20 to room temperature and further stirred for 24 h. After the completion of the reaction, sodium carbonate (124 g) was added and the resulting suspension was vigorously stirred for 3 hours. It is then filtered over glass filter and the filtrate is evaporated under vacuum. To the yellow residue, toluene (170 mL) is added whereupon crystallization occurred. The solid was filtered by suction, washed with hexanes/ethanol (9:1; 1 L), and dried to give yellowish solid 25 301 (99.1 g, 65 %). To a stirred suspension of 5,6-0-isopropylidene-L-gulono-1, 4 -lactone (70.0 g, 0.32 mol) in water (270 mL), sodium metaperiodate (123 g, 0.58 mol) was added portionwise at 0'C ovr 30 min maintaining pH 5.5 (adjusted by addition of 2 N NaOH). The suspension was stirred at room temperature for 2 hours, then saturated with sodium chloride and filtered. 30 The pH of the filtrate was adjusted to 6.5-7.0 and extracted with dichloromethane (5 times. 200 mL) and ethyl acetate (5 times 300 mL). The combined organic layer were dried with anhydrous magnesium sulfate, filtered and concentrated under reduced pressure (< 20 *C). 60 And then the resulting residue was distilled to give 302 (23.2 g, 69 %) as a colorless oil; b.p. 49-51 "C / 16 Torr. (a] 0 25 -66.4 (c 6.3, benzene). (E)/(Z)-Ethl-3-(R)-2,2-dimehyl-1,3-dioxolan-4-yl}-2-fnuoroacrylate (F-303 and Z-303). A solution of triethyl 2-fluorophosphonoacetate (39.2 g, 162 mmol) in THF (70 mL) was 5 cooled to -78 *C and a solution of sodium bis(trimethylsilyl)amide (1.0 M solution in THF, 162 mL, 162 nnol) was added dropwise. The mixture was kept for 30 min at -78 *C, then a solution of 303 (19.14 g, 147 mmnl) in THF (70 mL) was added. After being stirred for 1 h at -78 0 C, the reaction mixture was treated with aqueous NH 4 Cl and extracted with ether. The ether phase was washed with saturated NaCl, dried over MgSO, filtered and evaporated. The 10 residue was chromatographed on silica gel to give E-303 and Z-303 (9:1 by 'H NMR) as a pale yellowish oil (34.6 g, 97.9 %). 'H NMR (CDCl 3 ) 5 1.34. 1.36 (2t, J= 8 Hz, -CH 2

CU

3 ), 1.40, 1.45 (2s, -CH,), 3.69 (m, H,-5), 4.28 (m, Hr-5, -CU2CH 3 ), 5.02 (m, H-4), 5.40 (im, H-4), 6.02 (dd, 3 - 8, 20 Hz, H-3), 6.18 (dd, 3 8, 32 Hz, H-3). (R)-(+)-41(tert-Butyldimethylsilyloxy)methyll-2-luoro-2-buten-4-olide (307). A solution 15 of E-303 and Z-303 (19.62 g, 89.89 mmol) in 110 mL of anhydrous EtOH was treated with 30 mL of conc. HCI and stirred at room temperature for 2 hr. The solvent was removed in vacuo and the residue was coevaporated with Toluene (3*300 mL) to give the lactone 304 and uncyclized ester 305. The resulting yellowish syrup was used for next reaction without further purification. r-Butyldimethylsilyl chloride (27.1 g, 180 timol) was added to a mixture 20 of 304,305 and imidazole (12.3 g, 180 mmol) in CH 2

CI

2 (250 mL) and the reaction mixture was stirred for 4 h at room temperature. The resulting mixture was washed with water, dried (MgSOJ, filtered and concentrated to dryness. The residue was isolated by silica gel column chromatography using 4% EtOAc-hexanes as an fluent to give 307 (28.0g, 702% from compound 302) as a white crystalline solid, ip 48-50 "C; [a] 1 +105 .3 (c 1.60, CHCI,); 'H 25 NMR (CDCI 3 ) 5 0-07, 0.08 (2s, 2 x CR 3 ), 0.88 (s, Bu), 3.88 (m, 2M, H-5), 5.01 (m, 1H, H4), 6,73 (ps t, 1H, J 4 Hz); Anal. Calcd for C 10,,FOSi: C, 53.63; H, 7.77. Found: C, 53.70; H, 7.75. I-Acetyl- 4 -(tert-butyldimethylsilyloxy)methyl)-2-fluoro-2-buten-..olide (309). Lactone 307 (20.58 g, 83.54 mmnol) was dissolved in 200 mL of CHC1 2 under nitrogen atmosphere, 30 then the nixcture was cooled to -78 *C and 1.0 M solution of DIBAL-H in CH2CI, (125 mL) was added. The resulting mixture was stirred for 2 hours at -78 'C. The cold mixture was treated with dilute nitric acid, washed with water, and dried (Na 2 SOJ. Evaporation of the 61 solvent gave anomers of 308 as a pale yellow oil (16.6 g, crude yield 80 %), which was used for the next step without further purification. AcO (25 mL. 0.27 mo)) was added to a solution of 308 and pyridine (22 mL, 0.27 mol) in CHCl 2 (200 ml-) at 0 'C and the resulting mixture was stirred for 16 hours. The 5 reaction mixture was washed with dilute HCl, saturated NaHCO solution, and brine. The combined organic layer was dried, filtered, and concentrated to dryness. The residue was column chromatographed (6.5 % EtOAc/hexancs) to give 309 (12.6 g. 65 %) as a colorless oil. General procedure for condensation of acetate 309 with pyrimidine bases. 10 A mixture of uracil (420 mg, 3.75 mmol), hexamethyldisilazane (15 mL) and ammonium sulfate (20 mg) was refluxed for 3 hours under nitrogen. The clear solution obtained was concentrated to dryness in vacuo. TMSOTf (0.7 ml-, 3.14 nnol) were added to the solution of sugar 309 (728 mg, 2.50 mmol)) and the silylated base in dry DCE (20 mL) at 0 "C. The reaction mixture was stirred for 2 hours under nitrogen, poured into a cooled sat. NaHCO 15 solution (30 mL) and stirred for 15 min. The resulting mixture was washed, dried (Na 2

SO

4 ), filtered, and concentrated in vacuo. The crude product was purified by column chromatography (3 % MeO/CHCI) to give 310 (0.960 g, 2.73 mmol, 73 %) as an inseparable anomric mixture, which was used in the next step without separation. -1I5-O-(ert-Butyldimethylsilyl)-2,3-dideoxy-2-fluoro-L-gycero-pent-2enofuranosylurci 20 1(310). UV (CHCI,) ?, 257.5 m.; Anal. (C 3 JiFNO 4 Si) C, H, N. I-[5-O-(ert-Butyldimethylsi)yl)-2,3-dideoxy-2-fluoro-L-gycero-pent-2-enouranosylj thy mine (311). Silylated thymine (242 mg, 1.92 mrol), 307 (500 mg, 1.72 mmol), and TMSOTf (0-5 mL, 25 2.25 nunol) were reacted for 2 h to give a mixture of 311, which was purified by silica gel column chromatography ( 3 % MeOHI CI,) as an inseparable anomeric mixture ( 0.3 92 g, 1.10 mmol, 64%). UV (C C1 3 ) 4, 262.0 nm. Anal.(C 2 J1 2

IFN

2 0 4 Si) C, H, N. M-Benzoyl-1-[5-0-(tert-butyldimethylsilyl)-2,3-dideoxy-2-fluoro-(a,b)-1-glycero pent-2-enofuranosyljcytosine (312 and 313). 30 Silylated M-benzoyl cytosine (790 mg, 3.67 nunal), 307 (470 mg, 1.62 mmol), and TMSOTf (0.5 mL. 2.25 mmol) were reacted for 2 h to give mixtures of 312 and 313, which were purified by silica get column (30 % EtOAc/hexane) to afford f anomer 312 (0.34 g, 0.76 62 mmol, 471 %) as a white solid and a anomer 313 chromatography (0.23 g, 0.52 mmol, 31.8 %) as a white solid. 312: UV (CHCI 3 ) k 260.5 nm; Anal. (CJH1FN{0 4 Si) C, H, N.; 513: UV (CHC 3 ) X 260.5 nm.; Anal. (C 22

H

2

FN

3 0 4 Si) C, H, N. 5-Fluoro-1-I5-0-(rert-butyldimethylsilyl)-2,3-dideoxy-2-fluoro-(a,b--glycero-pent 5 -2-enofuranosylicytosine (314 and 315). Silylated 5-fluoro-cytosine (300 mg, 2.32 mmol), 309 (360 mg, 1.24 mmol), and TMSOTf (0.4 mL, 1.86 mmol) were reacted for 2 h to give a mixture of 314 and 315, which was purified by silica gel column chromatography (3 % MeOH/CH2Cl 2 ) to afford D anomer 314 10 as a white solid (168 mg, 37.8 %) and a anomer 315 (121 mg, 27.1 %) as a white solid. 314: UV (MeOH) X,. 281.5 nm; 315: UV (MeOH) A, 281-5 nm. l-(2,3-Dideoxy-2-fluoro-(aj)- L-gycero-pent-2-eno-furanosyl)uracii (316 and 317). Tetra-n-butylammoniurn fluoride (0.6 mL, 0.6 mmol) was added to a mixture of310 (177 mig, 0.52 mmol ) in THF (15 mL) and the reaction mixture was stirred at room temperature 15 for 15 min. The solvent was removed and the residue was purified by silica gel column chromatography (2 % MeOH/CHCI 3 ) to give p anomer 316 (52.8 mg, 0.23 mmol, 44.5 %) and a anomer 317 (35.1 mg, 0.15 mmol, 29.6 %). 316: UV (H2O) X,. 261.0 nam (pH 7); Anal. (C,H,FN 2 0.0.3H20) C, H, N. 317: UV (H20) X. 261. D nm (pH 7); Anal. (CHFN20,.0.2H 2 0) C, H, N. 20 ]-(2,3-Dideoxy-2-fluoro-(a,i )- L-gyero-pent-2-Cno-furanosyl)thymine (318 and 319). Tetra-n-butylammonium fluoride (0.8 mL, 0.8 mmol) was added to a mixture of 311 (240 mg, 0.67 mmol ) in THF (10 mL) at 0 *C and the reaction mixture was stirred at room temperature at rt for 15 min. The solvent was removed and the residue was purified by silica gel column chromatography (40 % THF/cyclohexane) to give P anomer 318 (66.5 mg, 0.28 25 mmol, 41 %) and a anomer 319 (52.8 rg, 0.23 mmol, 26 %). 318: UV (12O) k, 265.5 nn (pH 7); Anal. (C,OHFN 2 0 4 .0.4H 2 O) C, H, N. 319:tUV (H20) X. 266.0 nm (pH 7); Anal. (CHFNO 4 .03H 2 O) C, H, N. M-Benzoyl-1-(2,3-didory-2-fluoro-f-L-gyero-pent-2-enofurnosyl)cytosine (320). Tetra-n-butylammonium fluoride (I M in THF) (1 mL, I mmol) was added to a solution of the 30 @ anomer 312 (280 mg, 0-63 mmol) in THF (10 rmL) and the reaction was allowed to stir at room temperature for I h. The reaction mixture was concentrated under the reduced pressure 63 and the residue was purified by flash silica gel column using 2.5 % MeOH/CHCL: to give 320 (218 mg, 0.66 mmol, 75 %) as a white solid. UV (MeOH) k.. 260.5 nm. Anal. (C,,H 1

FN

3 0 4 ) C, H, N. N'Benzovl-1-(2,3-dideoxv-2-fluoro-a-t.-gycero-pent-2-enofuranosyl)cytosine (321). 5 Tetra-n-butylammonium fluoride (IM in THF) (1 mL, I inmol) was added to a solution of the a anomer 313 (280 mg, 0.63 mol) in THF (10 mL) and the reaction was allowed to stir at room temperature for lh. The reaction mixture was concentrated under the reduced pressure and the residue was purified by silica gel column chromatography using 2.5 % MeOH/CHCI, to give 321 ( 145.8 mg, 0.44 mmol, 69 %) as a white solid. 10 UV (MeOH) 4, 260.5 nm. Anal. (C,HI 4 FNO*.0.3H20) C, H, N. I-(2,3-dideoxy-2-fluoro-#-L-gycero-pent-2-euofuranosyl)cytosine (322). A solution of the P anomer (67.60 mg, 0.204 mmol) in MeOH (5 nL) was treated with NH/MeOH4 (10 mL saturated solution) and the reaction mixture was allowed to stir at room temperature until the disappearance of starting material was observed (10 h). The reaction mixture was 15 concentrated under reduced pressure and the residue was purified by preparative TLC using 12 % MeOH/CH2C 2 as an eluent. The material obtained from the plate gave 322 (43 mg, 93.1 %) as a solid on trituation with hexanes and diethylether. UV (H2O) X 266.5 nm (pH 7); Anal. (CH 0

FN

3 0 3 .0.41120) C, H, N. 1-(2,3-dideoxy-2-fluoro-a-L-gycero-pent-2-enofuranosyl)cytosine (323). A solution of the 20 a. anomer (65.90 mag, 0.199 mmol) in MeOH (5 mL) was treated with NH,/MeOH (10 mL saturated solution) and the reaction mixture was allowed to stir at room temperature until the disappearance of starting material was observed (16 h). The reaction mixture was concentrated under reduced pressure and the residue was purified by preparative TLC using 12 % McOH/CH 2 Cl as an eluent. The material obtained from the plate gave 322 (42.5 mg, 25 94.5 %) as a solid on trituation with hexanes and diethylether. UV (H20) X, 276.0 nm (pH 2), 267.0 nm(pi 7); Anal. (C4-,FN0,) C, H, N. 5-Fluoro-1-(2,3-dideoxy-2-fluoro-$i.L-gycero-pent-2-enofuranosyl)eytosine (324). Tetra-n-butylammonium fluoride (]M in THF) was added to a solution of the P anomer 314 in acetonitrilc and the reaction was allowed to stir at room temperature for lh. The reaction 30 mixture was concentrated under the reduced pressure and the residue was purified by flash silica gel column using 12 % MeOH/CHC 3 to give 324. 64 5-Fluore-1-(2,3-dideoxy-2-fluoro-a-L-gycero-pent-2-enofuranosylfrvtosine (325). Tetra-n-butylammonium fluoride (IM in THF) was added to a solution of the f anomer 315 in acetonitrile and the reaction was allowed to stir at room etperature for lh. The reaction mixture was concentrated under the reduced pressure and the residue was purified by flash 5 silica gel column using 12 % MeOHICHCI, to give 325. General procedure for condensation of acetate 309 with purine bases. A mixture of 6-chloropurine (I.20g, 7.75 mmol), hexamethyldisilazane (25 mL) and anmoniun sulfate (catalytic amount) was refluxed for 4 h under nitrogen. The clear solution was obtained was concentrated in vacuo and the residue was dissolved in dry DCE (10 mL) 10 and reacted with a solution of 307 (1.50 g, 5.17 mmol) in DCE (40 mL) and trimethylsilyl triflate (1.5 mL, 7.75 mmol) at room temperature. After stirring the mixture for I h at room temperature under nitrogen, the reaction solution was then poured into an ice cold saturated NaHCO) solution (20 mL) and stirred for 15 min. The organic layer was washed with water and brine, and dried over MgSO 4 . The solvents were removed under reduced pressure and 15 the residue was separated by silica gel column chromatography using 12.5 % ElOAc/hexanes to give anomeric mixture 326 (1.25 g, 62.9 %) as a syrup. 6-Chloro-9.[5-O-(rert-butyIdimethy1silyI)-2,3-dideoxy-2-fluoro-u-gycero-pent-2 enofuranosyllpuriue (326) 326: UV (MeOH) A. 265.0 nm; Anal. (CJH 22 C1FN 4 02Si) C, H, N. 20 6-Chloro-2-fluoro-9-[5-O-(er-butyldimethylsilyI)-2,3-dideoxy-2-fluoro (afl)-L-gycero-pent-2-enofuranosyl)purine (327 and 328). A mixture of silylated 2-fluoro-6-chloropurine [prepared from 1.170 g (6.78 mmol) of 2-fluoro-6-choropurine and dry DCE(40 mL) was stirred for 16 h at room temperature. After work-up similar to that of 326, purification by silica gel column chromatography (12 % 25 EtOAc/hexanes) gave 0 anomer 327 (685 mg, 1.70 mmol, 30.0 %/) as a white foam and a anomer 328 (502~mg, 1.25 mmol, 22.1 %) as an yellowish syrup. 327: UV (MeOH) X, 268.5 rnm. Anal. (C 6 1H1 2 1

F

2 C1 N,OSi) C, H, N., 328: UV (MeOH) A,, 269.0 nm. Anal. (C 1 6 H,,FZCI N 4 0 2 Si) C, H, N. 6-Chloro-9-(2,3-dideoxy-2-fluoro-(a, p)-L-gycero-pent-2-enofuranosyl)purine (329 and 30 330). A solution of 326 (12 g, 3.12 mmol) in dry CHCN (20 mL) was treated with TBAF (1 M solution in THF) (3.2 mL, 3.2 mmol) and stirred for I h. After evaporation of solvent, the 65 drvness was purified by column chromatography (3 % MeOH/CH Cl 3 ) to obtain 0 anomer 329 (296 mg. 35 %) as a white solid and o: anomer 330 (380 mg, 45 %) as a foam. 329: UV (MeOH) X. 265.0 un.; 330 IN (MeOH) 2, 265.0 nmn. (332). 5 6-Anino-2-fluoro- 9 -[5-0-(rerr-butyldimethylsily)-2,3-dideoxy-2-fluoro-,L-gyeero-pent 2 -enofuranosyl~purine (331) and 6--Cloro-2-amino-9-5--(rert-butyldimethysilyl)-2,3-dideoxy-2-fluoro-fL.-gycero-pent -2-enofuranosylipurine (332) Dry ammonia was bubbled into a stirred solution of 3217 (420 mg, 1.04 nunol) in dry DME 10 (35 mL) at room temperature ovemight. The salts were removed by filtration and the filtrate was evaporated under reduced pressure. The residue was purified by silica gel column chromatography (25 % ESOAc/hexanes) to give two compounds, 331 (114 mg, 0.30 mmol) as a white solid and 332 (164 mng, 0.41 mmnol) as a white solid. 331:JV (MeOH) X 268.5 nm. Anal. (C4i 2 3

F

2 N,0 2 SO.2Acetone) C, H, N, 332:LV 15 (MeOH) X,, 307.5 nm. Anal. (C,,HsiFN,0 2 CISi) C, H, N, Cl. 6 -Amin- 2 -fluoro-9-[5-O-(fert-butyldimethylsily)-2,3-dideoxy-2--fluoro-a -L-gycero-pent-2-enofuranosyllpurine (333) and 6 -Chloro-2-amino-9-1-5-0-(tert-butyldimethylsily)-2,3-dideoxy-2-fluoro.-a -L-gycero-pent-2-cofuranosyljpurine (334). 20 Dry ammonia was bubbled into a stirred solution of 333 (420 mg, 1.04 mmcl) in dry DME (35 mL) at room temperature overnight. The salts were removed by filtration and the filtrate was evaporated under reduced pressure. The residue was purified by silica gel column chromatography (25 % EtOAc/hexanes) to give two compounds, 332 (150 mg, 0.38 mmol) as a white solid and 333 (69.3 mng, 0 18 mmol) as a white solid. 25 333: UV (MeOH) X 269.0 nm. Anal. (CHF 2 N,02 Si-0.3Acetone) C, H, N, 334! UV (MeOH) k. 309.5 nm. Anal. (C 16 HF CIN,02Si) C, H, N. 9 -(2, 3 -dideoxy-2-fluoro-/.L-gycero-pent-2-enofuranosyl)adenine (335). A solution of 329 (100 mg, 0.369 mmol) and saturated NH 3 /MeOH (50 mL) was heated at 90 *C in a steel bomb for 24 h. After cooling to room temperature, the solvent was removed under reduced 30 pressure and the residual syrup was purified by column chromatography using 6 % MeOH/CHC, as an eluent to give 335 (70 mg, 75 %) as a white solid. 335: UV (H20) ,.G 66 '58 rnm (c 18,800) (pH 2), 258.5 nim (c 18.800) (pH 7), 258.5 nm (c 19,100) (pH 11). Anal.

(CIQN

10 FN,0,.0.2HO) C, H, N. 9-(2,3-dideoxy-2-fluoro-a-L-gycero-pent-2-enofuranosy l)adenine (336). A solution of 330 (99 mg, 0.366 mmol) and saturated NH 3 /MeOH (50 mL) was heated at 90 "C in a steel 5 bomb for 24 h. After cooling to room temperature, the solvent was removed under reduced pressure and the residual syrup was purified by column chromatography using 6 % MeOH/CHC1 3 as an fluent to give 336 (72 mng, 78 %) as a white solid. 336: UV (HO) ),. 258 nm (c 21,100) (pH 2), 259 ran (c 21,500) (pH 7), 259 nm (c 22,600) (pH 11). Anal. (C 1 oH 10

FN

5 0 2 .0.3MeOH) C, H. N. 10 9-(2,3-dideoxy-2-fluoro-$-L-gycro-pent-2-cnofuranosyl)hypoxantbine (337). A mixture of 329 (100 mg, 0.369 mmol), NaOMe (03 M solution in MeOH) (2.94 mL, 1.46 mmol) and

HSCH

2 C1HOH (0.1 mL, 1.46 mmol) in MeOH (20 mL) was refluxed for 4h under nitrogen. The reaction mixture was cooled, neutralized with glacial AcOH and evaporated to dryness under vacuum. The residue was purified by silica gel column chromatography (10 % 15 MeOH/CHCI 3 ) to afford 337 (74 mg, 80 %) as a white solid. 37: UV (H2O) X.. 247 mu (E12,400) (pH 2), 247.5 nm (E13,000) (pH 7), 253 nrm (E13,100) (pH 11). Anal.

(CQH,FN

4 O,.0.2H20) C, H, N. 9-(2,3~dideoxy-2-fluor-a-t-gyero-peint-2-enofuranosyl)hypoxanthinc (338). A mixture of 330 (100 mg, 0.369), NaOMe (0.5 M solution in MeOH) (2.94 mL, 1.46 mmol) and 20 HSCHCHLOH (0.1 mL, 1.46 nmol) in MeOH (20 niL) was refluxed for 4h under nitrogen. The reaction mixture was cooled, neutralized with glacial AcOH and evaporated to dryness under vacuum. The residue was purified by silica gel column chromatography (10 % MeOH/CHC 1 ) to afford 338 (70 ng, 80 %) as a white solid. 338: UV (H2O) X,. 247.5 rnm (& 12,700) (pH 2), 247.5 nm(c 13,700) (pH 7), 252.5 rn (E 13,100) (pHI 1I)- Anal, 25 (CI 0 HFN40 3 .0.3H2O) C, H, N. 2-Fluoro-6-amino-9-(2,3-dideoxy-2-flnoro-p-L-gycero-pent-2-euofuranosyl)purine (339). A solution of 31 (101 mg, 0.26 mmol) in dry acetonitrile (15 mL) was treated with TBAF (1 M solution in THF) (0.35 mL, 0.35 mmol) and stirred for 30 rmin. After evaporation of solvent, the dryness was purified by column chromatography (9 % CHCI2/MeOH) to obtain 30 339 (64.7 mng, 0.24 mmol, 92.3 %) as a white crystalline solid. UV (120) X,, 269.0 nm (pH 7). 67 2-Fluoro-6-smino-9-(2,3-dideoxy-2-fluoro-a -L-gycero-pent-2-en ofuranosvl)purine (340). A solution of 333 (73.4 mg, 0.19 mmol) in dry acexonitrile (10 mL) was treated with TBAF (1 M solution in THF) (0.25 mL. 0.25 mnmol) and stin-ed for 30 min. After evaporation of solvent, the dryness was purified by column chromatography (9 % 5 CH 7 CU/MeOH) to obtain 340 (46.2 mug, 0.17 mmol, 90.3 %) as a white crystalline solid. UV (1120) X.. 269.0 nm (pH 7). 2-Amino-6-chloro-9-(2, 3 -dideoxy-2-fluoro-#-L-gycrEro-pent-2-enofuranosyl)purine (341). A solution of 332 (143.5 mng, 0.40 rmol) in dry acetonitrile (15 mL) was treated with TBAF (1 M solution in THF) (0.6 mL, 0.60 immol) and stirred for 30 min. After evaporation of 10 solvent, the dryness was purified by column chromatography (5 % CHCL/MeOH) to obtain 341 (109 rag, 0.382 mrnol, 95.5 %) as a white crystalline solid. UV (H2O) . 308.5 rn (pH 7). 2-Arnino-6-chloro-9-(2,3-dideoxy.2-flnoro-a -L-gycero-pent-2-enofuranosyl)purine (342). A solution of 334 (145 mug, 0.36 mmol) in dry acetonitrile (10 mL) was treated with 15 TBAF (I M solution in THF) (0.5 mL, 0.50 mmol) and stirred for 30 min. After evaporation of solvent, the dryness was purified by column chromatography (9 % CH 2 Cl-/MeOH) to obtain 342 (99.9 mg, 0.35 mmol, 96.4 %) as a white crystalline solid UV (H20) 1 309.0 nm (pH 7). 9-(2,3-dideoxy-2-fluoro-#-L-Sycero-pent-2-enofuranosyl)guanine (343). A mixture of 20 341 (63.6 mg, 0.223 mmol), 2-mercaptoethanol (0.06 mL, 0.89 imol) and I N NaQMe ( 0.89 mL, 0.89 mmol) in MeOH (10 mL) was refluxed for 5 h under nitrogen. The mixture was cooled, neutralized with glacial AcOH and concentrated to dryness under reduced pressure. The residue was purified by column chromatography (12 % CH2CI/MeOH) to obtain 343 (30.1 mg, 0.113 mmol, 50.7 %) as a white solid. UV (H20) %,. 253.5 nm (pH 7). 25 9-(2,3-dideoxy-2-fluoro-a-L-gyccro-pent-2-enofuranoyl)guanine (344). A mixture of 342 (59.3 mg, 0.208 mmol), 2-mercaptoethanol (0.07 mL, 1.04 mmol) and I N NaOMe ( 1.04 mL, 1.04 mmol) in McOH (10 mL) was refluxed for 5 h under nitrogen. The mixture was cooled, neutralized with glacial AcOH and concentrated to dryness under vacuum. The residue was purified by column chromatography (12.5 % CH2CI2/MeOR) to obtain 344 (28.0 30 rug, 0.105 mmol, 50.5 %) as a white solid. UV (1-20) ,.,, 253.0 nm (pH 7). Synthesis of cis-(t)-Carbocyclic d4 Cytosine Nucleosides and their 5'Triphosphates 68 Referring to Scheme I I starting from diethyl diallylmalonate (701), the 4-carbethoxy-1,6 heptadiene (702) was synthesized in 78% yield (W. A. Nugent, J. Am. Chem. Soc,, 1995, 117, 8992-8998). Compound 703 was synthesized from compound 702 in 71% yield (L. E. Martinez, J. Org. Chem., 1996, 61, 7963-7966), and compound 705 was synthesized from 5 compound 704 in 43% yield (D. M. Hodgson, . Chem. Soc. Perkin Trans. 1, 1994, 3373 3378). The key intermediate cis-(±)-3-acetoxy-5-(aceroxymethyl)cyclopentene (708) can be alternatively synthesized from cyclopentadiene and formaldehyde in acetic acid using a Prins reaction (E. A. Saville-Stones, . Chem. Soc. Perkin Trans. I, 1991, 2603-2604) albeit it suffers low yield and inseparable problems; or from a bicyclic lactone which was synthesized 10 by multiple steps through 4 steps (F. Burlina. Bioorg. Med. Chem Leu, 1997, 7, 247-250). The latter methodology gave a chiral 7098 [(-)-enantiomerJ, although it needed to synthesized a chiral bicyclic lactone. N 4 -Acetyl-5-fluorocytosine was synthesized from 5-fluorocytosine and p-nitrophenyl acetate (A. S. Steinfeld, . Chem. Research (k), 19'79, 1437-1450). 69 Scheme 1] toCe copC cogEt wQCL/J E NaCN 2.6-dibTornophenol DMSo PbE oinene 701 702 703 -70 "C (PhSe)NaBH1, _ _ .- c2M EOHVO(acac 706 705 706 DMAP/CHCl 2 __C26 Id NaIIIMSOI Pd(PhP) 4 707 705 709 X i-F, T=H 710 -X-F.R-Ac 711 XuH.RwAc MeOH i) 2-chlorw-411. M 1,3,2-brmodioxa x phosphv*rir-onaf S 0 0 dkixan/DMF/Py a-04-0F-O N 0 i) Pyrophosphoic acidl 0'C- 0 BuN/DMF I IgiiN/O/PyITHF 714 X-F 712. X,, 715 X= H 713 X-H 70 Experimental Part GeneraL All reagents were used as received unless stated otherwise. Anhydrous solvents were purchased from Aldrich Chemical Co. Melting points (M-p.) were determined on an 5 Electrothermal digit melting point apparatus and are uncorrected, IH and 1 3C NMR spectra were taken on a Varian Unity Plus 400 spectrometer at room temperature and reported in ppm downfield from internal tetramethylsilane. 4-Carbethoxy-1,6-heptadiene (702). A mixture of diethyl diallymalonate (701; 50 g, 208 mmol), sodium cyanide (20.7 g, 422 mmol) and DMSO (166 mL) was heated at 160 *C for 6 10 h. After being cooled to r.t., the mixture was added to 400 mL of water and the product was extracted into hexane (4 x 100 niL). After evaporation of the solvent at reduced pressure, the residue was distilled (42-43 "C/ 1 Torr) to give 27.34 g (78%) of 702 as a colorless liquid. 4H NMR (400 MHz, CDCI 2 ) 5 5.80-5.70 (m, 2H, 2 CH=CH 2 ), 5.10-5.02 (m, 41, 2 CH=CR2), 4.14 (q, 211, J - 7.2 Hz, OCH 2 ), 2.54-2.48 (in, I H, CH), 2.41-2.34, 2.29-2.23 (2m, 411, 2 15 CH 2 ), 1.25 (t, J = 7.2 Hz, 3 H, CH 3 ). (*)-3-Cyclopentenecarboxylie Acid, Ethyl Ester (703). A flame-dried 500 mL flask was charged with 2,6-dibromophenol (1.20 g. 4.76 mmol), tungsten oxychloride (0.813 g, 2.38 nmol), and anhydrous toluene (25 mL). The resulting suspension was heated at reflux under nitrogen for I h, and then the solvent was evaporated in vacuo. The solid residue was broken 20 up with a spatula and dried in vacuo for 30 min. To the residue were added toluene (160 ML), Et4Pb (1.54 g, 4.76 mL), and 702 (22 g, 131.0 mmol). The mixture was heated at 90 *C under nitrogen for 1.5 h. After being cooled to r.t., the mixture was filtered through a celite, and the celite was rinsed with t-BuOMe. The combined filtrates were washed with 1% NaOH soln, water, and brine, and concentrated by evaporation at reduced pressure. The residue was 25 distilled (37-38 *C/I Torn) to give 13.06 g (71%) of 703 as a colorless liquid. IH NMR (400 MHz, CDC 3 ) 6 5.67 (s, 2H, CH=CH), 4.14 (q, 2H1 J 7.2 Hz, OCH 2 ), 3.11 (pentuplet, 3 7.6 Hz, IH, CH), 2.65 (d, 3 7.6 Hz, 411, 2 CH 2 ), 1.27 (t, J = 7.2 Hz, 3 H, CH 3 ). (*)-1-(Hydroxymethyl)-3.cyclopentene (704). To a cold (-78 *C) solution of 703 (7 g, 50 mmol) in dry THF (150 mL) was added LiAll1 (1 M soln in THF, 25 mL, 25 mmol), and the 30 reaction solution was stirred at -78 "C under argon for 4 h. Then the reaction solution was allowed to warm to 0*C, and 2.5 mL of water, 2.5 mL of 15% NaOH, and 7.5 mL of water were added sequentially. After warming to r.t., the precipitates were filtered through a cclite, 71 and the celite was washed with hot EtOAc. The combined filtrates were washed with 0.1 N NaOH, and brine. dried (MgSO 4 ), filtered, concentrated and dried in vacuo to give 4.294 g 184%) of 704 as a pale yellow liquid. I H NMR (400 MHz, CDC1 3 )6 5.68 (s, 2H, 2 CH=CH), 3.57 (d, J = 6.0 Hz, 2H. CH 2 OH), 2.54-2.48 (mt. 3H, CH + CH2), 215-2.10 (m, 2 H, CH 2 ). 5 cis-()-4-(Hydroxymethyl)-1,2-epoxycyclopentane (705). To a solution of 704 (930 mg, 9.1 mrnol), and vanadyl acetylacetonate (10 mg) in anhydrous CH 2 C1 2 (20 mL) was added ; BuO 2 H [3 M soln in CH 2 C1 2 , prepared from a mixture of i-BuO2H (70% by weight in water, 41 mL, 0-3 mol) and CH 2

C

2 (59 mL) by drying (2 x MgSO 4 ) and storage over 4A molecular sieve, 10 mL, -30 inol] dropwise. After stirring at r.t. for 24 h, aqueous Na 2

SO

3 (15% soln, 10 60 mL) was added, and the mixture was stirred at r.t. for 6 h. The organic layer was separated, washed with sat. NaHCO 3 , and brine, and concentrated. The residue was purified by flash chromatography on silica gel eluting with hexane/EtOAc (2:1) to give 460 Ing (43%) of 705 as a colorless liquid. 'H NMR (400 MHz, CDC 3 )d 3.54 (s, 2, (CH)20), 3.49 (t, J = 4.0 Hz, 2H, CI20H), 2.95 (bs, IH, OH) 2.44-2.40 (m, 1I, CH), 2.05-2.02 (In, 4 H, 2 CH 2 ). 13 C 15 NMR (100 MHz, CDCI 3 )3 66.9 (d, (CH)2O), 59.2 (t, CH 2 OH), 36.5 (d, CH), 31.4 (t, 2 CH 2 ). eia-(Q)-3-Acetoxy-S-(acetoxymethyl)eyclopentene (708). To a solution of diphenyl diselenenide (2.70 g, 8.65 minol) in anhydrous EtOH (100 miL) was added NaBH14 in portions. The solution was stirred until the yellow color turned to colorless, and then a solution of 705 (1.70 g, 14.4 mmol) in anbydrous THF (10 mL) was added. The reaction 20 solution was heated at reflux for I h under nitrogen, and then the solvent was evaporated in vacuo. To the residue was added EtOAc (80 mL) and water (30 nL). The organic phase was separated, washed with brine, dried (MgSO 4 ), filtered, concentrated and dried in vacuo. The obtained (t)-l-hydroxy-4-(hydroxymethyl)-2-(phenylselenenyl)-cyclopentane (706; light yellow oil) was used for the next reaction directly without further purification. To the crude 25 product 706 were added anhydrous CH 2

C

2 (60 mL), Ec 3 N (30 mL, 216 mmol), and DMAP (50 mg). The resulting solution was cooled to 0 "C, and Ac 2 O (14.7 g, 144 mmol) was added dropwise. After being stirred at r.t. under argon overnight, evaporation of the solvent provided (±)- I-acetoxy- 4 -(acetoxymethyl)-2-(phenylselenenyl)-cyclopentane (707; light yellow oil). To a cold (0 "C) solution of 707 in CH 2

C

2 (50 mL) containing 3 drops of 30 pyridine was added 30% H202 soln (20 mL) over a period of 5 min. After being stirred at 0 *C for 30 min and at r.t. for another 30 min, the reaction mixture was diluted by addition of CHC1 2 (50 mL). The organic phase was separated, washed with water, sat. NaHCO 3 , and 72 brine. dried (MgSO 4 ). filtered. and concentrated by evaporation in vacuo. The residue was punfied by flash chromatography on silica gel eluting with 0-10% EIOAc in hexane to give 2.254 g (79%, for the three steps) of 708 as a pale brown liquid. I H NMR (400 MHz,

CDCI

3 )6 6.01-6.00, 5.92-5.90 (2m, 2H, CH=CH), 5.66-5.64 (m, IH, H-3), 4.04 (d, J = 6.8 5 Hz, 2H, CH 2 Q), 2.98-2.92 (m, IfH, H-5), 2.53-2.46 (n, iH, H-4a), 2.08,2.04 (2s, 611, 2 CH 3 ), 1,60-1.54 (m, 2H, H-4b). 13 C NMR (100 MHz, CDCI 3 )8 171.1, 170.9 (2s, 2 C=O), 137.0, 131.4 (2d, CH-CH), 79.2 (d, C-3), 67A (t, CH 2 O), 43.7 (d, C-5), 33.4 (t, C-4), 21.3,20.9 (2q, 2 CH 3

)

eis-(t)-Carbocyclic 5'-O-acetyl- 2 ',3'-didehydro-Z',3'-dideoxy-S-fluorocytidine (709). A 10 suspension of 5-fluorocytosine (258 mg, 2 mmol) and NaH (58 mg, 2.4 mmol) in anhydrous DMSO (IS mL) was heated in a pre-warmed oil bath at 70 "C for 30 min. Then the resulting solution was cooled to r-t., and Pd(PPh 3

)

4 (73 mg, 0.063 mmol) and a solution of 708 (298 mg, 1.5 mmol) in anhydrous TRIF (2 mL) were added respectively. The reaction mixture was stirred at 70 "C under argon for 3 days. After removal of the solvent by evaporation in vacuo, 15 the residue was treated with CH 2

C

2 (50 mL). The precipitates were filtered through a celite, and the celite was washed with CH 2

C

2 . The combined filtrates were concentrated, and the residue was purified by flash chromatography on silica gel eluting with 0-5% MeOH in

CH

2 Cl 2 to give 40 mg (10%) of 709 as a light brown solid. Recrystallization from MeOH/CH 2 Cl 2 /hexane provided pure product as white powders. M.p. 182-184 C. 1 H NMR 20 (400 MHz, CDC1)6 7.43 (d, J = 6.0 Hz, IH, H-6), 6.18-6.16 (m, IH, H-3), 5.83-5,81 (m, IN, H-2', 5.73-5.71 (m, IH, H-1'), 4.23-4.21, 4.08-4.04 (2m, 2H, CH 2 O), 314-3.12 (m, 111, 14-4), 2.92-2.84 (m, IH, H-6a), 2.08 (s, 3H, CH 3 ), 1.41-1.35 (m, IH, H-6'b). cis-(d)-Carbocyclc N 4 ,5'-O-diacetyl-2',3'-didehydro--2',3'-dideoxy-5-fluorocytidine (710). In an analogy manner to the procedure for 709, the title compound 710 was prepared 25 from 708 (560 mg, 2.28 mmol) and Nkacetyl-5-fluorocytosine (726 mg, 4.24 mmol); 560 mg (64%, brown oil). This crude product was used directly for the next reaction without further purification. cis-()-Carbocyclic N4,5'-0-diacetyl-2',3'-didebydre-2',3'-dideoxycytidine (711). In an 30 analogy manner to the procedure for 709, the title compound 711 was prepared from 708 (272 mg, 1.37 mol) and M-aceryleytosine (316 mg, 2.06 rmol): 108 mg (27%) of white powders. M.p. 169.5-171.5'C. 'H NMR (400 MHz, CDC1)6 8.80 (bs, I H, NH), 7.72 (d, J= 73 6.8 Hz. IH, H-6), 7.39 (d, J 6.8 Hz, 1I. H-5), 6.19-6.17 (m, I H. h-3),5.86-5.81 (m 1H. B-2'), 5.77-5.75 (in, I H, H-l'), 4.17-4.13. 4.07-4.02 (2m, 211, CH 2 O), 3.18-3.10 (m. I H. H 4'), 2.96-2.88 (m, 1H, -6'a), 2.27, 2.06 (2s. 6H, 2 CH3), 1.43-1.37 (m, I H, H-6'b). 1 3 CNMR (100 MHz, CDCI 3 )6 170.8 (s, 2 C=0), 162.0 (s, C-4), 155.6 (s, C-2), 145.3 (d, C-6), 139.2 (d, 5 C-3'), 130.0 (d, C-2'), 96.8 (d, C-5), 66.3 (t, C-5'), 62.8 (d, C-i'), 44.2 (d, C-4'), 34.7 (t, C-6'), 25.0, 20.9 (2q, 2 CH 3 ). cis-(Q)-Carbocyclic 2 ',3'-didehydrn-2',3'-dideoxy-5-fluorocytidine (712). To a flask containing 709 (33 mg, 0.12 mmol) was added NaOMe (0.5 M soln in MeOH, 0.5 mL). The reaction solution was stirred at r.t. for I h, and then the solvent was evaporated in vacuo. The 10 residue was purified by flash chromatography on silica gel eluting with 5-10% MeOH in

CH

2 CI to give 17 mg (61%) of 712 as a light brown solid. Recrystallization from MeOHICH 2

CI

2 /hexane provided pure product as white powders. M.p. 205.5-206.0*C. 'H NMR (400 MHz, DMSO-d 6 )6 7.66 (d, J 6.0 Hz, IH, H-6), 7.60,7.40(2bs, 2H, NI 2 ), 6.06 6.05 (m. 1H, 11-3'), 5.68-5.65 (m, 1H, H-2'), 5.53-5.50 (n, IH, H-'), 4.77-4.75 (n, IH, H4'), 15 3.50-3.48, 3.41-3.37 (2m, 2H,H-5'), 2.79-2.77 (m, IN H-6a), 1.34-1.27 (m, IH,H-6'b). 13C NMR (100 MHz, DMSO-d6 157.0 (d, Jc.F = 11.9 Hz, C-4), 154.0 (s, C-2), 139.2 (d, C-3'), 135.8 (d, JC-F =241.3 Hz, C-5), 130.2 (d, C-2'), 126.8 (dC, JC-F 11.8 Hz, C-6), 63.5 (t, C-5% 61.3 (d, C-I'), 47.2 (d, C-4, 33.3 (t, C-6'). MS (FAB) n/e 226 (MH). Anal. (C 1

H

12

FN

3 0W caled C 53.33, H 5.37, N 18.66; found C 53.10, H 5.40, N 18.44. In an analogy manmer to the 20 above procedure, the title compound 712 was also prepared from 710 (750 mg, 2.42 mmol): 320 mg (59%, white powders). cis-(4)-Carbocyclic 2',3'-didchydro-2',3'-did soxycytidine (713). In an analogy manner to the procedure for 712, the title compound 713 was prepared from 711 (75 mg, 0.257 mmol): 48 mg (90%, white solid). M.p. 200-201"C. IH NMR (400 MHz, DMSO-d6)6 7.40 (d, J =7.2 25 Hz, IH, H-6), 7.03, 6.95 (2K, 2H, NH 2 ), 6.07-6.05 (m, 1H, H-3', 5.67 (d, J = 7.2 Hz, IH, 11 5), 5.65-5.64 (m, 1H, H-2), 5.55-5.52 (m, 1 H, H-l'), 4.71-4.68 (m, I H, H4'), 3.43-3.36 (m, 2H, H-5'), 2.78-2.76 (m, 1H1, H-6'a), 1.24-1.18 (m, 11-I, H-6'b). 1C NMR (100 MHz, DMSO d6)6 165.5 (s, C-4), 155.8 (s, C-2), 142.2 (d, C-6), 138.6 (d, C-3'), 130.5 (d, C-2'), 93.7 (d, C 5), 63.9 (t, C-'),60.8 (d, C-'), 47.3 (d, C4'), 34.0 (t, C-6). MS (FAB) m/e 208 (MH'). 30 Anal. (CH 13

N

3 , calcd D 57.96, H 6.32, N 20.28; found C 57.35, H 6.27, N 20.02. HRMS (FAB) caled for (CH 1 N,0 2 ):208.1086; found 208.1088. 74 cis-(tFCarbocyclic 2',3'-didehydro-2',3'-dideoxy-5-fluorocytidine 5'-triphosphate, triethylhydrogenamnmonium salt (714). To a solution of 712 (10 mg) in anhydrous DMF (0.3 mnL) and pyridine (0.1 mi) was added a I M solution of 2-chloro-4H--1 ,3,2 benzodioxaphosphorin-4-one in anhydrous 1,4-dioxane (0.05 mL). The reaction solution was 5 stirred at r.t. for 15 min. Then, a solution of I M pyrophosphoric acid-Bu 3 N in anhydrous DMF (0.12 ml), and BU3N (0.05 mL) was added sequentially. After stirring at r.t. for another 15 min, a solution of 1 2 /H2O/pyridine/THF was added to the above solution dropwise until the iodine color persisted (about 0.5 mL), and then the mixture was concentrated by evaporation in vacua. The residue was dissolved in water (2 mJL), washed with CH 2

CI

2 (3 x 1 10 mL), filtered, and purified by FPLC (column: HiLoad 26/10 Q Sepharose Fast Flow; buffer A: 0.01 M Et 3

NHCO

3 ; buffer B: 0.7 M Et 3

NHCO

3 ; flow rate: 10 mL/min; gradient: increasing buffer B from 0% at beginning to 10% at 4 min, then to 100% at 64 min). Collection and lyophilization of the appropriate fractions afforded 714 as a colorless syrup. HPLC [column: 100 x 4.6 mm Rainin Hydropore SAX ionic exchange; buffer A: 10 mM 15 NHjN 2

PO

4 in 10% MeOH/H 2 0 (pH 5.5); buffer B: 125 mM NH 4

H

2

PQ

4 in 10/1 MeOH/H 2 0 (pH 5.5); flow rate: 1.0 mUL/min; gradient: increasing B from 0% at beginning to 100% at 25 min} retention time: 1 .9 min. MS (FAB) mle 464 ([M-H]*). cis-(d)-Carbocyclic 2',3'-didehydro-2',3'-dideoxycytidine 5'-phosphate (715). In an analogy manner to the procedure for 714, the title compound 715 was prepared from 713. 20 HPLC (same conditions as above) retention time: 12.1 min. MS (FAB) m/e 446 ([M-H]'). Inhibitory effect of (i)-Carboxy-D4FC-triphospbate against HIV-1 reverse transcriptase. Extension assays were performed using a r(I)n-(dC) 12-18 homopolymer template primer (Pharmacia, Piscataway, NJ) and the HIV-1 heterodimer p66/51 reverse 25 transcriptase (RT, Biotechnology General, Rehovat, Israel). The standard reaction mixture (100 pl) contained 100 rmM Tris hydrochloride (pH 8.0), 50 mM KC1, 2 mM MgCl 2 , 0.05 units/ml r(1)r(dC)12-18, 5 mM DTT, 100 g/ml Bovine Serum Albumin, and 1 pM 3 -dCTP (23 Ci/mmol). 3TCTP (0.00 l-50 pM) was the positive control. Compounds were incubated I hr at 37 "C in the reaction mixture with I unit HIV-1 30 RT. The reaction was stopped with the addition of an equal volume of cold 10% TCA/0.05% sodium pyrophosphate and incubated 30 minutes at 4*C. The precipitated 75 nucleic acids were harvested onto fiberglass filter paper using a Packard manual harvester (Meriden. CT)- The radiolabel uptake in counts per minute (cpm) was determined using a Packard 9600 Direct Beta counter. IV. Anti-HIV Activity 5 in one embodiment, the disclosed compounds or their pharmaceutically acceptable derivatives or salts or pharmaceutically acceptable formulations containing these compounds are useful in the prevention and treatment of HIV infections and other related conditions such as AIDS-related complex (ARC), persistent generalized lymnphadenopathy (PGL), AIDS related neurological conditions, anti-HIV antibody positive and HPV-positive conditions, 10 Kaposi's sarcoma, thrombocytopenia purpurea and opportunistic infections. In addition, these compounds or formulations can be-used prophylactically to prevent or retard the progression of clinical illness in individuals who are anti-HIV antibody or HIV-antigen positive or who have been exposed to 1V. The ability of nucleosides to inhibit HIV can be measured by various experimental 15 techniques. One technique, described in detail below, measures the inhibition of viral replication in phytohemagglutinin (PHA) stimulated human peripheral blood mononuclear (PBM) cells infected with mV-1 (strain LAV). The amount of virus produced is determined by measuring the virus-coded reverse transcriptase enzyme. The amount of enzyme produced is proportional to the amount of virus produced 20 Antiviral and cytotoxicity asays: Anti-HIV-1 activity of the compounds is determined in human peripheral blood mononuclcar (PBM) cells as described previously (Schinazi, R. F.; McMillan, A.; Cannon, D.; Mathis, R.; Lloyd, R. M. Jr.; Peck, A.; Sommadossi, J.-P.; St. Clair, M.; Wilson, J.; Furman, P. A.; Painter, G.; Choi, W.-B.; Liotta. D. C. Antimicroh Agents Chemother. 1992, 36, 2423; Schinazi, R. F.; Sommadossi, J.-P.; 25 Saalmann, V.; Cannon, D.; Xie, M.-Y.; Hart, G.; Smith, G.; Hahn, E. Ansimicrob. Agents Chemother. 1990,34, 1061). Stock solutions (20-40 mM) of the compounds were prepared in sterile DMSO and then diluted to the desired concentration in complete medium. 3'-azido-3'-deoxythymidine (AZT) stock solutions are made in water. Cells are infected with the prototype HIV-1], at a multiplicity of infection of 0.01. Virus obtained from the cell 30 supernatant are quantitated on day 6 after infection by a reverse transcriptase assay using poly(rA),oligo(dT) 2

.

1 as template-primer. The DMSO present in the diluted solution (< 0.1%) should have no effect on the virus yield. The toxicity of the compounds can be 76 assessed in human PBM, CEM. and Vero cells- The antiviral EC, and cytotoxicity ]Ci is obtained from the concentration-response curve using the median effective method described by Chou and Talalay (Adv. Enzyme ReguL 1984. 22, 27) Three-day-old phytohemagglutinin-stimulated PBM cells 10* cells/mi) from hepatitis 5 B and HIV- I seronegative healthy donors are infected with HIV- I (strain LAV) at a concentration of about 100 times the 50% tissue culture infectious dose (TICD 50) per ml and cultured in the presence and absence of various concentrations of antiviral compounds. Approximately one hour after infection, the medium, with the compound to be tested (2 times the final concentration in medium) or without compound, is added to the flasks (5 10 ml; final volume 10 ml). AZT is used as a positive control. The cells are exposed to the virus (about 2 x 10' dpm/ml, as determined by reverse transcriptase assay) and then placed in a CO 2 incubator- HIV- I (strain LAV) is obtained from the Center for Disease Control, Atlanta, Georgia. The methods used for culturing the PBM cells, harvcsting the virus and determining the reverse transcriptase activity are those 15 described by McDougal et aL (J immun. Meth. 76, 171-183, 1985) and Spira et al. (J. Clin. Merh, 25, 97-99, 1987), except that fungizone was not included in the medium (sec Schinazi, et al., Antimicrob. Agents Chemother. 32, 1784-1787 (1988); Id., 34:1061-1067 (1990)). On day 6, the cells and supernatant are transferred to a 15 ml tube and centrifuged at about 900 g for 10 minutes- Five ml of supernatant are removed and the virus concentrated 20 by centrifugation at 40,000 rpm for 30 minutes (Beckman 70.1 Ti rotor). The solubilized virus pellet is processed for determination of the levels of reverse transcriptase. Results are expressed in dpmn/ml of sampled supernatant. Virus from smaller volumes of supernatant (I ml) can also be concentrated by centrifugation prior to solubilization and determination of reverse transcriptase levels. 25 The median effective (EC.) concentration is determined by the median effect-method (Antimicrob. Agents Chemother 30,491498 (1986). Briefly, the percent inhibition of virus, as determined from measurements of reverse transcriptase, is plotted versus the micromolar concentration of compound. The EC 0 is the concentration of compound at which there is a 50% inhibition of viral growth. 30 Mitogen stimulated uninfected human PBM cells (3.8 x 101 cells/ml) can be cultured in the presence and absence of drug under similar conditions as those used for the antiviral assay described above. The cells are counted after 6 days using a hdmacytometer and the 77 trypan blue exclusion method, as described by Schinazi et al.. Antimicrobial Agents and Chemotherapy, 22(3), 499 (1982). The IC, 0 is the concentration of compound which inhibits 50% of normal cell growth. Table 7 provides data on the anti-HIV activity of selected compounds. Using this 5 assay, it was determined that (+)-carbocyclic-D4FC-TP (2',3'.unsaturated-5-fluorocytidine) exhibited an EC, 0 of 0.40 pM, and (t)-carbocyclic-04C-TP (2',3'-unsaturated cytidine) exhibits an ECO of 0.38 pM. V. Anti-Hepatitis B Activity 10 The ability of the active compounds to inhibit the growth of hepatitis virus in 2.2.15 cell cultures (HepG2 cells transformed with hepatitis vision) can be evaluated as described in detail below. A summary and description of the assay for antiviral effects in this culture system and the analysis of 14BV DNA has been described (Korba and Milman, 1991, Antiviral Res., 15 15:217). The antiviral evaluations are optimally performed on two separate passages of cells. All wells, in all plates, are seeded at the same density and at the same time. Due to the inherent variations in the levels of both intracellular and extracellular HBV DNA, only depressions greater than 3.5-fold (for HBV virion DNA) or 3.0-fold (for HBV DNA replication intermediates) from the average levels for these HBV DNA forms in 20 untreated cells are considered to be statistically significant (P<0.05). The levels of integrated HBV DNA in each cellular DNA preparation (which remain constant on a per cell basis in these experiments) are used to calculate the levels of intracellular HBV DNA forms, thereby ensuring that equal amounts of cellular DNA are compared between separate samples. Typical values for extracellular HBV virion DNA in untreated cells ranged from 50 to 25 150 pg/im culture medium (average of approximately 76 pg/ml). Intracellular H1BV DNA replication intermediates in untreated cells ranged from 50 to 100 pg/pg cell DNA (average approximately 74 pg/pg cell DNA). In general, depressions in the levels of intracellular HBV DNA due to treatment with antiviral compounds are less pronounced, and occur more slowly, than depressions in the levels of HBV virion DNA (Korba and Milman, 1991, Antiviral Res, 30 15:217). The manner in which the hybridization analyses can be performed for these experiments resulted in an equivalence of approximately 1.0 pg of intracellular HBV DNA to 78 2-3 genomic copies per cell and 1.0 pg/mi of extracellular HBV DNA to 3 x 10' viral particles/ml. Toxicity analyses were performed to assess whether any observed antiviral effects are due to a general effect on cell viability. The method used herein are the measurement of the 5 uptake of neutral red dye, a standard and widely used assay for cell viability in a variety of virus-host systems, including HSV and HIV. Toxicity analyses are performed in 96-well flat bottomed tissue culture plates. Cells for the toxicity analyses are cultured and treated with test compounds with the same schedule as described for the antiviral evaluations below. Each compound are tested at 4 concentrations, each in triplicate cultures (wells "A", "B", and 10 "C"). Uptake of neutral red dye are used to determine the relative level of toxicity. The absorbance of internalized dye at 510 urn (A,) are used for the. quantitative analysis. Values are presented as a percentage of the average A. values in 9 separate cultures of untreated cells maintained on the same 96-well plate as the lest compounds. VL Anti-Hepatitis C Activity 15 Compounds can exhibit anti-hepatitis C activity by inhibiting HCV polymerase, by inhibiting other enzymes needed in the replication cycle, or by other known methods. A number of assays have been published to assess these activities. WO 97/12033, filed on September 27, 1996, by Emory University, listing C. Hagedorn and A. Reinoldus as inventors, and which claims priority to U.S.S.N. 60/004,383, 20 filed on September 1995, describes an HCV polymerase assay that can be used to evaluate the activity of the compounds described herein. This application and invention is exclusively licensed to Triangle Pharmaceuticals. Inc., Durham, North Carolina. Another HCV polymerase assays has been reported by Bartholoneusz, et al., Hepatitis C virus (HCV) RNA polymerase assay using cloned HCV non-structural proteins; Antiviral Therapy 1996:1(Supp 25 4) 18-24. VI. Treatment of Abnormal Cellular Proliferation In an alternative embodiment, the compounds are used to treat abnormal cellular proliferation. The compound can be evaluated for activity by testing in a routine screen, such as that performed cost by the National Cancer Institute, or by using any other known screen, 30 for example as described in WO 96/07413. The extent of anticancer activity can be casily assessed by assaying the compound according to the procedure below in a CEM cellar other tumor cell line assay. CEM cells are 79 human lymphorna cells (a T-lymphoblastoid cell line that can be obtained from ATCC, Rockville. MD). The toxicity of a compound to CEM cells provides useful information regarding the activity of the compound against tumors. The toxicity is measured as IC, micromolar). The IC6 0 refers to that concentration of test compound that inhibits the growth 5 of 50% of the tumor cells in the culture. The lower the ICm, the more active the compound is as an antitumor agent, In general, 2'-fluoro-nucleoside exhibits antimor activity and can be used in the treatment of abnormal proliferation of cells if it exhibits a toxicity in CEM or other immortalized tumor cell line of less than 50 micromolar, more preferably, less than approximately 10 micromolar, and most preferably, less than 1 micromolar. Drug solutions, 10 including cycloheximide as a positive control, are plated in triplicate in 50 p1 growth medium at 2 times the final concentration and allowed to equilibrate at 37"C in a 5% C2 incubator. Log phase cells are added in 50 g1 growth medium to a final concentration of 2.5 x I0) (CEM and SK-MEL-28), 5 x I0 (MMAN, MDA-MB-435s, SKMES-1, DU-I 45, LNCap), or I x 10 (PC-3, MCF-7) cells/well and incubated for 3 (DU-145, PC-3, MMAN), 4 (MCF-7, SK 15 MEL-28, CEM), or 5 (SK-MES-1, MDA-MB-435s, LNCaP) days at 37"C under a 5% CO 2 air atmosphere. Control wells include media alone (blank) and cells plus media without drug. After growth period, 15 pl of Cell Titer 96 kit assay dye solution (Promega, Madison, WI) are added to each well and the plates are incubated 8 hr at 37"C in a 5% CO, incubator. Promega Cell Titer 96 kit assay stop solution is added to each well and incubated 4-8 hr in the 20 incubator. Absorbance is read at 570 nm, blanking on the medium-only wells using a Biotek Biokinetics plate reader (Biotek, Winooski, VT). Average percent inhibition of growth compared to the untreated control is calculated. ICo, IC, slope and r value are calculated by the method of Chou and Talalay. Chon T-C, Talalay P, Quantitative analysis of dose-effect relationships: The combined effects of multiple drugs or enzyme inhibitors. Adv Enzyme 25 Regul 1984,22:27-55. The active compound can be administered specifically to treat abnormal cell proliferation, and in particular, cell hyperproliferation. Examples of abnormal cell proliferation include, but are not limited to: benign tumors, including, but not limited to papilloma, adenoma, firoma, chondroma, osteoma, lipoma, hemangioma, lymphangioma, 30 leiomyoma, rhabdomyoma, meningioma, neuroma, ganglioneuroma, nevus, pheochromocytoma, neurilemona, fibroadenoma, teratoma, hydatidifonn mole, granuosa theca, Brenner tumor, arrhenoblastoma, hilar cell tumor, sex cord mesenchyme, interstitial 80 cell tumor. and thyoma as well as proliferation of smooth muscle cells in the course of development of plaques in vascular tissue; malignant tumors (cancer), including but not limited to carcinoma, including renal cell carcinoma, prostatic adenocarcinoma, bladder carcinoma, and adenocarcinoma, fibrosarcona. chondrosarcoma, osteosarcoma, liposarcoma, s hemarngiosarcoma, lymphangiosarcoma, leiomyosarcoma, rhabdomyosarcoma, myelocytic leukemia, eryth-oleukemia, multiple myeloma, glioma, meningeal sarcoma, thyoma, cystosarcoma phyllodes, nephroblastoma, teratoma choriocarcinoma, cutaneous T-cell lymphoma (CTCL), cutaneous tumors primary to the skin (for example, basal cell carcinoma, squamous cell carcinoma, melanoma, and Bowen's disease), breast and other tumors 10 infiltrating the skin, Kaposi's sarcoma, and premalignant and malignant diseases of mucosal tissues, including oral, bladder, and rectal diseases; preneoplastic lesions, mycosis fungoides, psoriasis, dermatomyositis, rheumatoid arthritis, viruses (for example, warts, herpes simplex, and condyloma acuminata), molluscum contagiosum, premalignant and malignant diseases of the female genital tract (cervix, vagina. and vulva). The compounds can also be used to 15 induce abortion. In this embodiment, the active compound, or its pharmaceutically acceptable salt, is administered in an effective treatment amount to decrease the hyperproliferation of the target cells. The active compound can be modified to include a targeting moiety that concentrates the compound at the active site. Targeting moieties can include an antibody or antibody 20 fragment that binds to a protein on the surface of the target cell, including but not limited to epidermal growth factor receptor (EGFR), c-Esb-2 family of receptors and vascular endothelial growth factor (VEGF). VI. Pharmaceutical Compositions Humans suffering from any of the disorders described herein can be treated by 25 administering to the patient an effective amount of the active compound or a pharmaceutically acceptable derivative or salt thereof in the presence of a pharmaceutically acceptable canier or diluent. The active materials can be administered by any appropriate route, for example, orally, parenterally, intravenously, intradermally, subcutaneously, or topically, in liquid or solid form. A preferred dose of the compound for all of the 30 abovementioned conditions will be in the range from about I to 50 mg/kg, preferably I to 20 mg/kg, of body weight per day, more generally 0.1 to about 100 mg per kilogram body weight of the recipient per day. The effective dosage range of the pharmaceutically 81 acceptable derivatives can be calculated based on the weight of the parent nucleoside to be delivered. If the derivative exhibits activity in itself. the effective dosage can be estimated as above using the weight of the derivative. or by other means known to those skilled in the art. The compound is conveniently administered in unit any suitable dosage form, 5 including but not limited to one containing 7 to 3000 mg, preferably 70 to 1400 mg of active ingredient per unit dosage form. A oral dosage of 50-1000 mg is usually convenient. Ideally the active ingredient should be administered to achieve peak plasma concentrations of the active compound of from about 0.2 to 70 pM, preferably about 1.0 to 10 pM. This may be achieved, for example, by the intravenous injection of a 0.1 to 5% solution 10 of the active ingredient, optionally in saline, or administered as a bolus of the active ingredient. The concentration of active compou d in the drug composition will depend on absorption, inactivation, and excretion rates of the drug as well as other factors known to those of skill in the art. It is to be noted that dosage values will also vary with the severity of 15 the condition to be alleviated. It is to be furt her understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person adxr inistering or supervising the administration of the compositions, and that the concentration rar ges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed composition. The active ingredient 20 may be administered at once, or may be div ded into a number of smaller doses to be administered at varying intervals of time. A preferred mode of administration >f the active compound is oral. Oral compositions will generally include an iner. diluent or an edible carrier. They may be enclosed in gelatin capsules or compressed nto tablets. For the purpose of oral therapeutic 25 administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nat ; a binder such as microcrystalline cellulose, 30 gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a s eetening agent such as sucrose or saccharin; or a g2 flavoring agent such as peppermint. methyl salieviate, or orange flavoring. WhCn the dosage unit form is a capsule, it can contain, in addition to material of the above type, a liquid carrier such as a fatty oil. In addition, dosage unit forms can contain various other materials which modify the physical form of the dosage unit, for example, coatings of sugar, shellac, or other 5 enteric agents. The compound can be administered as a component of an elixir, suspension, syrup, wafer, chewing gum or the like. A syrup may contain, in addition to the active compounds, sucrose as a sweetening agent and certain preservatives, dyes and colorings and flavors. The compound or a pharmaceutically acceptable derivative or salts thereof can also be 10 mixed with other active materials that do not impair the desired action, or with materials that supplement the desired action, such as antibiotics, antifungals, anti-inflammatories, or other antivirals. including other nucleoside compounds. Solutions or suspensions used for parenteral, intradermal, subcutaneous, or topical application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, 15 polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. The parental preparation can be enclosed in ampoules, disposable 20 syringes or multiple dose vials made of glass or plastic. If administered intravenously, preferred carriers are physiological saline or phosphate buffered saline (PBS). In a preferred embodiment, the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release 25 formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation. 30 Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodics to viral antigens) are also preferred as pharmaceutically acceptable carriers. These may be prepared according to methods known to those skilled in the art, for 83 example. as described in U.S. Patent No. 4,522.811 (which is incorporated herein by reference in its entirety). For example, liposome formulations may be prepared by dissolving appropriate lipid(s) (such as stearoyl phosphatidyl ethanolamine, stearoyl phosphatidyl choline, arachadoyl phosphatidyl choline, and cholesterol) in an inorganic solvent that is then 5 evaporated, leaving behind a thin film of dried lipid on the surface of the container. An aqueous solution of the active compound or its monophosphate, diphosphate, and/or triphosphate derivatives is then introduced into the container. The container is then swirled by hand to free lipid material from the sides of the container and to disperse lipid aggregates, thereby forming the liposomal suspension, 10 Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not to the exclusion of any other element, integer or step, or group of elements, integers or steps. It will be appreciated by persons skilled in the art that numerous variations and/or modifications = may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive. All publications mentioned in this specification are herein incorporated by reference. Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed in Australia before the priority date of each claim of the application. 84

Claims (3)

1. 85. The claims defining the invention are as follows; 1. A method for the treatment of a HCV infection in a host in need thereof comprising administering an effective treatment amount of a #-D-2'-fluoronuclcoside, or a pharmaceutically acceptable salt or prodrug thereof, optionally in a pharmaceutically 5 acceptable carrier or diluent. 2. The method of claim 1, wherein the -D-2'-fluoronucleoside has a pyrimidine base. 3. The method of claim 2, wherein the pyrimidine base is selected from the group consisting of thymine, uracil, 5-halouracil, 5-fluorouracil, cytosine, 5-fluorocytosine, 5-methylcytosine, 6-aza-pyrimidine, 6-azacytosine, 2- and/or 4-mercaptopyrimidine, 10 C 5 -alkylpyrimidinc, C5-benzylpyrimidinc, C 5 -halopyrimidine, C 5 -vinylpyrimidine, C5-acetylenic pyrimidine, C'-acyl pyrimidinc, C 5 -hydroxyalkyl purine, C 5 aniidopyrimidine, C 5 -cyanopyrimidine, C 5 -nitropyrimidine, and C 5 aminopyrimidinc. 4. The method of claim 2, wherein the pyrimidine base is thyminie. 15 5. The method of claim 2, wherein the pyrimidine base is uracil. 6. The method of claim 2, wherein the pyrimidine base is 5-halouracil. 7. The method of claim 2, wherein the pyrimidine base is cytosine. 8. The method of claim 2, wherein the pyrimidine base is 5-fluorocytosine. 9. The method of claim 1, wherein the j-D-2'-fluoronucleoside has a purine base. 20 10. The method of claim 9, wherein the purine base is selected from the group consisting of N 6 -alkylpurine, N 6 -acylpurine (wherein acyl is C(O)(alkyl, aryl, alkylaryl, or arylalkyl), N 6 -benzylpurine, N 6 -halopurinc, N 0 -vinylpurine, N-acetylenic purine, N-acyl purine, N'-hydroxyalkyl purine, N 6 -thioalkyl purine, N 2 -alkylpurines, N2_ alkyl-6-thiopurines, N 2 -alkylpurine, N 2 -alkyl-6-thiopurine, 5-azacytidinyl, guanine, 25 adenine, hypoxanthine, 2,6-diaminopurine, and 6-chloropurine.
2. 86. 11- The method of claim 1, wherein the 1-D-2'-fluoronucleoside has a triazolopyrininyl, imidazolopyridinyl, pyrrolopyrimidinyl, or a pyrazolopyriminidinyl base. 12. The method of claim 1, wherein the f-D-2'-fluoronucleoside is in substantially pure form. 5 13. The method of claim 1, wherein the #-D-2'-fluoronucleoside is at least 90% by weight of the t9-D-isomer. 14. The method of claim 1, wherein the #-D-2'-fluoronucleoside is at least 95% by weight of the s-D-isomer. 15. The method of claim 1, wherein the O-D-2'-fluoronucleoside is administered in the 10 form of a dosage unit. 16. The method of claim 15, wherein the dosage unit contains 50 - 1000 mg of the compound, 17. The method of claim 15, wherein the dosage unit is in the form of a tablet or capsule. 18. The method of claim 1, wherein the pharmaceutically acceptable carrier is suitable 15 for oral delivery. 19. The method of claim 1, wherein the pharmaceutically acceptable carrier is suitable for intravenous delivery. 20. The method of claim 1, wherein the pharmaceutically acceptable carrier is suitable for parenteral delivery. 20 21. The method of claim 1, wherein the pharmaceutically acceptable carrier is suitable for intradermal delivery. 22. The method of claim 1, wherein the pharmaceutically acceptable carrier is suitable for subcutaneous delivery. 23. The method of claim 1, wherein the pharmaceutically acceptable carrier is suitable 25 for topical delivery.
3. 87. 24. The method of any one of claims 1 - 11, wherein the host is a human,