Recent Advances in Antiviral Nucleosides

CHAPTER 1 RECENT ADVANCES IN ANTIVIRAL NUCLEOSIDES GIUSEPPE GUMINA, YONGSEOK CHOI and CHUNG K. CHU 1.1. Introduction During the last two decades, treatment of viral infections has advanced remarkably, thanks to the heroic efforts of chemists and pharmacologists, the rapid progress of molecular virology as well as the cumulative knowledge of more detailed mechanism of action of antiviral agents.^ In recent years, we are facing an outburst of new and emerging viral diseases, such as new strains of hepatitis and herpes viruses, Ebola virus, West-Nile virus, plus a number of exotic viruses which, although still isolated in small areas of the world, have the potential for pandemic outbreak. Besides, the threat that viruses and other microorganisms could be used as biological weapons in warfare or bioterrorism has become a reality. Although vaccination is a valuable tool to fight viral diseases and in some cases is available and successful, the difficulty associated with state- or worldwide vaccination programs makes antiviral chemotherapy a more practical approach in the fight to epidemic viral infections. Among the most successful antiviral agents, nucleoside analogs have been the drugs of choice in the treatment of a number of deseases caused by herpes simplex virus (HSV), human cytomegalovirus (HCMV), varicella zoster virus (VZV), human immunodeficiency virus type 1 (HIV-1) and human hepatitis B (HBV) and C (HCV) vuns. Since 1980, a variety of biologically interesting and promising nucleosides have been discovered, some of which are being used clinically or are undergoing preclinical or clinical development. Currently, eighteen nucleosides are clinically being used for the treatment of HIV-1, herpes virus, HBV, RSV and HCV infections (Table 1). Despite these achievements, continued discoveries of novel nucleoside analogs are needed in order to overcome common problems in antiviral chemotherapy, such as toxicity, metabolic instability and, above all, the emergence of resistant viral strains as well as of new and emerging viral diseases. So far, a number of reviews have been published, regarding specific nucleoside classes,^ general aspects of nucleosides^ and their chemistry^ as well as their antiviral activity spectrum and target of actions.^ In view of these reviews, the purpose of this chapter is to give a brief overview on the most recent advances in antiviral nucleosides focusing on the structure-activity relationships with particular regard to the biochemical mode of action of the most promising nucleosides. Antiviral Nucleosides: Chiral Synthesis and Chemotherapy, Ed. by C.K. Chu. 1 — 76 © 2003 Elsevier B.V. All rights reserved. G. Gumina, Y. Choi and C. K. Chu Table 1. Generic name Antiviral nucleosides used in clinics Acronyms Target viruses Mode of action Anti-HIV agents Zidovudine AZT HIV-1 Didanosine ddl HIV-1 Zalcitabine ddC HIV-1 Stavudine d4T HIV-1 Lamivudine 3TC HIV-1 Abacavir 1596U89 HIV-1 Tenofovir disoproxil PMPA HIV-1 3TC HBV Idoxuridine IdU HSV-1/2 Trifluridine TFT HSV-1/2 Acedurid EdU HSV-1/2 Vidarabine araA HSV-1/2 Acyclovir ACV HSV-1/2, vzv Selective viral DNA polymerase inhibitor Valaciclovir val-ACV HSV-1/2, vzv Valine ester prodrug of acyclovir Penciclovir PCV HSV-1/2, vzv Selective viral DNA polymerase inhibitor; topical use Famciclovir FCV HSV-1/2, vzv Oral prodrug of PCV Ganciclovir DHPG HCMV Cidofovir (S)-HPMPC HCMV Virazole Ribavirin RSV, HCV • ~ \ >- Reverse transcriptase inhibitor/ chain terminator J Anti-HBV agent Lamivudine Reverse transcriptase inhibitor/ chain terminator Anti-Herpetic agents "\ > DNA polymerase inhibitor; topical use ,) } Selective viral DNA polymerase inhibitor Viral RNA polymerase inhibitor Recent Advances in Antiviral Nucleosides 1.2. Structural features of nucleosides as antiviral agents Nucleoside analogs as inhibitors of viral replications usually act by interaction of their triphosphates with viral polymerases. As structural units of nucleic acids, the nucleoside triphosphates (NTPs) are the substrates for polymerase enzymes, which catalyze the polymerization of the NTPs. The biosynthesis of the NTPs is controlled by nucleoside kinases. The structural requirements of nucleosides to interact with kinases and polymerases have important implications in the design of potential antiviral nucleosides (Figure 1). The 5'-hydroxymethyl group and the base moiety of nucleosides interact with kinases and their complementary nucleotides on the DNA template. The sugar moiety of the nucleoside can be considered as a spacer to connect the hydroxymethyl group and the base moiety.^ Therefore, modification of the sugar moiety has provided opportunities in the design of biologically active nucleosides. Some viruses, such as herpes viruses, encode their own nucleoside-phosphorylating enzymes, which offers the potential for a therapeutic target.^ Nucleosides, which are preferably phosphorylated by viral enzymes rather than by the cellular homologue, are only activated in infected cells and can have high selectivity against these viruses. This is, for example, the main factor in the success of acyclovir (ACV). However, other viruses, such as HIV and HBV, do not encode nucleoside kinases. In order to be active against these viruses, nucleoside analogs have to be phosphorylated by cellular kinases. Thus, the selectivity between antiviral activity and cellular toxicity depends on the substrate specificity of the NTPs for viral and host polymerases, and often the therapeutic exploitation of active nucleosides is compromised by the toxicity resulting from inhibition of the host enzymes or incorporation in the host nucleic acids. In general, enzymes act on one enantiomer of a chiral substrate, the specificity of which is related to the unique structure of the enzymes.^ However, recent findings have indicated that there are some exceptions to this rule among enzymes involved in the phosphorylation of nucleosides.^'^'^'^° For instance, herpes virus thymidine kinases (TKs) phosphorylate both D- and L-enantiomeric forms of several uracil analogs as well as acyclic nucleosides, cellular deoxycytidine (dCyd) kinase phosphorylates both enantiomeric forms of several dCyd analogs, and some viral DNA polymerases, such as herpes viruses, HIV-1 RT and HBV DNA polymerase, are inhibited by the triphosphates of a number of L-nucleosides. These findings offer new opportunities for antiviral chemotherapy, although, at the molecular level, it is not completely understood how kinases phosphorylate both D- and L-nucleosides. In recent years, a growing number of nucleoside analogs have been discovered which exert their antiviral activity by inhibiting enzymes different from polymerases, such as inosine monophosphate dehydrogenase, 5'-adenosylhomocysteine hydrolase, orotidine 5'-monophosphate decarboxylase and CTP synthetase.^^ Such compounds may prove useful because, by targeting different enzymes, they may offer synergistic action with classic polymerase inhibitors. G. Gumina, Y. Choi and C K. Chu O HO-i II B •Q-P-O 1^ -Q-P-O-P-O-P-O B 0 O "O-P-O-P-O 6' 6' Interaction with viral DNA polymerases B [ O ^ Antiviral activity J and/or Interaction with cellular DNA polynnerases a, p, y, e cytotoxicity, mitochondrial toxicity, antitumor activity A: virus-encoded TK, cellular TK, dCyd kinase, dPyr kinase, dGua kinase, 5'-nucleotidase, adenosine phosphotransferase, or UL97 gene product, etc. B: Nucleotide kinases or 5-phosphoribosyl-1-pyrophosphate synthetase, etc. C: NDP kinase, phosphoenolpyruvate carboxykinase, phosphoglycerate kinase or creatine kinase, etc. Figure 1. General mode of action of nucleoside analogs. 1.3. 2'-Deoxy nucleosides and related analogs 2'-Deoxy nucleoside analogs have proved effective against DNA viruses, such as HSV, VZV, EBV and HBV. Some nucleosides of this class show poor selectivity between the viral polymerases and the host polymerases due to their structural resemblance to natural substrates. However, modification of the base moiety or the sugar portion as well as the use of the unnatural L-enantiomer have been shown to reduce cellular toxicity, as in the case of 2'-fluoro-5-methyl-p-L-arabinofuranosyluracil (L-FMAU).^^ Recent Advances in Antiviral Nucleosides Since the discovery of the first antiherpes compound, 5-iodo-2'-deoxyuridine (1, IdU)/^^ a modifications of the 5-position of the pyrimidine moiety have produced a number of active antiviral compounds (Figure 2)P Several 5-substituted 2'-deoxy-uridines, such as 5-trifluoromethyl-2'-deoxyuridine (2, TFT)!^^ and 5-ethyl-2'-deoxyuridine (3, EdU)/^^ have been approved for the treatment of herpetic keratitis. IdU and TFT are phosphorylated to their triphosphates by the virus-encoded TK. The triphosphates inhibit HSV DNA polymerase as well as, even though to a lesser extent, cellular polymerases. EdU has higher affinity for the herpesvirus-induced TK than for the cellular TK, and its triphosphate is incorporated to a large extent into the viral DNA.^^'^^"^ BVdU (4, brivudin) was originally synthesized by Walker and co-workers and shown to be a potent and selective anti-herpes agent. ^"^ It is specifically phosphorylated by virusencoded TK and nucleoside diphosphate (NDP) kinase to give BVdUTP, which may act as either an inhibitor of or a substrate for viral DNA polymerase. However, BVdU is cleaved by pyrimidine nucleoside phosphorylases to (£)-5-(2-bromovinyl)uracil (BVU), which is cytotoxic.^^ The marked loss of activity of BVdU in thymidine kinase-deficient (TK) HSV-1 or VZV strains has been bypassed by its incorporation into phosphoramidate prodrugs {vide infra)}^ The inhibitory effects of several 5-alkynyl-2'-deoxyuridine analogs on virus replication, host cell metabolism and tumor cell proliferation have been investigated, among which 5-ethynyl-2'-deoxyuridine (5) is the most cytotoxic against L1210 cells.^^ 5-Heteroaryl-substituted 2'-deoxyuridines,^^'^^ i.e. 5-(3-Bromoisoxazol5-yl)-2'-deoxyuridine (6), 5-(5-bromothien-2-yl)-2'-deoxyuridine (7) and 5-(5-chlorothien-2-yl)-2'-deoxyuridine (8) also share with BVdU a common antiviral spectrum against various strains of HSV-1 and VZV, but not HSV-2, HCMV or TK" HSV-l.^'^ 0 HO—1 '^N^O HO 1 2 3 4 5 6 7 8 o O'^N" |—OH OH (IdU, X = l) (TFT,X = CF3) (EdU, X = Et) (BVdU, X = (E)-bromovinyl) (X = CsCH) (X = 3-Br-isoxazol-5-yl) (X = 5-Br-thien-2-yl) (X = 5-Cl-thien-2-yl) 9 (L-ldU, X = 1) 10 (L-BVdU, X = (E)-bromovinyl) 11 (L-dT,X = CH3) Figure 2. 2'-Deoxyuridine analogs. Focher et al. have demonstrated that L-IdU (9), L-BVDU (10) and L-thymidine (11, L-dT) (Figure 2) are not recognized by human cytosolic TK in vitro, but function as a substrate for HSV-1 TK and inhibit HSV-1 proliferation in infected cells.^ L-dT is selectively phosphorylated in vivo to L-dTMP by HSV-1 TK. L-dTMP is further phosphorylated to the di- and triphosphate forms by non-stereospecific cellular kinases. L-dTTP not G. Gumina, Y. Choi and C. K. Chu only inhibits HSV-1 DNA polymerases in vitro, but also human DNA polymerases a, y, 5 and 8, HIV-1 RT, E. coli DNA polymerase I and calf thymus terminal transferase, although DNA polymerase (3 is resistant. Spadari et al. have also reported that HSV-1 TK shows no stereoselectivity and phosphorylates both D- and L-dT to their corresponding monophosphates with identical efficiency, with a K. value of 2 |LIM, almost identical to the K^^ for the natural substrate thymidine (2.8 |LIM).^ L-IdU and L-BVdU inhibit HSV-1 TK with activities comparable to those of their corresponding D-enantiomers. In addition, the L-isomers of IdU and BVdU have no effect on human thymidylate synthase and are fully resistant to hydrolysis by nucleoside phosphorylase.^ However, Chu and co-workers reported that L-BVdU and L-BVaraU show no activity against herpes viruses.^^ Furthermore, L-dT and L-2'-deoxycytidine (L-dC) do not show any inhibitory effect against HIV, HSV-1, HSV-2, EBV, VZV and vaccinia virus, whereas they show selectively potent anti-HBV activity with an EC^^ value of 0.05-0.26 |iM in 2.2.15 cells and duck HBV and with an ECg^ value of 0.05 |LiM in primary duck hepatocytes without any cellular toxicity (CC3Q >2000 |iM).^^'^^'^^ L-dT and L-dC do not inhibit the growth of human bone marrow progenitor cells, although L-dT is a substrate of cytosolic TK and mitochondrial TK, and L-dC is phosphorylated by dCyd kinase and mitochondrial TK (Figure 3).^^'^^ L-dC is not a substrate of dCyd deaminase, but its L-dCMP is deaminated to form L-dUMP, which is further converted to L-dUTP. L-dTTP and L-dCTP inhibit woodchuck hepatitis virus DNA polymerase with an IC^Q value of 0.34 and 2.0 juM, respectively, whereas none of them is a substrate for the HIV RT or for human DNA polymerases a, (3 or 8 up to 100 jiM. Moreover, L-dT and L-dC do not cause any reduction in mitochondrial DNA content, any lactic acid accumulation nor the alteration in mitochondrial morphology or function up to 10 |LiM.^^'^^ L-dT and L-dC are currently undergoing clinical trials as anti-HBV agents. cytosolic TKor L-dT ^ L-dTMP ^ L-dTDP L-dTTP I mtTK Inhibition of WHBV polynnerase No inhibition of human DNA polymerase a, p or £ dCyd I kinase ' L-dCTP L-dC ^ L-dCMP ^ L-dCDP ^ L-dUTP ^ mtTK dCyd deaminase L-dU L-dUMP Figure 3. Metabolic pathway of L-dT and L-dC.^ Recent Advances in Antiviral Nucleosides During the synthesis of 5-alkynyluridine analogs (12), 3-glycosyl-6-substituted-furano[2,3-<flpyrimidine-2-one derivatives (13) have been obtained as cyclic by-products (Scheme 1). These furanopyrimidine derivatives exhibit potent and selective in vitro inhibition against VZWP In this series, the 6-octyl derivative is the most potent, followed by the 6-decyl and 6-nonyl derivatives. Shorter chains (<Cg) led to antiviral activity similar to that of ACV and C^-C^Q led to higher antiviral activity. Longer chains {>C^^ reduced the potency against VZV, probably due to low water solubility. Interestingly, the furanopyrimidine derivatives (13) exhibited only anti-VZV activity. It seems that these analogs may be phosphorylated by VZV TK, as the complete loss of antiviral activity in the VZV TK assays seems to support. I HO—I [I "N^^O HO HO-i [ N'^^O HO 1 12 (R = C5H11 - C12H25) Scheme 1. Synthesis of furanopyrimidine derivatives. Substitution of the oxygen on the furanose ring by a sulfur or methylene group also retains comparable antiviral activity with increased stability of the glycosidic bond. Among 2'-deoxy-4'-thiouridine analogs, 4'-thio BVdU (14, S-BVdU) is the most interesting compound (Figure 4), showing potent activity against HSV-1, HSV-2 and VZV (EC3Q 0.6, 10 and 0.08 |iM, respectively) with no cytotoxicity and improved in vivo stability.^"^ Other 5-substituted analogs in this series also have good activity against HSV-1 and VZV in vitro without any apparent cytotoxicity {e.g. 5-ethyl, 5-vinyl and 5-chloroethyl).^^ Among them, the 5-ethyl analog has the broadest antiherpetic spectrum, being active against HSV-1, HSV-2 and VZV. Furthermore, the isopropyl and cyclopropyl analogs have significant activity in vitro against HSV-1 and VZV, whereas no activity is observed for their oxygen counterparts.^^ Among 2'-deoxy-4'-thio-n7?(9 purine analogs, the 2-amino-6-(cyclopropylamino)purine derivative (15) is the most potent and selective agent against HCMV and HBV replication in vitro (EC^^ 0.2 and 0.0072 |LiM in 2.2.15 cells, respectively), but it is also nephrotoxic in vivo.^^ tjenishi et al. have reported the synthesis and biological activity of D- and L-2'-deoxy-4'-thiouridines and their 5-trifluoromethyluridine analogs, D- and L-4'-thiothymidine, and D- and L-2'-deoxy-4'-thiocytidine.^^ D-Thymine, D-cytosine and D-5-trifluoromethyluracil derivatives are also potent inhibitors of the growth of LI210 cells. On the other hand, none of the L-nucleosides showed any cytotoxicity toward L1210 and KB cells except for the L-thymidine analog, which was slightly toxic toward L1210 cells. G. Gumina, Y. Choi and C. K. Chu A Br <tx NH HO HO N NH2 HO HO 14 (S-BVdU) 15 Figure 4. Biologically active 2'-deoxy-4'-thionucleosides. The racemic carbocyclic analogs of several 2'-deoxyribofuranosides are also active against HSV-1 and HSV-2 replication in cell culture. Among them, the carbocyclic analogs of IdU (16, C-IdU) and BVdU (17, C-BVdU) show similar selectivity and potency to their parent compounds (Figure 5).^^ Racemic C-BVdU and its analogs as well as C-IdU are equally selective, albeit slightly less potent in their antiherpes action than their parent compounds. Although resistant to degradation by pyrimidine nucleoside phosphorylases, C-BVdU is no more effective than BVdU in systemic (oral, intraperitoneal) or topical treatment of HSV-1 infections in mice. However, both (-)- and (+)-enantiomers of C-IdU (18) and C-BVdU (19) are active against HSV-1, which indicates that both may act as substrates for HSV-1 TK.^° O O NH OH N^O HO OH HO 16(C-ldU, X = l) 17 [C-BVdU, X = (E)-bromovinyl] HO N NH, 18(L-C-ldU.X=l) 19 [L-C-BVdU, X = (E)-bronnovinyl] IJC> H2N OH N OH HO 20 (CdG) 21 (L-CdG) Figure 5. Biologically active carbocyclic 2'-deoxy nucleosides. Recent Advances in Antiviral Nucleosides Among purine analogs, (±)~carbocyclic 2'-deoxyguanine (2'-CdG, 20) shows the most potent antiherpetic activity.^^ Secrist et al. resolved the enantiomers, and reported that D-2'-CdG is as active and potent as (±)-2'-CdG against HSV-1 and HSV-2, whereas L-2'-CdG displays only modest activity against HSV-1.^^ According to the metabolic study of (±)-2'-CdG, D-2'-CdG (20) and L-2'-CdG (21, Figure 5), D-2'-CdG seems to be a good substrate for the virus-encoded kinase and a very poor substrate for cellular phosphorylating enzymes.^^ Besides, both D- and L-2'-CdG are phosphorylated by dCyd kinase from MOLT-4 cells, 5'-nucleotidase from Hep-2 cells, and mitochondrial deoxyguanosine (mt-dGua) kinase from human platelets and CEM cells.^"^ For both dCyd kinase and mt-dGua kinase, L-CdG is a better substrate with K^^ values of 0.63 and 4.9 mM, respectively v^. 1.98 and 1.2 mM for D-CdG. In the case of 5'-nucleotidase, D-CdG is a better substrate with a V /K,, value of 0.02 for L-CdG and 0.05 for D-CdG.^^ max M In addition, D-CdG shows a 50% inhibition of HBV DNA polymerase activity at 5 ng/mL in 2.2.15 cells, and at 25 ng/mL the complete disappearance of HBV replication has been observed.^^ D-CdG is phosphorylated to its triphosphate (although the exact identity of the enzymes responsible for this phosphorylation is not clear), which can be efficiently incorporated into HBV DNA. D-CdGTP is a competitive inhibitor of dGTP for both HBV DNA polymerase and eukaryotic DNA polymerase 5, with a 6-fold lower K. for the viral enzyme.^^ Unfortunately, D-CdG is toxic with a 50% inhibition of cell growth (HepG2 2.2.15 cells at 32 |iM).^^ Replacement of the oxygen with an ethenyl group produces compounds with potent and selective anti-HBV activity in 2.2.15 cells.^^ Entecavir (BMS-200475,22), originally synthesized as an anti-herpesvirus agent, displayed also moderate activity against HSV-1, HSV-2 and VZV (Figure 6). O HO N ^ N ^ N H j O H^N^N^N 11 HO pOH OH 22 (entecavir) 23 Figure 6. BMS-200475 and its L-enantiomer. Activity was also seen with HCMV, a herpes virus lacking TK, but no activity was detected against RNA viruses such as HIV or influenza. Further studies have established that entecavir is one of the most potent anti-HBV nucleosides discovered in vitro as well as in vivo, (EC^^ 3 nM, IC^^ 30 |LiM) in 2.2.15 cells. Treatment with entecavir results in no apparent inhibitory effects on mt-DNA content.^"^ Furthermore, daily oral treatment at doses ranging from 0.02 to 0.5 mg/kg of body weight for 1 to 3 months effectively reduces the level of woodchuck hepatitis virus (WHV) viremia in chronically infected woodchucks as measured by reductions in serum WHV DNA levels and endogenous 10 G. Gumina, Y. Choi and C. K. Chu hepadnaviral polymerase activity. However, WHV viremia in BMS-200475-treated WHV carriers eventually returns to pretreatment levels after therapy is discontinued.^^ In vitro biochemical studies indicate that entecavir can be efficiently phosphorylated by cellular enzymes to its triphosphate, which is a potent inhibitor of HBV DNA polymerase, inhibiting both priming and elongation steps of HBV DNA replication."^^ The enantiomer (23) of entecavir as well as the adenine, thymine and iodouracil analogs, are much less active against HBV.^"^ Entecavir is currently undergoing clinical trials as an anti-HBV agent. 9-P-D-Arabinofuranosyl adenine (24, ara-A, vidarabine) has been known to have significant antiviral activity in vitro against herpes and vaccinia virus"^^ and is also a potent inhibitor of HBV DNA polymerase (Figure 7)."^^ Due to its low water solubility, its 5'-monophosphate (ara-AMP) is administered intramuscularly, and ara-AMP has been extensively studied for treating chronic HBV infections in humans.^^ Although a 8-week treatment has been shown to effect the loss of HbeAg and HBV DNA, in many cases serious neurotoxicity is evident after 4 weeks. Ara-A is phosphorylated by cellular enzymes to its triphosphate, which interfere with viral nucleic acid replication. Unfortunately, vidarabine is deaminated rapidly by adenine deaminase to arabinosyl hypoxanthine, which has weak antiviral activity.^^ Its carbocyclic analog, cyclaradine (25), synthesized in efforts to develop deaminase-resistant ara-A derivatives, exhibits significant anti-HSV-1 and anti-HSV-2 activity.^^ \ NHo NH2 N^../^M N..^.^M ^ HO 24 (ara-A) ^ H O ^ HO 25 (cyclaradine) O ^'^^-^>r ^ ^NH ^ HO 26 (BVaraU) HO 27 (zonavir) Figure 7. Biologically interesting arabinofuranosyl nucleosides. Substitution of an arabinofuranose for the ribose moiety of BVdU leads to BVaraU (26), the most potent anti-VZV agent discovered so far which, for this reason, had been registered in Japan for the treatment of herpes zoster (shingles)."^^ However, several patients who had been treated with BVaraU along with 5-fluorouracil (5-FU) died because of the drugs interaction. In fact, 5-bromovinyluracil, released by phosphorolytic cleavage of the glycosylic bond, is a potent inhibitor of dihydropyrimidine dehydrogenase, whose inhibition results in elevating 5-FU to lethal levels."^ The L-enantiomer of BVaraU does not exhibit antiviral activity against any herpes viruses, including VZV.^^"^^ Introduction of an alkenyl or alkynyl group at 5-position of the uracil base also produces potent anti-herpetic activity."^^ (i-D-Arabinofuranosyl 5-propynyluracil (27, zonavir) is a good substrate for viral kinases, particularly for VZV TK, which converts it to the monophosphate whereas cellular cytosolic thymidine kinase is not effective.'*^ 11 Recent Advances in Antiviral Nucleosides The monophosphate is then specifically converted to the diphosphate by the thymidylate kinase activity of VZV TK. The triphosphate of zonavir is a potent inhibitor of the VZVspecific DNA polymerase and this inhibition is probably the major mechanism of the antiviral activity. 4'-Thio derivatives of BVaraU and related analogs have selective antiviral activities against HSV-1, HSV-2 and VZV, but not superior to the 4'-oxo nucleosides.^^ 4'-Thioarabinofuranosyl guanine and diaminopurine have the most potent anti-HCMV and anti-proliferative activities, whereas arabinosyl guanine and diaminopurine show only marginal antiviral activity."^^^ The L-enantiomer of 4'-thioarabinofuranosyl cytosine does not exhibit significant antiviral activity.'^^^ Introduction of a fluorine atom at the 2'-position of nucleoside analogs has produced a variety of interesting antiviral agents (Figure 8). 2'-Fluoro-P-D-arabinofuranosyl pyrimidine nucleosides are potent agents against herpes virus.^^ 1-P-D-Arabinofuranosyl5-iodocytosine (FIAC) and l-P-D-arabinofuranosyl-5-iodouridine (FIAU, 28) are phosphorylated in HSV-1 infected cells by virus-encoded TKs.^^ The 5-alkenyl analogs were also found to be active against HSV-1, HSV-2 and VZV.^^'^ Furthermore, FIAC, FIAU, FMAU (29) and FEAU (30) have significant anti-HBV activity.^^ Studies in HepG2 cells indicate that FIAU is activated by host cell enzymes including cellular TK, thymidylate kinase and pyrimidine diphosphate kinase. o ^v^ „ HO-i ^NH N ^O HO 28 (FIAU, X = I) 29 (FMAU, X = CH3) 30 (FEAU, X = Et) o 13 HN O^ N [—OH HO 31 (L-FMAU) Figure 8. 2'-Deoxy-2'-fluoro-arabinofuranosyl nucleosides. Unfortunately, problems associated with the toxicity of potential therapeutic compounds have been demonstrated by the results of the FMAU^^ and FIAU (fialuridine) cHnical trials (Figure 9).^'^^ Although initial trials in humans showed very good efficacy in terms of reducing the plasma levels of HBV as measured by viral DNA concentrations or viral polymerase activity, longer trials, in which the duration of drug treatment was extended, had to be curtailed when serious toxic effects became apparent. ^ These included myopathy, lactic acidosis, peripheral neuropathy, pancreatitis and liver failure, and the severity of the toxic effects was such that several patients died. ^ The primary cause of this delayed toxicity is due to the incorporation of the drug into mitochondrial DNA (mt-DNA), which causes damage to the mitochondrial function.^^ Studies in HepG2 2.2.15 cells indicate that FIAU is activated by host cell enzymes 12 G. Gumina, Y. Choi and C. K. Chu including cellular TK, thymidylate kinase and pyrimidine diphosphate kinase to FIAUTP, which inhibits the viral DNA polymerase. However, FIAUTP is also efficiently used as a substrate by DNA polymerase y, which incorporates it into mt-DNA, causing disruption in the replication of DNA, resulting in either decreased production of proteins or the production of defective proteins.^^ mitochondrial DNA Figure 9. Mitochondrial toxicity caused by FIAU: internalization of FIAU into mt-DNA, which cannot be repaired by exonucleases.^^ Since the broad spectrum of biological activity of 2'-F-arabinofuranosyl nucleosides was discovered, a number of structural modifications of these analogs have been carried out. Chu and co-workers have demonstrated that the enantiomer of FMAU, L-FMAU (clevudine, 31, Figure 8) has potent anti-HBV as well as anti-EBV activity.^^ Most importantly, L-FMAU has low cytotoxicity in a variety of cell lines, including MT2, CEM, HI and 2.2.15 and bone marrow progenitor cells. L-FMAU is phosphorylated stepwise to L-FMAUMP, L-FMAUDP and L-FMAUTP in 2.2.15 cells by cytosolic TK, dCyd kinase or mt-dPyd kinase, respectively (Figure 10), acting as a potent inhibitor of HBV DNA polymerase.^"^ However, it is not utilized as a substrate by human DNA polymerase a, P, y or 5. In addition, L-FMAU exhibits potent anti-EBV activity. The metabolic studies suggest that EBV-specific TK in HI cells can phosphorylate L-FMAU to its mono, di- and triphosphates.^"^ Interestingly, L-FMAUTP is not a substrate for HBV or EBV DNA polymerases unlike other antiviral nucleosides, which suggests that the anti-HBV and anti-EBV activity of L-FMAU may not be due to its incorporation into HBV and EBV DNA.^"^ Currently undergoing clinical trials against chronic hepatitis B virus infection, L-FMAU has been found to be one of the most potent anti-HBV agents so far in woodchuck as well as in humans, since no significant viral rebound was observed in woodchucks or humans. The 4'-thio substitution of 2'-fluoro nucleosides also retains their antiviral activity (Figure 11). Machida et al. have reported that 2'-fluoro-4'-thioarabinofuranosyl nucleosides (4'-thio-F-araNs) are active against HSV-1, HSV-2, VZV and HCMV.^^ 4'-Thio-FaraG (32) and 4'-thio-F-araDAP (33) have particularly potent activity against all herpes viruses tested, equipotent to arabinosyl guanine and diaminopurine. These compounds also have a 6-fold lower EC^^ value than ganciclovir against clinical isolates of HCMV. In addition, 4'-thio-F-araA (34) shows biological activities similar to that of araA. 2'Fluoro-5-methyl-4'-thio-p-L-arabinofuranosyluracil (35, L-SFMAU), the enantiomer of 4'-thio-FMAU (36), also has moderate activity against HSV-1 and HSV-2.^^ Recent Advances in Antiviral 13 Nucleosides Inhibition of HBV and EBV DNA polymerases, but no incorporation into HBV DNA TK L-FMAU dCyd kinase L-FMAUDP L-FMAUMP L-FMAUTP no inhibition of human DNA polymerases a, p, 5 and y I DNA L-FMAU L-FMAUDP L-FMAUMP dPyd kinase L-FMAUTP DNA polymerases y mt-DNA mitochondrial compartment Figure 10. Proposed metabolism of L-FMAU.^^ O <tL HO HO ^^^-- H N ' -^ - ^ " ^ O^N^ r-OH OH 32 (S-FaraG, X = OH, Y = NH2) 33(S-FaraDAP,X = Y = NH2) 34 (S-FaraA, X = NH2, Y = H) 35 (L-S-FMAU) HO-1 ^NH ^N^O HO 36 (S-FMAU) Figure 11. 2'-Deoxy-2'-fluoro-4'-thioarabinofuranosyl nucleosides. Among carbocyclic derivatives of FMAU (Figure 12), C-FMAU (37) showed moderate anti-HSV-1 activity although it is less active than FMAU.^^ The 2'-ara-fluoroguanosine derivatives^ is potent against HSV-1 and HSV-2 and poorly active against VZV, but it 14 G. Gumina, Y. Choi and C. K. Chu has also showed cytotoxicity. The 2'-fluoro analog of cyclaradine (38) is 10 times more active than cyclaradine itself against HSV-1 and HSV-2, and more active than ACV against HSV-2 in mice.^^ NH2 HO- W 1 HO 37 (C-FMAU) V^ 1 HO 38 (C-F-araA) Figure 12. Carbocyclic 2'-deoxy'2'-fluoro-arabinofuranosyl nucleosides. Introduction of geminal fluorine atoms at the 2'-position has resulted in the discovery of 2'-deoxy-2',2'-difluorocytidine (39, gemcitabine),^^ which has been approved by the FDA for the treatment of pancreatic cancer (Figure 13). Gemcitabine shows a complex mechanism of action, inhibiting the synthesis of DNA and RNA as well as inhibiting ribonucleotide reductase.^ Its guanosine analog also shows similar activity.^^ Among the series of L-enantiomers of gemcitabine including L-gemcitabine (40), only the adenine analog shows marginal anti-HIV-1 activity without cytotoxicity (EC^^ 3.4 \\M in PBM cells).^^ The 4'-thio analog of gemcitabine (41, 4'-thiogemcitabine)^^ also shows moderate antineoplastic activity. However, its enantiomer, L-4'-thiogemcitabine (42), shows neither antiviral nor antitumor activity against a panel of five different tumor cell lines.^ NH2 N HO F 39 (gemcitabine) NH2 N ^ F OH 40 (L-gemcitabine) NH2 rp^N HO F 40 (S-gemcitabine) NH N F OH 42 (L-S-gemcitabine) Figure 13. 2'-Deoxy-2'-fluorocytidine derivatives. The methylidene substitution of 2'-deoxycytidine provides 2'-deoxy-2'-methylidenecytidine (43, DMDC, Figure 14),^^ endowed with anti-neoplastic activity against several solid tumor cell lines as well as leukemia. DMDC is resistant to cytidine deaminase^^ 15 Recent Advances in Antiviral Nucleosides and its diphosphate is a potent inhibitor of ribonucleotide reductase.^^ DMDC and 2'deoxy-2'-methylidene-5-fluorocytidine (DMDFC) (44) are potent inhibitors of HSV-1, HSV-2, VZV and HCMV with significant anti-proliferative activity.^^ The (£)-5-(2bromovinyl)uracil analog (BV-DMDU) has moderate antiviral activity against HSV-1, HSV-2, VZV and HCMV. Among the 2'-deoxy-2'-methylidene pyrimidine nucleoside analogs, BV-DMDU showed the most potent and selective anti-VZV activity, which was more potent than ACV, but less active than BVaraU.^^ Also the 4'-thio analog of DMDC (45, Figure 14) has potent antineoplastic properties in vitro with an IC5^^ value of 0.0091 and 0.12 |ig/mL in CCRF-HSB-2 and KB cells, respectively.^^ Its enantiomer, L-4'-thio-DMDC (S-DMDC, 46), does not show antitumor activity against different cell lines.^^ In addition, various 5-substituted 4'-thio-DMDUs show potent anti-HSV-1 activity (EC^^ 0.016-0.096 |ig/mL). 5-Ethyl- and 5-iodo-4'-thioDMDUs are also active against HSV-2 (EC^^ 0.17 and 0.86 |Xg/mL, respectively), and 5-bromovinyl-4'-thio-DMDU has efficacy against VZV with an EC^^ value of 0.013 |ig/mL without significant cytotoxicity.^^ NH? N NH2 NH2 ''V^N ff^N NH; N ''"W^' ''"W^' ''"W^' '^^t^'" HO ^ 43 (DMDC) HO ^ 44 (DMDFC) HO ^ 45 (S-DMDC) OH 46 (L-S-DMDC) Figure 14. 2'-Deoxy-2'-methylidenecytidine derivatives. Introduction of a functional group at the 4'-a-position of 2'-deoxynucleosides elicits potent antiviral activity. For example, 4'-azido analogs exhibit potent anti-HIV activity in A3.01 cell cultures with EC^^ values of 0.003-0.8 \iMJ^ The guanine analog is the most potent compound, but it is also cytotoxic. Further evaluations of 4'-azidothymidine (47, ADRT) in H9, PBL and MT-2 cells infected with HIV have demonstrated a similar inhibitory profile to that of AZT. Interestingly, ADRT retains its activity against HIV mutants that are resistant to AZT. In the 4'-methoxy series, adenosine, thymidine and guanosine analogs are also inhibitors of HIV, but 2-3 orders of magnitude less active than their azido counterparts.^^ A metabolic study has revealed that ADRT is not a substrate for thymidine phosphorylase, but is metabolized by kinases (Figure 15).^^ Thymidine kinase (TK) phosphorylates ADRT to its monophosphate with a K. value of 5.2 |LLM, and a K^^ value of 8.3 |LiM, in comparison to a K^^ value of 0.7 |iM for thymidine. ADRTMP has a low affinity toward thymidylate kinase and thymidylate synthase, which suggests that ADRT can be activated effectively by other cellular kinases without significant interference of normal thymidine metabolism. In cultured human lymphocytes (A3.01, H9 and U937 cells), ADRT is phosphorylated efficiently to ADRTTP, which is a poor competitive inhibitor 16 G. Gumina, Y. Choi and C. K. Chu against dTTP toward DNA polymerases a and P with K. values of 62.5 and 150 |iM, respectively. However, ADRTMP is incorporated into cellular DNA, which can lead to mutations.^^ Because of these toxicity issues, the development of ADRT as an anti-HIV agent was discontinued. TK HO HO 47 (ADRT) ADRTMP ADRTDP Incorporation into cellular DNA: possible mutation ADRTTP Chain termination ofHIV-1 DNA Figure 15. Metabolism of 4'-azidothymidine (ADRT).' 4'-a-Substituted 2'-deoxycytidines also exhibit potent anti-HIV-1 activity (Figure 16), although they are cytotoxic to the host cells (MT-4).^^ In the series, the cyano derivative (48) is the most potent against HIV-1 with an EC^^ value of 1.2 nM in MT-4 cells followed by ethynyl, ethenyl, ethyl, methyl and chloroethenyl derivatives. The methyl and cyano derivatives show moderate activity against HSV-1 and HSV-2. 2'-Deoxy-4'-C-methyl pyrimidine nucleosides show potent anti-HSV-1 and anti-VZV activity in vitro.''^ 4'-Methyl-BVdU (49, Figure 16) displays more potent activity than BVdU against VZV, and it is cytotoxic against human T-cell leukemia, CCRF-HSB-2. The arabino analog, 4'methyl-BVaraU, shows a weaker antiviral activity than that of 4'-methyl-BVdU without any cytotoxicity. The 4'-hydroxy C-deoxyguanosine analog (50, 4'-0H-CdG) has antiviral activity equivalent to ACV against HSV-1 and HSV-2, but is inactive against VZV^3b,75 4'-C-Ethynyl-substituted 2'-deoxyribonucleosides (Figure 16) have shown impressive anti-HIV activity, with EC^^ in the nano- or subnanomolar range, but many also display severe toxicity, with IC^^ in the micromolare range.^^ Thus, the diaminopurine (51), guanosine (52) and cytosine (53) derivatives show an EC^^ of 0.3, 1.4 and 4.8 nM, respectively, and an IC^^ of 0.82, 1.5 and 0.92 |iM, respectively, in MT-4 cells. The thymidine (54), 5-bromouridine (55), 5-iodouridine (56), 5-fluorocytidine (57) and guanidine (58) analogs, although less active, do not show toxicity in MT-4 cells, with the best therapeutic index found in the cytidine analog 54 (EC^^ 0.030 |LIM, IC^^ > 1 0 0 |LIM, TI >3333).^6 Recent Advances in Antiviral 17 Nucleosides HO—1 NH2 Br NH NH z^^' :?^^' i^' HO HO N NH2 HO 50 (4-OH-CdG) 49 48 B HC 51 52 53 54 55 56 57 58 HO (B = 2,6-di-aminopurine) (B = guanosine) (B = cytosine) (B = thymine) (B = 5-Br-uracil) (B = 5-l-uracil) (B = 5-F-cytosine) (B = guanine) Figure 16. 4'-Substituted-2'-deoxy nucleoside analogs. Marquez and co-workers have synthesized conformationally locked nucleosides with bicyclo[3.1.0]hexane templates to investigate the correlation between puckering of the sugar ring and biological activity (Figure 17).^^ The antiherpetic activity of these nucleosides is associated with the northern conformation of the thymidine analog, (N)-methanocarba-T (59), which is more active than ACV against HSV-1 and HSV-2 with EC^Q values of 0.03 and 0.09 |ig/mL, respectively. The cytosine analog has anti-HSV-1 activity (EC^^ 0.14 |ig/mL) and the adenine analog is active against HCMV. On the other hand, the southern conformation of the thymidine analog, (S)-methanocarba-T (60) does not show any antiherpetic activities. Unlike other nucleoside analogs, it seems that the diphosphorylation of 59 in HSV-1-infected cells is the rate-limiting step in the activation to the active triphosphate.^^ Conformationally restricted nucleosides have also been synthesized by Chu and co-workers, who reported the complete D- and L-series of 2',3'-dideoxy-2',3'-^«J6>methylene nucleosides (61 and 62, respectively).^^ None of the synthesized compounds, however, showed anti-HIV activity. -1 HO HO 59 B 61 i—OH B 62 60 [(N)-methanocarba-T] [(S)-methanocarba-T] Figure 17. Conformationally locked carbocyclic thymidine analogs. Computer-based conformational studies and biological evaluation of cyclohexenyl nucleosides have demonstrated that a cyclohexene and a furanose ring can be considered 18 G. Gumina, Y. Choi and C. K. Chu as bioisosters.^^'^^ Thus, both enantiomers of cyclohexenyl-G (63 and 64, Figure 18) show potent antiviral activity against HSV-1, HSV-2, VZV, HCMV and HBV. The antiviral activities of the two isomers are comparable, although the D-isomer is slightly more potent in all the tested systems. The fact that both isomers show reduced activity in TK HSV-1 suggests that intracellular phosphorylation plays an important role in the bioactivation of these compounds.^^'^^ //I NH N HN OH OH 63 D-cyclohexenyl-G 64 L-cyclohexenyl-G Figure 18. D- And L- cyclohexenyl-G. 1.4. 2',3'-Dideoxy nucleosides and related analog The discovery of dideoxynucleosides, such as 2',3'-dideoxycytidine (ddC),^^ 2',3'-dideoxyinosine (ddl)^^ and 3'-azido-3'-deoxythymidine (AZT)^^ as potential therapeutic agents for the treatment of acquired immunodeficiency syndrome (AIDS) has triggered an extensive development of this class of compounds to identify active anti-HIV agents, which inhibit the virus-associated RT reaction by terminating DNA chain elongation. A number of selective HIV-1 inhibitors have now been approved for the treatment of HIV infections, such as AZT, ddC, ddl, d4T,^4 3TC,^^ abacavir^^ and tenofovir disoproxil.^^'^^'^^'^^ Some of these, particularly 3TC, are also active against HBV. Given that the DNA polymerase of HBV is also a reverse transcriptase, it is not surprising that such degree of overlap exists as a chain terminator of DNA synthesis. The finding that (±)-dioxolane-thymine^^ and (±)-BCH-189^^ (see below) are potent anti-HIV agents and that the L-isomer of BCH-189 is more potent and less toxic than its D-isomer have opened the new era of L-nucleosides. Since then, a number of L-nucleoside analogs have been synthesized and biologically evaluated, and the importance of chirality and its influence on the antiviral activity of the L-nucleosides has been recognized.^^'^^ ddl (65, Figure 19) is a potent and selective anti-HIV agent in ATH8 cells.^^ It is phosphorylated to ddIMP by cytosolic 5'-nucleotidase, then aminated to ddAMP by adenylosuccinate synthase/lyase enzymes, and converted to ddADP and ddATP by cellular nucleotide kinases.^^ ddATP is the active agent against HIV-RT.^'* Peripheral neuropathy and pancreatitis are the major side effects of ddl. 19 Recent Advances in Antiviral Nucleosides cytosolic 5'-nucleotidase adenylosuccinate synthase/lyase ddIMP ^ ddAMP nucleotide kinases 65 (ddl) Inhibition of HIV-1 RT/ Chain termination nucleotide kinases ddATP ddADP Figure 19. Metabolism of the anti-HIV-1 agent ddl (didanosine).' In various cell lines, ddC (66, Figure 20) is phosphorylated to ddCMP, ddCDP and ddCTP by dCyd kinase, CMP/dCMP kinase and NDP kinase, respectively.^^ The affinity of ddCTP for DNA polymerase a is poor, and intermediate for DNA polymerase |3 and high for DNA polymerase y.^^^ ddC exerts delayed cytotoxicity and reduces the cellular content of mt-DNA^^^ and, at higher concentrations, causes a delayed distortion of mitochondrial ultrastructure.^^ It also exhibits a significant inhibitory effect on the replication ofHBVDNA. NHo N HO-i IN N ^O CMP/dCMP kinase dCyd kinase ddCDP ddCMP NDP kinase 66 (ddC) Inhibition of HIV-1 RT/ HBV/Chain termination ddCTP Inhibition of DNA polymerase y Figure 20. Metabolism of the anti-HIV-1 agent ddC (zalcitabine). A number of L-2',3'-dideoxy nucleosides also show moderate to potent anti-HIV and anti-HBV activities (Figure 21)/^'^^'^^ Among these analogs, P-L-2',3'-dideoxy-5fluorocytidine (67, L-FddC) is the most active against HIV-1, approximately 3- to 4-fold more potent than ddC in vitro.^'^ In addition, L-FddC and L-ddC (68) are potent anti-HBV 20 G. Gumina, Y. Choi and C. K. Chu agents with an EC^^ value of 0.01 |iM without any toxicity up to 100 |LIM against host mtDNA synthesis. L-ddA shows moderate anti-HIV-1 and anti-HBV activity in PBM and 2.2.15 cells, respectively.^^ An enzymatic study of D-ddA (69) and L-ddA (70) has been performed with respect to adenosine kinase, dCyd kinase, adenosine deaminase (ADA) and purine nucleoside phosphorylase (Figure 22).^^ Adenosine deaminase was strictly enantioselective and favored D-ddA, whereas adenosine kinase and purine nucleoside phosphorylase had no apparent preference for the D- or L-enantiomers.^^ Human dCyd kinase showed a remarkable inversion of the expected enantioselectivity, with L-ddA having better substrate efficiencies than its corresponding D-enantiomer.^^ p-2',3'-LDideoxy-5-azacytidine shows potent anti-HIV activity at approximately the same level as ddC. However, unlike ddC, it has no antiviral activity against HBV. NHo NHo N r—OH M 67 (L-FddC) 1 NI O^ NHo NHo OH r—OH 68 (L-ddC) 70 (L-ddA) 69 (ddA) Figure 2L 2',3'-Dideoxy nucleosides with antiviral activity. D-ddIMP D-ddl ADA D-ddA D-ddAMP D-ddADP D-ddATP L-ddAMP L-ddADP L-ddATP dCyd kinase L-ddA =1= ADA * D-ddl better substrate of HIV RT. but ineffective inhibition of HBV polynnerase Figure 22. Comparative metabolism of D-ddA and L-ddA.^* Replacement of a 4'-oxygen atom of both D- and L-2',3'-dideoxynucleosides with a methylene group, a sulfur atom or amino groups fails to elicit any significant antiviral activity except for 2',3'-dideoxy-4'-thiocytidine (71) (Figure 23), which displays modest Recent Advances in Antiviral 21 Nucleosides activity in vitro against HIV (EC^^ 1.0 and 38 iig/mL in CEM and MT-2 cells, respectively).99 NHo N HO-i IN 71 (S-ddC) Figure 23. 4'-Thio-ddC (S-ddC). Among the 3'-azido-2',3'-dideoxy nucleosides, AZT (72, Figure 24), which is the first anti-HIV-1 agent approved for use against HIV-1, has remained one of the most potent and selective anti-HIV agents.^^ The uracil analog of AZT (73, AZdU)^°° and the 5-methylcytosine analog (74, AZdMeC)^^^ are less potent but also less toxic than AZT. The guanine (AZdG) and diaminopurine (AZdDAP) analogs of AZT have also been reported to be more effective against HIV than ddA.^^^'^°^ o H3C HO-i NHo NH " N ^ O H3C NH HO—i ^N'^O HO—I ^0^ N3 72 (AZT) N " N ^ O ^O^ N3 N3 73 (AZdU) 74 (AZdMeC) Figure 24. 3'-Azido-2',3'-dideoxy nucleosides. AZT is phosphorylated to its monophosphate (AZTMP), diphosphate (AZTDP) and triphosphate (AZTTP) by TK, TmdK and NDP kinase, respectively (Figure 25).^^ AZTTP acts as a competitive inhibitor or alternate substrate of HIV RT leading to viral DNA chain termination and has much less affinity to cellular DNA polymerase a.^^^'^^"^ In the presence of 2 |iM AZTTP, the activities of HIV-RT and cellular DNA polymerase y were inhibited by more than 80 and 90%, respectively. ^^^ However, AZT is associated with several toxicities, particularly bone marrow suppression including anemia and leukopenia. The hematopoietic toxicity of AZT is generally due to high intracellular levels of AZTMP.^^^ Moreover, a number of AZT-resistant HIV strains have been isolated from AIDS patients, which stimulated an extensive search for new anti-AIDS (Jj.^g33a,107,108 22 G. Gumina, Y. Choi and C. K. Chu Among "unnatural" enantiomers of this class of compounds, L-AZT is about 10,000 times less active than its D-counterpart.^^^ The finding that L-AZTTP inhibits HIV reverse transcriptase (RT), as well as HBV DNA polymerase, at sub-micromolar concentration, suggests that L-AZT is devoid of antiviral activity because it is not efficiently phosphorylated intracellularly in lymphocytes or hepatocytes.^^^ Inhibition of HIV-1 RT/chain termination or incorporation into HIV-1 DNA IK AZT TmdK ^ AZTMP NDP kinase ^ AZTDP ^ AZTTP (rate-limiting) Inhibition of cellular DNA polymerase or incorporation into cellular DNA Figure 25. Cellular metabolism of AZT." AZdU was the first nucleoside analog with a uracil base found to have anti-HIV activity at submicromolar concentrations. It is phosphorylated to AZdUTP, which acts as both an HIV-RT inhibitor and a proviral DNA chain terminator.^*''^^^'^^^ Although its anti-HIV activity is less potent than that of AZT, its toxicity on bone marrow cells is also significantly lower than that of AZT.^^^ In human PBM cells, AZdUMP is the predominant intracellular metabolite and levels of AZdUMP are two orders of magnitude greater than AZdUTP. AZdUMP is also converted to its 5'-0-diphosphohexose and 5'-0-diphospho-A^-acetyl-glucosamine. This unique metabolism may explain the lower toxicity of AZdU.^^'* However, AZdU shows cross-resistance with AZT-resistant HIV virus.^^ The clinical trials of AZdU were discontinued due to its extensive metabolism to 5'-glururonide. AZdMeC shows potent anti-HIV activity in human PBM cells and macrophages and is less toxic than AZT in human bone marrow cells.^^'^^^ Metabolic studies indicate that this compound is slowly converted intracellularly to AZT.^^^ The major metabolite of AZdMeC is AZTMP with no formation of AZdMeCMP. The low toxicity of this compound is related to the lack of formation of AZTTP in human bone marrow cells. AZdMeCTP efficiently inhibits HIV-RT, competing with dCTP while binds to human DNA polymerase a with much lower affinity (< 6000-fold).^^ AzdMeC is deaminated to AZT in monkeys, but not in humans. Additional modifications of the sugar moiety of AZT have failed to produce any compounds with antiviral activity, including 2'-|3-fluoro-AZT,^^^ 4'-thio-AZT^^^ and carbocyclic AZT.^^^ 23 Recent Advances in Antiviral Nucleosides Conformationally locked AZT analogs, (H)-methano-c2ixhdi-KLT (75) and (S)methano-carbsi-AZT (76) (Figure 26) have been reported. ^^^ The chemically synthesized 5'-triphosphates of the two isomers have been evaluated as RT inhibitors using both a recombinant enzyme and an enzyme purified from wild-type viruses.^^^ Inhibition of RT occurs only with the conformationally locked ^E (N)-methano-C2ivbsi-AZT (75) triphosphate. This inhibition is equipotent to and kinetically indistinguishable from that produced by AZTTP. On the other hand, the antipodal ^E (Symethano-carba-AZT (76) triphosphate does not inhibit RT. [(N)-methanocarba-AZT] [(S)-methanocarba-AZT] Figure 26. Conformationally locked carbocyclic AZT analogs. Introduction of a fluorine atom at the 3'-a-position of 2',3'-dideoxy nucleosides (ddNs) increases their anti-HIV activity (Figure 27), whereas 3'-p-fluoro derivatives do not show significant anti-HIV activity.^^ Particularly, in MT-4 and CH3 cells 3'-a-fluoro2',3'-dideoxyuridine (FddU) has significantly increased anti-HIV activity than ddU,^^° and in MT-4 cells 3'-fluoro substitution of ddG has greater anti-HIV activity than the parent nucleoside. The fluoro-substituted diaminopurine derivative (FddDAP) has higher anti-HIV activity than ddG.^^^ Analogously, FLT (77) shows potent anti-HIV activity in various cell lines.^^^'^^^ FLT is phosphorylated intracellularly to FLTTP,^^^ which is one of the most potent inhibitors of HIV RT in vitro.^^"^ Unfortunately, FLT produces toxic effects similarly to AZT in cultures of normal human hematopoietic progenitor cells. ^^^ Thus, despite the promising in vitro anti-HIV profiles of FLT, clinical trials with this nucleoside analog failed due to its severe hematological toxicity.^^'^^^ O HO-n ^N^O 77 (FLT) HO 78 (FddCIU) Figure 27. 3'a-F-2',3'-dideoxy nucleosides. 24 G. Gumina, Y. Choi and C. K. Chu FddClU (78) also shows significant antiviral activity against HIV and remarkably low cytotoxicity in human leukemic cells and bone marrow progenitor cells.^^'^^^ Furthermore, FddClU induces very little resistance in HIV-1 and is active against strains of HIV which are resistant to AZT, ddl, ddC and 3TC as well as many non-nucleoside RT inhibitors.^^^ FddClU is metabolized to its mono-, di- and triphosphate in human cells, and the monophosphate is the predominant metabolite. The triphosphate selectively inhibited HIV-RT and DNA polymerase y while it had little effects on DNA polymerases a and p. 128,129 Among other L-3'-a-fluoro-ddNs, the cytidine analog shows moderate anti-HBV activity in 2.2.15 cells, but none of them possess anti-HIV activity.^^^ Also carba-2',3'dideoxy-3'-fluorothymidine, the carbocyclic analog of FLT,^^° and related carbocyclic nucleosides^^^ failed to elicit significant antiviral activity against HIV. Introduction of a fluorine atom at the 2'-P-position of 2',3'-dideoxy purine nucleosides retains their anti-HIV activity. Moreover, both 2'-F-ara-ddA (79, Figure 28) and 2'-F-ara-ddI are stable in acidic conditions under which ddA and ddl decompose instantaneously by acid-catalyzed glycosylic bond cleavage.^^'^^^ Both 2'-F-ara-ddA and 2'-F-ara-ddI retain the same anti-HIV activity as their parent drugs in ATH8 cells but seem to be slightly more cytotoxic.^^^ Phosphorylation of 2'-F-ara-ddA by dCyd kinase forms 2'-F-ara-ddAMP, which is then sequentially phosphorylated to 2'-F-ara-ddADP and 2'-F-ara-ddATP. In MT-4 cells, the levels of 2'-F-ara-ddADP and 2'-F-ara-ddATP are 20- and 5-fold higher than the levels of ddADP and ddATP under the same incubation conditions.^^^ As a potent ADA inhibitor, 2'-deoxycoformycin significantly increased the levels of 2'-F-ara-ddATP when 2'-F-ara-ddA was incubated in ATH8 cells.^^^ 2',3'-Dideoxy-2'-fluoro-P-D-arabinofuranosyl cytosine (80, 2'-F-ara-ddC) is effective against several strains of HIV in a number of different cell lines, but the in vitro therapeutic index of this compound is considerably lower than that of ^2^J 116,135,136 PQJ. |-j^jg reason, initial clinical trials of 2'-F-ara-ddC were discontinued. On the other hand, the ribo derivatives of 2'-F-ara-ddNs including the cytidine analog do not retain the antiviral activity of the parent compounds.^^^ Additional introduction of a fluorine atom at the 3'-position either in the ribo or in the arabino configuration also abolishes the anti-HIV activity of the parent drug.^^^ \ ^ \j^ 79 (2'-F-ara-ddA) 80 (2'-F-ara-ddC) tE:/ 81 (L-2'-F-ara-ddC) Figure 28. Biologically active 2',3'-dideoxy-2'-fluoro nucleosides. Chu and co-workers have reported synthesis and antiviral activity of L-2'-F-ara-ddNs, against HIV and HBV.^^^ Among the synthesized compounds, the cytosine analog (81) shows moderate anti-HIV activity (Figure 28). 2',3'-Dideoxy-2',2'-difluoro-P-L-ribofu- 25 Recent Advances in Antiviral Nucleosides ranosyl nucleosides have also been reported, but none of them showed significant activity or toxicity.^^^ Fluorination at the 2' or 3'-position of 2',3'-dideoxy-4'-thionucleosides provides fluorinated nucleosides of four different configurations, of which only the cytidine analog displays weak activity against HIV in ATH8 cells.^"^^ Substitution of a hydroxymethyl group in ddNs has also produced some anti-HIV activity. Originally, the 3'-hydroxymethyl branched nucleosides of 2-deoxyribofuranose were synthesized as anti-tumor agents.^"^^ Both a- and P-thioguanine analogs with 2,3-dideoxy-3-(hydroxymethyl)-D-^ry^/ir(9-pentofuranose show an inhibitory effect on the growth of WI-L2 human lymphoblastoid cells and are phosphorylated and incorporated into the DNA to the same extent of Mecca lymphosarcoma in mice, proving more effective than the parent analog, a-2'-deoxythioguanosine. Since the pandemic of AIDS, a number of 2',3'-dideoxy-3'-C-hydroxymethyl nucleosides have been prepared and evaluated against HIV.^"^^ Among them, the adenine derivative (82, Figure 29) appears to be the most effective in inhibiting viral replication in H9 cells with an activity comparable to ddl and AZT. The cytosine derivative is also a potent inhibitor of HIV-1 in vitro and a broad-spectrum antiviral agent. The 4'-thio-^'^^^ and carbocyclic^"^^^ analogs of 2',3'-dideoxy-3'-C-hydroxymethyl nucleosides are devoid of anti-HIV activity. NHo it) HO-i N HO 82 Figure 29. 2',3'-Dideoxy-3'-C-hydroxymethyladenosine. Various C-branched functionalities, such as hydroxymethyl, fluoromethyl, azidomethyl and aminomethyl, have also been introduced to the 2,3-dideoxy-^ryf/iro-pentofuranose, 3-deoxyribofuranose and 3-deoxyarabinofuranose moieties.^'^'^ Among the derived nucleosides, 2',3'-dideoxy-3'-C-hydroxymethylthymidine has significant anticancer activity against L1210, P388, S-180 and CCRF-CEM cells with ED^^^ values of 50, 5, 10 and 1 |iM, respectively. However, none of these compounds show any significant antiviral activity against HSV-1, HSV-2 or HIV. Evaluation of these compounds against thymidine kinases derived from HSV-1 (strain KOS), HSV-2 (strain 333) and mammalian (K562) cells shows that TK from HSV-1 is inhibited significantly by both 3'-deoxy-3'C-(hydroxymethyl) and 3'-deoxy-3'-C-(fluoromethyl)thymidines. Introduction of a fluorine atom in the 2'-|3-position of 2',3'-dideoxy-3'-C-hydroxymethyl nucleosides has also been considered. The thymine, 5-iodouracil and cytosine analogs showed weak anti-HSV-1 activity.^"^^ Their carbocyclic analogs are inactive against HIV-1 and HSV-1.^^^ 26 1.5. G. Gumina, Y. Choi and C. K. Chu 2',3'-Unsaturated nucleosides and related analog Although for nucleoside analogs it has not been possible to elaborate a pharmacophore, 2',3'-unsaturated sugars are probably the most effective moieties for the inhibition of HIV and HBV replication. Among compounds with this feature, 2',3'-didehydro-2',3'dideoxythymidine (83, d4T, stavudine)^'* and its carbocyclic 2-amino-6-cyclopropylaminopurine analog (84, 1592U89, abacavir)^^ have been approved for the treatment of HIV infection (Figure 30). A O 83 (d4T) HN 84 (abacavir) Figure 30. Anti-HIV-1 agents d4T and abacavir. The phosphorylation of d4T to its monophosphate is the rate-limiting step of the sequential conversion to d4TTP, which inhibits HIV-RT equipotently with AZTTP.^^'^^^ d4TTP inhibits DNA polymerase y and is incorporated into the viral DNA, thus terminating DNA synthesis at the incorporation site (Figure 31). The 3',5'-exonuclease £ cannot remove d4TMP from the 3'-end of DNA once it is incorporated into cellular DNA, whereas, in the case of AZTTP, the enzyme maintains about 20% of its normal deoxynucleotide excision capability. ^"^^ It has been shown that d4T causes peripheral neuropathy. d4T, however, shows 10-fold less toxicity to human hematopoietic progenitor cells compared to AZT.^"^^ After exposure of human bone marrow cells to similar extracellular levels of parent drugs, steady-state level of d4TMP incorporated into cellular DNA was 10- to 50-fold less than that of AZTMP.^^^ In CEM cells, d4T decreased mt-DNA synthesis with higher potency than that of AZT.^^^ L-d4FC (85) and L-d4C (86, Figure 32) have also been reported to have potent antiviral activities.^^2 L-d4FC showed potent anti-HBV activity (EC^^ 2 nM in 2.2.15 cells) and anti-HIV activity (EC5Q 0.09 |iM in CEM cells), whereas L-d4C was less potent against both viruses (8 nM and 1.0 |LiM for HBV and HIV, respectively). However, both compounds inhibited cell growth at concentrations below 20 |iM. Nevertheless, L-d4FC did not exhibit significant inhibition of mt-DNA at 100 |LiM. The fluorinated derivative D-d4FC (87, Figure 32) has potent anti-HIV activity in vitro with an EC^^ value of 0.05 jiM in PBM cell and anti-HBV activity with an EC^^ value of 3 nM in 2.2.15 cells without cytotoxicity up to 100 jiM in both cell lines.^^^'^^"^ A comparison of the antiviral activity of D-d4FC and L-d4FC shows that the latter is active against HIV (EC^^ 0.034 |iM) and HBV replication (EC^^ 0.01 |LiM), but has significant cytotoxicity in various cell lines.^^^ Another comparison of the antiviral activity Recent Advances in Antiviral 27 Nucleosides of the two enantiomers has been reported, in which L-d4FC was active against HB V in 2.2.15 cells and HIV in MT-2/IIIB cell line with an EC50 value of 0.008 and 0.2 |iM, respectively, while D-d4FC showed anti-HBV and anti-HIV activity with an EC^^ value of >0.3 and 0.2 jLiM, respectively.^^^ These results are different from those previously published.^^^ In any case, as reported, L-d4FC appears to be more toxic than D-d4FC.^^^ An important feature of D-d4FC is its activity against 3TC- and AZT-resistant viral strains.^^^ The combination of its resistance profile, rapid uptake and conversion to the active triphosphate, and intracellular half-life of 13 to 17 h^^^ make D-d4FC a promising anti-HIV candidate. Both D- and L-d4FC are currently undergoing clinical trials. Inhibition of HIV-1 RT/ chain ternnination TK d4T d4TTP d4TDP d4TMP ratelimiting Incorporation into cellular DNA or inhibition of DNA polymerase y Figure 31. Cellular metabolism of d4T. ^' NHp NHo N NHo N k^ 85 (L-d4FC) OH O^ M N 86 (L-d4C) r-OH HO—i N 87 (D-d4FC) Figure 32. Anti-HIV-1 and anti-HBV agents L-d4FC, L-d4C and D-d4FC. In a series of purine analogs, L-d4A has shown potent anti-HIV-1 activity with an EC^^ value of 0.38 and 0.54 |LiM in PBM and CEM cells, respectively, and moderate anti-HBV activity with an EC^^ value of 1.2 JLLM in 2.2.15 cells.^^^ L-d4I and L-d4G exhibit moderate anti-HIV-1 activity with EC^^ of 5.5 and 14.1 jLiM in PBM cells, respectively.^^^ 28 G. Gumina, Y. Choi and C. K. Chu Although D-2',3'-didehydro-2',3'-dideoxyguanosine d4G (88, Figure 33) was found to be inactive against HIV-1,^^^ recent transient kinetic studies with HIV-1 RT showed that its triphosphate could potentially be an inhibitor of the viral enzyme. ^^^ The reason for the lack of activity of d4G was found to be its solution instability. In fact, the stable prodrug cyclo-d4G (89) is active against HIV-1 (EC3Q 8.6 |LiM in MT-2 cells), with increased stability, lipophilicity and solubility, as well as decreased toxicity compared to H N ^ NH y^-rT 88 (d4G) ^NH 89 (cyclo-d4G) Figure 33. d4G and its prodrug cyclo-d4G. 2'- or 3'-Substituted d4N analogs also retain their antiviral activity. In particular, introduction of a fluorine atom at the 2'-position produces compounds with anti-HIV and anti-HBV activity (Figure 34). Among the 2'-fluorinated d4N analogs, cytosine derivative D-Fd4C (90) shows moderate anti-HIV activity with significant toxicity. ^^^ A complete SAR study of the D-series has also shown that the 5-F-cytosine analog (91) possesses potent anti-HIV and anti-HBV activity.^^'^^^ Most of the purine analogs have moderate to potent anti-HIV activity. In addition, the adenosine and inosine derivatives show no cross-resistance against 3TC/FTC-resistant strains.^^^ Chu and co-workers demonstrated that a series of L-Fd4N has an interesting biological profile.^^^ Among these compounds, cytosine (92) and 5-F-cytosine (93) exhibit potent anti-HIV-1 and anti-HBV activity. In addition, L-Fd4A is moderately active against HIV and HBV. Further study of this series revealed that L-Fd4C and L-Fd4FC are among the most potent anti-HBV agents (EC^^ 2 and 4 nM in 2.2.15 cells, respectively).^^^ NH2 HO—1 ^N-^O 90 (D-Fd4C, X = H) 91 (D-Fd4FC, X = F) NH2 O^H^ p-OH 92 (L-Fd4C, X = H) 93 (L-Fd4FC, X = F) Figure 34. D- and L-2' -fluorinated-2' ,3' -unsaturated cytidines. 29 Recent Advances in Antiviral Nucleosides Among 3'-fluoro-2',3'-unsaturated D-nucleosides (Figure 35), 3'-Fd4C (94) and 3'-Fd4A (95) show modest anti-HIV activity in H9 cells.^^"^'^^^ The thymidine analog is marginally active against HIV in MT-4 cells.^^^ The 3'-fluoro-2',3'-unsaturated-L-cytosine (96) is a potent anti-HIV agent, with EC^^ of 0.03 |iM in PBM cells with little or no significant toxicity. ^^^ NH2 NH2 F' 94 (D-3'-F-d4C) NH2 F ^F 96 (L-3'-F-d4C) 95 (D-3'-F-d4A) Figure 35. 3'-Fluorinated d4Ns, The 4'-thio analogs of d4C also show marked anti-HBV and anti-HIV activity (Figure 36).^^^ Particularly, L-4'-thio-d4C (97) and L-4'-thio-d4FC (98) exhibit significant anti-HIV (EC^^ 0.8 and 0.4 |iM in HeLa CD4 cells, respectively) and anti-HBV (EC^^ 0.8 and 3.5 |LiM in HepG2 cells, respectively) activity without toxicity. No other antiviral activity of these compounds has been detected up to 100 |iM against HSV-1, HSV-2, VZV, HCMV and influenza.^^*' NH2 NH2 .F O^N-^ r-OH ^ ^ 97 (S-d4C) (-0H O^H^ ^ ^ 98 (S-d4FC) Figure 36. Biologically active L-4'-thio-d4Ns. The recently reported D- (99) and L- (100) 2'-fluorinated 4'-thio-2',3'-unsaturated cytidines (Figure 37) also show potent anti-HIV activity, with EC^^ values of 0.37 and 0.47 |LiM, respectively, and no significant toxicity up to 100 ^M.^^^'^^^'^^^ 30 G. Gumina, Y. Choi and C. K. Chu NHo NH2 1 O^H^ (I L r-OH HO-1 ^N^O F F 99 (D-2'-F-S-d4C) 100 (L-2'-F-S-d4FC) Figure 37. D- And L-2'-fluorinated-4'-thio-2',3'-unsaturated cytidines. Replacement of the furanose ring with a cyclopentyl ring yields selective HIV inhibitors. Structure-activity relationship studies indicate that the optimal anti-HIV activity requires a 2-amino-6-substituted purine and a 2',3'-unsaturated carbocyclic sugar moiety.^^'^^^ Racemic carbovir (CBV) was first reported to show anti-HIV activity with low toxicity in H9 cells. (-)-CBV (101) was found to be the biologically active isomer against HIV, having a 75-fold higher activity than its (+)-counterpart (102, Figure 38), although HIV-RT is equally sensitive to (-)-CBVTP and (+)-CBVTP.^^^ The difference in the anti-HIV activity of CBV enantiomers appears to result from the preferential stereoselective phosphorylation of (-)-CBV over its (+)-counterpart. CBV is anabolized intracellularly to its mono-, di- and tri- phosphates rather inefficiently. The enzyme mediating the monophosphorylation is cytosolic 5'-nucleotidase, while the diphosphorylation is catalyzed by GMP kinase (Figure 39).^^^ Both these enzymes show preferential selectivity for (-)-CBV over (+)-CBV.^^3 In contrast with AZTTP, CBVTP is an inhibitor of HIV-RT, but essentially has no effect on DNA polymerase a, p and y.^^^i^^ (_)_CBVTP inhibits HIV-1 RT with an apparent K. similar to that of AZTTP and, in addition, (-)-CBVMP is also incorporated into the proviral DNA and acts as a chain terminator. ^^^ CBV and AZT do not affect each other with regard to their intracellular anabolism. The cytotoxicity of CBV may be due to the inhibition of the DNA synthesis, and DNA polymerase a would be responsible for the majority of the incorporation of CBV into DNA in CEM cells.^'^^ (-)-CBV has no delayed adverse effect on mt-DNA synthesis,^^'^ however, its poor solubility has prevented the development of CBV as a drug. For this reason, abacavir was synthesized to improve its solubility and pharmacokinetic profile. o . " - / • . HO-i N^N^NH, 101[(-)-CBV] N H^N^N^N 102[(+)-CBV] Figure 38. Carbovir and its (+) isomer. pOH Recent Advances in Antiviral 31 Nucleosides Incorporation into proviral DNA/chain termination or Inhibition of HIV RT/ chain termination (-)-CBV (+)-CBV (-)-CBV-MP ^ (-)-CBV-DP \ / GMP \ / 5-nucleotidase w/ kinase vy ^ (+)-CBV-MP (+)-CBV-DP (-)-CBV-TP No interaction with DNA polymerase a, p and y Figure 39. Cellular metabolism of (-)- and (+)-CBV.'' Abacavir (84, Figure 40), the 6-cyclopropylamino analog of CBV, shows significant inhibition of HIV in PBL cultures with the potency equivalent to AZT, and has synergistic anti-HIV activity in combination with AZT.^^ Its cytotoxicity is low in various human T-cells and bone marrow cells. In addition, toxicity common to other dideoxynucleosides such as peripheral neuropathy and hematopoietic toxicity has not been detected during preclinical studies. The intracellular activation of abacavir sequentially included its monophosphorylation by adenosine phosphotransferase, deamination to (-)-CBVMP by cytosolic deaminase, and two further phosphorylation steps to form (-)- CBVDP and (-)CBVTP (Figure 40).^^^ Therefore, abacavir overcomes the pharmacokinetic and toxicological deficiencies of CBV while maintaining potent and selective anti-HIV activity. ^^^ For this reason, it has been approved by the FDA for the treatment of HIV infection, and has also been incorporated in a combination with AZT and 3TC (trizivir®).^^^'^^^ HN HO OCX N - ' ^ ^ M ' ^ ^ .J n ^ _ . N NH2 84 abacavir adenosine phosphotransferase cytosolic deaminase ^ abacavir-MP (-)-CBV-MP (-)-CBV-TP (-)-CBV-DP Figure 40. Cellular metabolism of abacavir.^ 32 G. Gumina, Y. Choi and C. K. Chu Among L-carbocyclic 2',3'-didehydro-2',3'-dideoxy nucleosides, only the adenine analog (-)-BCA (103) has potent in vitro anti-HBV activity (EC^Q 0.9 jLiM in 2.2.15 cells) as well as moderate anti-HIV activity (EC^^ 2.4 jiM in PBM cells) without cytotoxicity up to 100 |LiM. Its D-counterpart, (+)-BCA (104) is devoid of anti-HIV activity (Figure Aiy^'^'^^ NHo NHo HO^ HO < I J 104[(+)-BCA] 103[(-)-BCA] Figure 41. 2',3'-Didehydro-2',3'-dideoxy-4'-C-hydroxymethyl carbocyclic nucleosides. In order to obtain more potent antiviral compounds by increasing the bioavailability of the drug or bypassing critical steps such as the first phosphorylation, a number of prodrugs of 2'-deoxy and 2',3'-unsaturated nucleosides have been prepared.^J In the attempt of obtaining higher intracellular levels of d4T, a number of prodrugs have been developed which can deliver d4TMP. Phosphoramidate derivatives of d4TMP (105)^^^ efficiently deliver the monophosphate (106), according to the mechanism showed in Figure 42. The same approach has proved successful in delivering 3TCMP,^^'^ AZTMP,^^^ ddAMP^^^ and d4AMP.^^6^^^ P^-OThd NH CH. esterase H3C -0-P-O X'^=^ NH \ H3C-CH 0=C OCH: NH HO- P-OThd NH esterase ^ H O - P - O T h d H3C-CH OH 0=C 106(d4TMP) OH ^ 105 chemical hydrolysis HO-P-OThd NH H3C-CH 0=C OH Figure 42. Phosporamidate prodrugs of d4T and their mechanism of action. 33 Recent Advances in Antiviral Nucleosides In the cjc/oSal-pronucleotide approach, nucleotides are deHvered intracellularly thanks to pH-driven selective chemical hydrolysis of the prodrug (Figure 43).i88,i89,i9o, The tandem cleavage originates with the hydrolysis of a phenyl ester (107) followed by hydrolysis of a benzyl ester in the resulting phosphotriester (108) with liberation of the nucleotide (109). This concept is based upon the principle that selection of phenyl, benzyl and alkyl phosphate esters can influence the hydrolysis steps of the tripartate approach. The phenyl ester is cleaved first, because of stabilization caused by delocalization of the negative charge in the aromatic ring, affording the 2-hydroxybenzylphosphodiester. This concept has been applied to anti-HIV and antitumor agents such as d4T, 191,192,193 5_FU,i94 AZT,''''''''''' 2',3'-dideoxyadenosine (ddA),''''''' d4Ai99 and 2'-fluoro2',3'-dideoxyadenosines (F-ara-ddA and F-ribo-ddA).^^^ O selective chemical ^T^^'^^^^^O" V - spontaneous cleavage Y 107 ^W ^^^ V^OH 108 9 ^ONu 109 Y Figure 43. Proposed decomposition of c};c/6>Sal-pronucleotides 1.6. Nucleosides with a heterocyclic sugar ring moiety This important class of nucleosides will be extensively discussed in Chapter 3. Since the discovery of (±)-dioxolanethymine (110)^^ and (±)-BCH-189 (111)^^ as potent anti-HIV agents as racemic mixtures, all the four possible diastereomers have been synthesized (Figure 44). From extensive structure-activity relationships studies of these isomers, a number of compounds have made considerable impact on HIV and HBV chemotherapy.^^^'^^^ Currently, among this series of nucleosides, 3TC (lamivudine) has been approved by the FDA for the treatment of HIV-1 and chronic HBV infections, and 2',3'-dideoxy-5-fluoro-3'-thiacytidine (FTC)^^^ and |3-D-2,6-diaminopurine dioxolane (DAPD)^^^'' are under clinical development as anti-HIV-1 and anti-HBV agents, and P-L-dioxolane cytosine (L-OddC)^^^"^ as an anti-cancer agent. 0 TT iin (±)-dioxolane-thynnine NH2 HO-i ^N"^( m (±)-BCH-189 Figure 44. Racemic dioxolane-thymine and BCH-189. 34 G. Gumina, Y. Choi and C. K. Chu Belleau and co-workers originally reported that (±)-BCH-189 exhibited potent in vitro anti-HIV activity in T-cells as well as human peripheral lymphocytes. (±)-BCH-189 was found to be less toxic than AZT, inhibited AZT-resistant virus, and was non-toxic at 100 mg/kg given orally over 14 days in rats.^^ Moreover, it showed potent anti-HBV activity in 2.2.15 cells.^^^ Since then, various approaches for the asymmetric synthesis of optically active compounds have been reported including enzymatic resolution.^^'^^"^ Of significance was that while both enantiomers have potent anti-HIV activity, cytotoxicity resides mainly with the natural (+)-D-isomer (Table 2)}^^ Table 2. Comparison of antiviral activities and cytotoxicities of DL- and LL-oxathiolane cytosine analogs.^^^ Anti-HIV-1 (EC50, ^iM) Anti-HBV (EC50, ^iM) 2.2.15 Cytotoxicity (IC50, ^iM) PBM GEM PBM GEM (+)-D-BCH-189 0.2 0.1 0.5 2.7 >100 (-)-L-BCH-189 (3TC) 0.002 0.007 0.001 >100 >100 The first asymmetric syntheses of enantiomers of (±)-BCH-189 and their trans-i^omers were described by Chu and co-workers from D-mannose, D-galactose or L-gulose as starting materials. ^^^ An extensive study of structure-activity relationships has made clear that the unnatural L-2',3-dideoxy-3'-thiacytidine (112, 3TC, Figure 45) is more potent against HIV-1 in human PBM cells as well as against hepatitis B virus (HBV) in 2.2.15 cells than its racemate or its D-enantiomer (113). Most importantly, the comprehensive S AR of the enantiomerically pure D- and L-isomers revealed that most of the nucleosides, among which the 5-fluoro analogs 114 and 115, exhibited not only good to excellent anti-HIV-1 activity, but also low toxicity in PBM as well as Vero cells.^^^ NH2 \ 0^'^N NH; \\ [I r-OH 112 [(-)-3TC, X = H)] 114 [(-)-FTC, X = F] HO—1 L N^'^O 113 [(+)-BCH189.X = H] 115 [(+)-FTC, X = F] Figure 45. 3TC, FTC and their enantiomers. Regarding the cellular metabolism of the optically pure isomers of (±)-BCH-189, 3TC is resistant to deamination or enzymatic hydrolysis,^^^ whereas the D-isomer is de- 35 Recent Advances in Antiviral Nucleosides aminated to 2'-deoxy-3'-thiauridine, although no hydrolysis of glycosyl bond is observed (Figure 46). dCyd kinase is the enzyme responsible for the monophosphorylation of 3TC,2^^ which is a better substrate than the D-counterpart.^^^ Further phosphorylation of 3TC or (+)-BCH-189 to its di- and triphosphate is accomplished by deoxycytidylate and NDP kinase, respectively. Inhibition of DNA polymerases (5, y D-SddU: inactive dCyd deaminase (+)-BCH 189 (+)-BCH189MP dCyd kinase 3TC t (+)-BCH189DP NDP kinase deoxycytidylate kinase 3TCMP -H- (+)-BCH189TP 3TCDP 3TCTP dCyd deaminase L-SddU: inactive Competitive with dCTP against HIV^1 and HBV DNA polymerase: Incorporation into viral DNA chain and chain termination Figure 46. Metabolism of (+)-BCH-189 and 3TC. 3TCTP is a competitive inhibitor (with respect to dCTP) of the RNA-dependent DNA polymerase activity with apparent K. = 10.6 ± 1.0 to 12.4 ±5.1 |iM, depending on the template and primer used.^^^ DNA-dependent DNA polymerase activity is inhibited by 50% by a 3TCTP concentration of 23.4 ± 2.5 |iM when dCTP is present at a concentration equal to its K^^ value. 3TCTP is a rather weak inhibitor of DNA polymerase y, but (±)-BCH-189 is about 650 times more inhibitory than 3TC, due to the activity of the D-isomer. This observation might explain why 3TC is more potent and less toxic than its D-counterpart against HIV-1 in vitro}^ Furthermore, 3TCTP is not an inhibitor of DNA polymerase P, whereas (±)-BCH-189 has a significant inhibitory effect on this enzyme (Figure 46).^^° Chain elongation studies with 3TC show that 3TCTP is incorporated into newly synthesized DNA and that transcription is terminated in similar fashion as seen with ddCTP. As mentioned before, 3TCTP also shows a potent inhibitory effect against HBVassociated DNA polymerase, and is a better inhibitor of HBV DNA polymerase than its D-counterpart.^^^ Schinazi et al. have reported the anti-HIV activity of the racemates as well as the single enantiomers of the 5-fluoro congener of 3TC, FTC (emtricitabine, 114, 36 G. Gumina, Y. Choi and C K. Chu Figure 45).2oi'2i 1,212,213 p ^ ^ ^^ows potent in vitro anti-HIV-1, HIV-2, SIV, and FIV activity in various cell cultures. Like 3TC, FTC exhibits 20-fold more potency against HIV-1 in human PBM cells and less toxicity in myeloid progenitor cells than its D-enantiomer (115). It also shows anti-HBV activity in hepatoma cell lines (HepG2 cells), whereas its D-enantiomer is significantly less potent.^^^ However, both enantiomers do not show significant cytotoxicity in human bone marrow progenitor cell assays and any detectable hepatotoxic effects at concentrations above their antiviral activities. Currently, (-)-FTC (coviracil) is undergoing phase III clinical trials against HIV and HBV infection and racemic FTC (racevir) is in phase I clinical trials as an anti-HIV agent. Enzymatic studies of FTC show a similar profile to 3TC, in which the D-enantiomer of FTC is a substrate for dCyd deaminase, and FTC is resistant to deamination by the same enzyme.^^^^ dCyd kinase and NDP kinase phosphorylate FTC to its triphosphate, which functions as chain terminator of viral DNA synthesis, similarly to 3TC. Schinazi et al. have also reported that highly 3TC/FTC-resistant HIV-1 variants dominate the replicating virus population after two or more cycles of infection in the presence of 3TC or FTC.^'^^ These variants are cross-resistant to 3TC and FTC but are susceptible to ddC, AZT, and ddl. DNA sequence analysis of the RT gene amplified from resistant viruses consistently identified a mutation at codon 184 from Met (ATG) to Val (GTG or GTA) or He (ATA). Synthesis of the 3'-deoxy-3'-oxa-thymidine analog (±)-dioxolane-T and other natural pyrimidine base analogs has given promising leads for the inhibition of HIV-1 as well as HBV and herpes virus replication. Originally, (±)-dioxolane cytosine was reported by Belleau and co-workers as an anti-HIV agent and subsequently, (±)-dioxolane thymidine was also reported as moderately active against HIV-1.^^'^^ Extensive studies of the structure-activity relationships has led to the synthesis of p-L-dioxolane cytosine (116, L-OddC) and its 5-fluoro congener (117, L-F-OddC), which exhibit potent in vitro anti-HIV-1 and HBV activities (Figure Al)}^^ However, L-OddC is quite toxic and stable to degradation by cytidine deaminase and deoxycytidine deaminase. O-" N f—OH -o 116(L-0ddC) 117(L-F-0ddC) Figure 47. P-L-Dioxolane cytosine and 5-fluorocytosine. L-OddC is metabolized in cells by dCyd kinase to its monophosphate, and subsequently to the di- and triphosphate, which inhibits DNA polymerase a, (3, and y (Figure 48).^^^ L-OddC exhibits potent antitumor activity against various solid tumor cell lines, including prostate, renal, hepatoma, and colon.^^^ Thus, L-OddC is the first Recent Advances in Antiviral 37 Nucleosides L-nucleoside analog ever shown to have anticancer activity, and also the first true chain terminator capable of inhibiting tumor growth.^^^ L-OddC is currently undergoing phase II clinical trials against leukemia and solid tumors. L-OddC t dCyd kinase L-OddC-MP - ^ L-OddC-DP L-OddC-TP dCyd deaminase L-OddU: inactive Inhibition of DNA polymerase a, p, y: Incorporation into HepG2 DNA chain and chain termination antitumor activity against various solid tumor cell lines Figure 48. Metabolism of L-OddC.^'^b Among purine derivatives, p-2,6-diaminopurine dioxolanes, DAPD (118) and its enantiomer, L-DAPD (119), display potent anti-HIV and anti-HBV activities (Figure 49). 202^^ Interestingly, L-DAPD is more potent against HIV-1 (EC^^ 0.014 |LiM) than DAPD (EC^Q 0.7 |iM) in human PBM cells, while DAPD shows more potent anti-HBV activity (EC5Q 0.009 |LiM) than its L-isomer (EC^^ 8.3 juM) with a favorable toxicity profile. NHo OH 118 (DAPD) 119 (L-DAPD) Figure 49. Enantiomers of P-2,6-diaminopurine dioxolane. Pharmacokinetic studies suggest that DAPD is the prodrug of the corresponding guanine derivative, dioxolane-guanine (120, DXG). DAPD and P-D-2-amino-6-chloropurine dioxolane (121, ACPD) are converted to DXG by ADA, and p-D-2-aminopurine dioxolane (122, APD) is converted to DXG by xanthine oxidase (Figure 50).^^^ As discussed extensively in Chapter 3, DAPD and DXG are active against 3TC-resistant HIV and HBV strains, which provides DAPD a promising therapeutic potential. 38 G. Gumina, Y. Choi and C. K. Chu NH2 itx "°>_OJ'"N NHo N 118(DAPD) NH2 122 (APD) xanthine oxidase ADA CI «-11. -V0^^^N--NH. ADA , ^ ° n ^ ^ ^ ^ ^ N - - ^ 0- 121 (ACPD) 120 (DXG) Figure 50. Pharmacokinetics of DAPD, APD, and ACPD.^ Mansour and co-workers reported synthesis and activity of BVU analogs with a dioxolane moiety, among which the (i-L-dioxolane nucleoside shows significant activity against HSV-1 {EC^^ 0.3 fig/mL) and HCMV {EC^^ 5 jig/mL). The p-D-oxathiolane nucleoside demonstrated potent activity against HSV-2 (EC^^ 2.9 |Xg/mL).^^^ In an extensive SAR study, Chu and co-workers recently reported the activity of a series of dioxolane and oxathiolane (F)-5-(2-halovinyl)uracil nucleosides (Figure 51) against a number of viruses.^^ The P-L-dioxolane nucleosides show potent anti-VZV and anti-EBV activities, which can be related to the size of the halogen atoms [chlorovinyl (123) < bromovinyl (124) < iodovinyl (125) against VZV and iodovinyl < bromovinyl < chlorovinyl against EBV]. p-L-(£)-5-(2-Iodovinyl)uracil dioxolane (125, L IV-OddU) is 60-fold more potent against VZV than ACV. No inhibition of CEM cell growth or mt-DNA synthesis is observed for any compounds at concentrations up to 200 jiiM. This selectivity has been explained, in the case of p-L-(£)-5-(2-bromovinyl)uracil dioxolane (124, L-BV-OddU), with selective phosphorylation by viral TK, but not human TK.^^^ Unlike other D-configuration BVU analogs, such as BVdU and BVaraU, L-BV-OddU is metabolized only to its corresponding monophosphate instead of the dior triphosphate, which suggests a unique inhibitory mechanism other than DNA chain termination. As mentioned above, L-dioxolane derivatives with 5-substituted uracil show potent anti-EBV activities (Figure 51).2i^ p-L-5-Iodouracil dioxolane (128, L-I-OddU) is the most potent anti-EBV agent with an EC^^ value of 0.03 |iM without any cytotoxicity up to 100 \\M. Also in this series, their activities can be related to the size of the halogens [EC^^ CI (126) 0.15; Br (127) 0.07; I (128) 0.033 |LiM]. L-I-OddU is an efficient substrate for EBV TK, but not for human cytoplasmic dThy or mt-dPyd kinases, with L I-OddUMP being the major metabolite.^^^ L-I-OddU and L-Br-OddU are currently undergoing preclinical studies as potential anti-EBV agents. 39 Recent Advances in Antiviral Nucleosides O NH ^VNH 123 (L-CV-OddU, X = CI) 124 (L-BV-OddU, X = Br) 125 (L-IV-OddU. X = I) 126 (L-CI-OddU. X = CI) 127 (L-Br-OddU. X = Br) 128 (L-l-OddU, X = I) Figure 51. Structure of L-I-OddU, L-BV-OddU and related nucleosides. Reversed oxathiolane nucleosides, such as 2'-deoxy-3'-oxa-4'-thiocytidine (dOTC, Figure 52), have also been found active against HSV-1, HSV-2, HBV and HIV-1 in a panel of cell lines. The BVU analog has demonstrated potent activity against HSV-2 and the cytosine and 5-F-cytosine derivatives have exhibited appreciable antiviral activity in cord blood mononuclear cells (CBMCs) and U937 (human monocyte) cell lines.217 221 (+)_ciOTC is moderately active against HBV in 2.2.15 cells. (±)-dOTC (129) is phosphorylated within cells via the dCyd kinase pathway and approximately 2 to 5% is converted into the racemic triphosphate derivatives (Figure 52).^^^ Both 5'-triphosphate derivatives (TP) of (±)-dOTC are more potent than 3TCTP at inhibiting HIV-1 RT in vitro. In cell culture experiments, (±)-dOTC is a potent inhibitor of primary isolates of HIV-1 with an IC^^ for viruses resistant to 3TC and viruses resistant to 3TC and AZT of 2.53 and 2.5 |LiM, respectively.^^^ After 14 days of continuous culture, at concentrations up to 10 |iM, no measurable toxic effect on HepG2 cells or mitochondrial DNA replication within these cells has been observed. dOTC is currently undergoing phase I clinical trials as an anti-HIV agent. NHo HO—I ^N^O dCyd kinase (±)-dOTC-MP ^ (±)-dOTC-DP 129 [(±)-dOTC] ( Inhibition of HIV RT ) ^ (±)-dOTC-TP Figure 52. Metabolism of (±)-dOTC.2 Prepared as bioisosters of 3TC and FTC, oxaseleno compounds (±)-Se-ddC (130) and (±)-F-Se-ddC (131, Figure 53), have also been found to exhibit potent anti-HIV-1 40 G. Gumina, Y. Choi and C. K. Chu (EC^Q 2.7 and 0.73 |LIM, respectively) and anti-HBV (EC^^ 1.2 and 1.2 |LiM) activities.^^^ Resolution of the racemic mixtures showed that most of the anti-HIV activity of 130 and 131 resides with the (-)-isomers (EC^^ 0.9 \iM for 130 and 0.2 |aM for 131).224 Substitution of 3'-sulfur in 3TC with an amino group does not produce antiviral activity against HIV.^^^ NH2 NH2 N ""^r^N I HO—I "N'^0 Se—-^ 130 [(±)-Se-ddC] [ I I HO-n N"^0 S e — ^ 131 [(±)-F-Se-ddC] Figure 53. Oxaselenonucleosides. Isodideoxynucleosides,^^^ in which the base is transposed from the 1' to the 2' position of the sugar, have been reported by Huryn et al}^'^ and Nair et al?^^ (Figure 54). The isomeric form of ddA, (/?,/?)-iso-ddA (132), is as active against HIV-1 as ddA, with a better hydrolytic stability.^^^ The anabolism of (/?,/?)-iso-ddA has been studied in CEM cell cultures.^^^ The formation of (/?,/?)-iso-ddATP is significant and increases almost linearly upon incubation for 24 h. In comparison, phosphorylation of ddA yields three to four times the amount of ddATP as (/?,/?)-iso-ddATP, but the amount of ddATP does not increase much after 10 h. (/?,/?)-iso-ddATP competitively inhibits the incorporation of dATP into a synthetic polynucleotide primer. Nitrogen or sulfur analogs of iso-ddA do not show significant antiviral activity.^^^^^^ (5,5')-Iso-ddA (133), which can be viewed as an L-related ddN, also shows significant antiviral activity against HIV in MT-4 and PBL cell cultures and is also active against AZT-resistant HIV strains.^^^ Synergistic effects are observed in combination with AZT, ddl or FTC. (5',5')-IsoddA has little cytotoxicity in leukemic cell lines and lower inhibition on human bone marrow cells than AZT. In CEM cells, it is metabolized, although rather inefficiently, to (5',5')-isoddATP, which is a potent inhibitor of HIV-RT. The metabolism of (5',5')-IsoddA is unique. The nucleoside is neither phosphorylated by adenosine kinase nor oxidized by adenosine deaminase. Instead, the first step in its metabolic activation seems to be phosphorylation by dCyd Kinase.^^^ Compared to ddATP, (iS,5')-isoddATP is a weaker inhibitor of DNA polymerase (3 and y, but a stronger inhibitor of DNA polymerase a.^^^ Another series of isoddNs is related to the natural sugar, D-apiose, and its enantiomer, L-apiose, which can be considered as regioisomers of the natural nucleosides through transposition of the hydroxy methyl group from the normal 4'-position to the 3'-position (134 and 135, Figure 54). A comprehensive study of these dideoxynucleosides has been accomplished by Sells and Nair^^^ However, these compounds do not show any antiviral activities, and even further modifications of the apiosyl moiety, with the in- 41 Recent Advances in Antiviral Nucleosides troduction of ring substituents such as 4'-hydroxymethyl,^^'^ 3'-fluoro,^^^ 3'-azido^^^ and 3'-amino,^^^ have failed to induce significant antiviral activity. 132 133 [(R, R)HSO-dd A] [(S, S)-iso-dd A] HO—1 Base Base r—OH ' \ : : ^ ^ D-apionucleosides L-apionucleosides 134 135 Figure 54. Enantiomers of iso-ddA and apionucleosides. A series of branched-chain sugar isonucleosides has been reported to exhibit significant antiviral activity against herpes viruses (Figure 55)?^^ The hydroxymethylguanosine analog (136) displays potent and selective anti-HSV-1 and anti-HSV-2 activity. Although the antiherpetic activity in vitro of this compound is lower than that of ACV, it displays superior efficacy in mouse infections. The BVU analog (137) also shows selective activity against HSV-1 and VZV, with no cytostatic effect on WI-38 cell growth at >800 |iM. Several other substituted isonucleosides have been prepared, including 3'-hydroxymethyl and 3'-azidomethyl derivatives, but none of them has shown any significant antiviral activity.^^^ O HO-x o . N'-^N'^^NH2 ^ 0 - A O HO 136 Figure 55. 3'-C-Hydroxymethylisonucleosides. . N ^O 42 G. Gumina, Y. Choi and C. K. Chu 1.7. 3- or 4-Membered ring nucleosides Transformation of the furanose ring to a 3- or 4-membered ring produces compounds with interesting biological activity. Oxetanocin A, 9-(2-deoxy-2-hydroxymethyl-P-D-erythrooxetanosyl)adenine (138) (Figure 56), is an antibiotic produced by Bacillus megaterium, which inhibits infection of T cells by HIV-1 in vitro.^^"^ Chemical and enzymatic modifications of oxetanocin A (OXT-A) have afforded 2,6-diamino (2-amino-OXT-A), guanine (OXT-G), hypoxanthine (OXT-H) and xanthine (OXT-X) analogs.^^o In MT-4 cells, OXT-A markedly reduces the expression of HIV antigens, and 2-amino-OXT-A, OXT-G and OXT-H also show significant anti-HIV activity. In addition, OXT-G is very potent and selective in inhibiting the replication of HCMV in vitro (EC^^ 0.1 jig/mL) and against HSV-2 (EC3, 3.5 \iglmL)}^' The thymidine analog of oxetanocin, A-73209 (139), is a potent in vitro and in vivo inhibitor of HSV-1, HSV-2 and VZV.^^^ A-73209 is two logs more potent against five TK^ strains of VZV in vitro and one log more potent against TK^ HSV-1 strains than ACV. A-73209 is more effective than ACV against lethal systemic or intracerebral HSV1 infections in mice. L-oxetanocin (L-OXT-A, 140) is inactive against HIV.^"^^ Early reports of racemic carbocyclic analogs of OXT-A and OXT-G have described the protective effect of both carbocyclic analogs on CD4^ ATH8 cells against the infectivity and cytopathic effect of HIV-1, suppressing pro viral DNA synthesis.^'^'^'^^'^'*^ In addition, carbocyclic OXT-G showed excellent activity against HSV and it was suggested that it is phosphorylated by virus-encoded TK prior to exerting its antiviral effect. In contrast, the adenine congener, carbocyclic OXT-A, is a good inhibitor of HCMV in vitro and in vivo. However, severe cytotoxicity to host cells has prevented further development of this compound as an anti-HCMV agent. NH2 V NH2 O NH HO-n ^ N ^N^ HO—' HO-i Q^ N HO—' 138 (OXT-A) 139 (A-73209) O ^N^ N ^ ^-OH "—OH 140 (L-OXT-A) H0-| ^ ^ "N NHj HO141 (LBV) Figure 56. Oxetanocin A (OXT-A), its thymine analog A-73209 and lobucavir (LBV). The active enantiomer of carbocyclic OXT-G (141, lobucavir, LBV, Figure 56) displays an impressive broad-spectrum antiviral activity against a wide variety of herpesviruses and HBV as well as HIV.^"^^ The mechanism of action of LBV against HSV-1, HSV-2 and VZV consists in the inhibition of the viral polymerases after phosphorylation by the virally encoded TK (Figure 57).^"^^ However, HCMV, HBV and HIV do not encode enzymes which are capable of mediating LBV phosphorylation. Recent Advances in Antiviral 43 Nucleosides It is known that HCMV has homologs of a herpesvirus-encoded protein kinase (UL97 gene), which mediates the phosphorylation of ganciclovir (GCV). In the case of VZV, both the herpesvirus TK and protein kinase may independently enable the phosphorylation of LBV. Furthermore, LBV is phosphorylated to its triphosphate intracellularly in both HCMV-infected and uninfected cells, with phosphorylated metabolites levels 2- to 30-fold higher in infected cells. These studies^^^ suggest that LBVTP can halt HCMV DNA replication by inhibiting the viral DNA polymerase and that LBV's phosphorylation can occur in the absence of viral factors including the UL97 protein kinase. In addition, LBV may be effective in the treatment of GCV-resistant HCMV. LBV has undergone clinical trials as an anti-HBV agent. viral TK for HSV-1/2 and VZV LBV LBV-DP LBV-MP LBV-TP protein kinase for HCMV Broad spectrum antiviral activity Figure 57. Metabolism of lobucavir. 3'-Fluorocarbocyclic oxetanocin A (142, Figure 58) exhibits a broad spectrum of antiviral activity especially against HCMV with an EC^^ of 0.18 |ig/mL, which is 4-fold more potent than that of ganciclovir.^"^^ However, this compound is slightly cytotoxic at higher concentrations (100 |ig/mL) in HEL or MT-4 cells, although this toxicity is minor compared with that of carbocyclic OXT-A (CC^^ for HEL; 8 |ag/mL, CC^^ for MT-4; 12 |ig/mL).248 NH2 N HO F HO 142 Figure 58. Carbocyclic 3'-fluoro-oxetanocin A. (±)-9-{[(Z)-2-(Hydroxymethyl)cyclopropyl]methyl}guanine (143) (Figure 59) displays significant antiherpetic activity in vitro, and the (£)-adenine analog has a modest antiviral activity despite an apparent inability to be enzymatically phosphorylated.^"^^ 44 G. Gumina, Y. Choi and C. K. Chu Enzymatic phosphorylation studies indicate that in the (cyclopropyl)methyl derivatives, both the cis- and trans-(hydroxymcihyl) derivatives are reasonably good substrates for the HSV-1 TK in comparison with ACV and its carba analog. The cis-isomer is converted efficiently to the triphosphate, but it inhibits the HSV-1 DNA polymerase poorly. The trans-isomcY accumulates as the diphosphate with little triphosphate detected, but the triphosphate appears to be a better inhibitor of HSV-1 DNA polymerase.^"^^ O NH NHo HO 143 Figure 59. (±)-9-{ [(Z)-2-Hydroxymethyl)-cyclopropyl]methyl}guanine. Recently, Chu and co-workers also reported the synthesis of enantiomeric l-[2-(hydr oxymethyl)cyclopropyl]methyl nucleosides, among which the adenosine and guanosine analogs show moderate antiviral activity against HIV-1 and HBV in PBM and 2.2.15 cells, respectively, without significant cytotoxicity.^^^ 9-{[d5'-r,2'-Bis(hydroxymethyl)cycloprop-r-yl]methyl}guanine (A-5021, 144, Figure 60) is an extremely potent anti-HSV-1 agent without significant cytotoxicity.^^^ Both enantiomers were prepared from chiral epichlorohydrins, and only one A-5021 (r5,2'/?-configuration) exhibits strong antiherpetic activity (EC^^ of 0.020 |ig/mL against HSV-1 Tomioka vs 0.81 |Lig/mL for ACV). A-5021 is more inhibitory than ACV against HSV-2 and VZV but ineffective against HIV. Its enantiomer (145) has modest anti-HSV-1 activity. A-5021 is monophosphorylated by viral TKs.^^^ A-5021 triphosphate accumulates more than ACVTP but less than penciclovir (PCV) triphosphate in MRC-5 cells infected with HSV-1 or VZV, whereas HSV-2 infected MRC-5 cells show comparable levels of A-5021 and ACV triphosphates. A-5021TP competitively inhibits HSV DNA polymerases with respect to dGTP (ACVTP > A-5021TP > PCVTP) and is incorporated into DNA instead of dGTP terminating elongation, although limited chain extension has been observed. Thus, the stronger antiviral activity of A-5021 appears to depend on a more rapid and stable accumulation of its triphosphate in infected cells than that of ACV as well as on stronger inhibition of viral DNA polymerase by its triphosphate than that of PCV. A number of 5-substituted uracil nucleoside derivatives with a 1-(VS,2'R)-[1\2'bis(hydroxymethyl)cyclopropyl]methyl group have also been reported to exhibit antiherpetic activity.^^^ Among them, the BVU analog (146, Figure 60) is the most potent (EC^Q 0.027 iLig/mL), 40 to 60-fold more than ACV (EC3Q 3.4 |Lig/mL) against clinical isolates of VZV. Catabolism of 146 does not produce BVU, responsible for toxic drug interactions (vide supra)}^^ Recent Advances in Antiviral 45 Nucleosides N ^O HO 144(A-5021) 145 Figure 60. Cis-1' ,2'-bis(hydroxymethyl)-cyclopropyl]methyl nucleosides. Other types of cyclopropyl-containing nucleosides (147,148 and 149), where the base is directly linked to the cyclopropyl ring, have been prepared, but none of them showed significant antiviral activity (Figure 61).^^'^ HO HO OH B 147 148 149 Figure 61. 2-(Hydroxymethyl)cyclopropyl nucleosides. Nucleoside analogs based on a methylenecyclopropane structure show broad-spectrum antiviral activity against HCMV, EBV, human herpes virus type 6 (HHV-6), VZV and HBV (Figure 62)?^^ (Z)-2-{[(Hydroxymethyl)cyclopropylidene]-methyl}adenine (150, synadenol), -2-amino-6-chloropurine (151) and -guanine (152, synguanol) are the most effective agents against HCMV (EC^^ 1-2.1, 0.04-2.1 and 0.8-5.6 |iM, respectively) and EBV in H-1 cells (EC^^ 0.2, 0.3 and 0.7 |LiM, respectively). Synadenol is moderately active against HIV, HBV, VZV and HHV-6. It is a substrate for ADA from calf intestine and is also deaminated by AMP deaminase from Aspergillus sp. HO N ^ ^ 150 (synadenol) o CI NHo HO iti v-- N NH N L NH2 HO 151 Figure 62. Methylenecyclopropyl nucleosides. N 152 (synguanol) NHo 46 G. Gumina, Y. Choi and C. K. Chu Conversion of the modestly active analogs to their methyl phenyl phosphoro-L-alaninate esters results in potentiation of their anti-HIV-1 activity.^^^ Among these prodrugs, the 2,6-diaminopurine (153) and adenine derivatives (154, Figure 63) are the most potent against HIV-1 in vitro with EC^^ of 0.034 and 0.0026 jiM, respectively in MT2 cell-based assays. Both compounds are interestingly active against AZT-resistant, ddl-resistant and multi-dideoxynucleoside-resistant infectious clones in vitro. Analogously, synguanol phosphoralaninate prodrug Q Y L - 6 7 8 (155) inhibits EBV with EC^Q (in the viral capsid antigen expression assay) of 0.05 |iM vs 5.6 \iM for synguanol and 6.3 jLiM for ACWP' -C-COoMe H3C-C-C02Me NH I P h O-P -P-0-^ N. N ^ . .L. ^ ^. H3C-C-C02Me _N N'^N-^^Y Hnu-r-u-y N - II O 153(X = NH2, Y = H) 154(X = NH2, Y=NH2) 155(X = OH. Y = NH2) 156 Figure 63. Phosphoralaninate prodrugs of methylenecyclopropyl and spiropentane nucleosides. Spirocyclic analogs of 2'-deoxynucleosides have been synthesized and evaluated by Zemlicka and co-workers. Among these novel derivatives, the phosphoralaninate 156 (Figure 63) is an effective inhibitor of HCMV (EC^^ 0.38 \iM vs 2.9 ^M for GCV in HFF cells). It also shows interesting activity against HSV-1 (EC^^ 7.0 jlM in BSC-1 and 20 ^iM in Vero cells), HSV-2 {^C^^ 31 |iM in Vero cells), VZV (EC^^ 1.4 |LIM in the cytopathic effect inhibition effect assay), EBV (EC^^ 8.4 jLiM in Daudi cells), HIV-1 (EC30 3.5 |LiM in CEM-SS cells) and HBV (EC^^ 3.1 |iM in HepG 2.2.15 cells). Unfortunately, this compound also shows varying degrees of toxicity in cell lines (e.g. EC.„ 27 ^iM in Vero cells).^^^ 1.8. Acyclonucleosides Acyclonucleosides are characterized by the absence of a cyclic sugar moiety and, thus, a higher conformational flexibility than other nucleoside analogs. A consequence of this flexibility is that acyclonucleosides possess biological properties despite their lack of chirality. Whenever chirality or prochirality (as in ganciclovir and penciclovir) is present, quite surprisingly the S configuration at the 4'-equivalent position gives the only active enantiomer.^s Acyclovir (ACV, 157, Figure 64) was one of the first antiviral agents that showed potent and selective viral inhibition and is still one of the most effective anti-herpetic 47 Recent Advances in Antiviral Nucleosides dmgs,^^^'^^° although its use is limited by poor oral bioavailability (20-25%).^^^ This is much improved in its prodrug valaciclovir {vide infra). Ganciclovir (GCV, 158) is the drug of choice for the treatment of HCMV retinitis in AIDS patients.^^^'^^^'^^ Like acyclovir, its systemic use is limited by poor oral bioavailability (2-7%).265 Penciclovir (PCV, 159), the carba-Similog of GCV, has a broad-spectrum antiviral activity, being active against HSV-1, HSV-2, VZV, EBV and HBV.^^^'^^^'^^^ It is currently approved for the treatment of herpes zoster infections.^^^ Like its congeners, it has a low oral bioavailability. 158 (ganciclovir) O A. N^N H X " "O NH2 HsC^O OH 159 (penciclovir) O 160 (famciclovir) Figure 64. Acyclovir and related compounds. Famciclovir (FCV, 160) is a prodrug of PCV which is converted to its parent drug by three metabolic steps, two hydrolytic and one oxidative.^^^ Bioavailability of PCV is 77% upon oral administration of FCV.^^^ PCV is used for the treatment of VZV retinitis. As mentioned above, a common drawback of most acyclonucleosides is their low oral bioavailability. This is mainly due to the low solubility of these compounds.^^^ In order to overcome this problem, many modifications of the parent structures have been tried to give prodrugs with favorable absorption/distribution properties. The conversion of a nucleoside into its phosphonate offers several advantages. In particular, a phosphonate can mimic a phosphate group and can be converted to the triphosphate-like analog, thus bypassing the first phosphorylation necessary for the acti- 48 G. Gumina, Y. Choi and C. K. Chu vation of the drug. As a result, inactive drugs can be converted into active phosphonates if their lack of activity is due to a non-efficient initial phosphorylation step. Furthermore, unlike a phosphate, a phosphonic group is not cleaved by chemical or enzymatical hydrolysis, therefore it is metabolically stable. Adefovir, 9-(2-phosphonylmethoxyethyl)adenine (PMEA, 161, Figure 65) is a broad-spectrum antiviral agent, active against retroviruses, hepadnaviruses, and herpesviruses.^^^'^^^'^^^ However, the possibility of PMEA of becoming an orally administered drug is limited by its poor bioavailability,^^^ due to the negative charge of the phosphonate functionality at physiological pH, which limits its gastroenteric absorption. In order to increase the low bioavailability and decrease the toxicity of PMEA, a number of prodrugs have been prepared,^^^ and the bis-pivaloyloxymethyl derivative (bis-POM-PMEA, adefovir dipivoxil, 163)^^^ has recently been approved as an anti-HBV agent. In vitro studies show that adefovir dipivoxil is able to increase the intracellular concentration of PMEA by 2 logs.^^^ It has comparable antiviral activity in HIV-1 infected CEM cells and HCMV-infected MRC-5 cells, and it is even more potent than the parent compound on HSV-1 and HSV-2-infected Vero cells.^^^ In clinical trials, oral bioavailability was found to be greater than 40%.^^^ However, the bis-POM functionality has been found to confer cytostatic effects, probably due to the liberation of formaldehyde and pivalic acid.^^^ In clinical trials, adefovir has shown modest anti-HIV activity with nephrotoxicity at dose levels of 60-120 mg once daily.^^° However, adefovir does not show cross-resistance with lamivudine,^^^ and a combination of the two drugs has proved effective in the therapy of HB V-HIV-coinfected patients.^^^ In this combination, the use of adefovir dipivoxil at a suboptimal concentration for HIV activity (10 mg once daily) prevents the occurrence of mutations at codons 65 and 70 of HIV RT, responsible for HIV resistance to adefovir.^^^ 9-[2-(R)-(phosphonomethoxy)propyl]adenine (PMPA, tenofovir 162)^^^ is an effective anti-HIV agent. In a phase I/II clinical study, it showed a 1.1 log reduction in HIV RNA levels after administration of only eight doses.^^^ Tenofovir is less toxic towards erythroid progenitor cells than AZT, 3TC and d4T.^^'^ Its resistance to phosphorolysis and nucleotide-dependent chain-terminator removal is greater than AZT or 3TC.^^^ As in the case of adefovir, also tenofovir displays low bioavailability in animals. This problem has been overcome with the bis-isopropyloxycarbonyloxymethyl derivative (bis-POCPMPA, tenofovir disoproxil, 164),^^^^ the first nucleotide analog approved by the FDA for the treatment of AIDS.^^'^ Bis-POC derivatives were designed in order to eliminate the side effects of the POM group. Tenofovir disoproxil retains the antiviral activity of the parent drug, showing an oral bioavailability of 30% with minimal toxicity.^^'^^'^^'^^'^^^ Its efficacy on antiretroviral-experienced patients makes it one of the best choices in salvage therapy.^^ Cidofovir (HPMPC, 165) exhibits potent in vitro and in vivo activity against a broad spectrum of herpes viruses, including HCMV,^^^ and has been approved for the treatment of HCMV retinitis in AIDS patients.^^^ Its adenine congener HPMPA (166) has anti-HBV activity in both duck hepatocytes and 2.2.15 cells with an EC^^ of 1.2 |LiM.^^^ The cyclic prodrug of cidofovir, cHPMPC (167) has antiviral activity similar to the parent compound with reduced toxicity.^^ 49 Recent Advances in Antiviral Nucleosides NHo NH2 '^' O HO-P., HO N o 161 (adefovir, X = H) 162 (tenofovir, X = CH3) 163 (adefovir dipivoxil) NHo O .A. i\^ O 0-P^ I o H3C ho NHo O HO HO N N-^O HO- 165 (cidofovir, B = Cytosine) 166(HPMPA, B = Adenine) 04 HO-" "O167(cHPMPC) 164 (tenofovir disoproxil) Figure 65. Acyclonucleoside phosphonates and their "bis-POM" and "bis-POC" prodrugs. As discussed above, the major drawback of acyclovir and its phosphonates is their poor oral bioavailability, due to low water solubility. The valine conjugate valacyclovir (VCV, 168, Figure 66) has more than twice-greater bioavailability compared to that of ACV. This is due to the increased water solubility as well as the presence of an aminoacid moiety, which probably allows VCV to be absorbed in the intestin via the saturable dipeptide transporter system.^^^ The lipophilic prodrug l-O-hexadecylpropanediol-3-phosphoacyclovir (HDP-PACV, 169) also has improved bioavailability compared to the parent drug. Besides, unlike ACV, it is active against HBV in 2.2.15 cells.^^^ This is due to the fact that, unlike herpes viruses, HBV does not encode for a TK, which catalyses the conversion of ACV to ACVMP. HDP-P-ACV delivers ACVMP, which can then be further phosphorylated to the active triphosphate form. The ganciclovir prodrug HDP-P-GCV (170) has given promising results in the therapy of HSV-1 or HCMV retinitis. In the rabbit model, intravitreal injections with resultant 0.2 |LiM intravitreal concentration of prodrugs allowed a 4 to 6 weeks complete protection of the retina against HSV-1 with an IC^^ of 0.6 |LiM.293.294 H D P - P - G C V has also been evaluated in HCMV-infected human lung fibroblasts. Its IC^^ was 0.6 |LiM. 50 G. Gumina, Y. Choi and C. K. Chu -0(CH2)i5CH3 O (XX. R 168 (VCV) 169 (HDP-P-ACV), R = H 170 (HDP-P-GCV), R = CH2OH Figure 66. Prodrugs of acyclovir and ganciclovir. Recently, the same liphophilic prodrug approach has been applied to the synthesis of prodrugs of cidofovir and cyclic cidofovir.^^^ The alkoxyalkyl esters 1-O-hexadecyloxypropyl-cidofovir (HDP-CDV, 171, Figure 67) and 1-0-octadecyloxyethyl-cidofovir (ODE-CDV, 172) and their cyclic analogs HDP-cCDV (173) and ODE-cCDV (174) were more active against HSV-1, HSV-2, HCMV, VZV, EBV and human herpes virus type 6 (HHV-6) and 8 (HHV-8) than the parents compounds, with the cidofovir analogs generally more active than the cyclic ones. ^^^^^ All the prodrugs were active against several ganciclovir- and cidofovir-resistant HCMV strains, particularly.^^^^ An in vitro study using ^"^C-labeled HDP-CDV and cidofovir showed that the cellular drug content of HDP-CDV increased progressively to 24 hours, whereas the content of cidofovir reached a peak after 1-4 hours, remaining stable or slightly declining at 24 hours. The cellular content of cidofovir after 24 hours was 73-fold higher after incubation with the prodrug, which may explain the increased activity.^^^^ Cidofovir is an effective inhibitor of variola, monkeypox, cowpox and vaccina viruses. In a study on the use of cidofovir for the treatment of smallpox, a single intravenous dose fully protected mice against a lethal cowpox virus aerosol.^^^'^ In the search for orally active anti-smallpox agents, alkoxyalkyl esters of cidofovir and cCDV proved able to deliver, upon oral administration in mice, plasma levels of drugs in the low micromolar range, that is 10-fold the EC^^ for smallpox.^^^'^ Analogous pharmacokinetic studies on HDP-CDV for the treatment of CMV retinitis showed plasma levels of drug that should allow antiviral activity. ^^^^ Recently, 5-(l-azidovinyl)-substituted acyclic pyrimidine nucleosides (175, 176 and 177, Figure 68) have shown potent and selective anti-HBV activity, with EC^^ values of ranging from 0.01 to 0.1 |LIM in duck hepatitis B virus-infected primary duck hepatocytes, without significant toxicity.^^^ 1.9. Ribofuranosyl nucleosides Because of their close resemblance with natural nucleosides, the class of ribofuranosyl analogs has not produced many useful antiviral agents. In recent years, however, a number of carbocyclic analogs have shown promising antiviral activity, particularly against DNA virus. Recent Advances in Antiviral HO. 51 Nucleosides NHo NHo N N N p ^0 HO, /O CH3(CH2)i70>.,^/-.Q>t^O^ CH3(CH2)i50' H0-' 172 (ODE-CDV) 171 (HDP-CDV) CH3(CH2)i50'^ " - " " O " CH3(CH2)i7' O" 174(ODE-cCDV) 173(HDP-cCDV) Figure 67. Liphophilic prodrugs of cidofovir and cyclic cidofovir. N3 O HOR 175X = 0 , R = H 176X = 0 , R = CH20H 177X = C,R = CH20H Figure 68. 5-(l-Azidovinyl)-substituted acyclic pyrimidine nucleosides. The most important analog bearing a ribose sugar moiety is ribavirin (178, Figure 69), one of the first discovered compounds endowed with anti-respiratory syncytial virus, which has been shown to be effective as an anti-HBV and anti-HCV agent. It is currently approved, in combination with interferon-a, for the treatment of chronic hepatitis C.^^^ 52 G. Gumina, Y. Choi and C. K. Chu O VNH2 HO 0~ OH OH 178 (ribavirin) Figure 69. Ribofuranosyl nucleoside analogs The unusual ring-expanded ("fat") ribonucleosides 179-182 (Figure 70) are endowed with anti-HBV activity in 2.2.15 cells, with EC^^ in the micromolar range.^^^ Analog 180 also displayed potent and selective antitumor activity against a number of leukemia, non-small cell lung cancer, colon cancer, CNS cancer, melanoma, ovarian cancer, renal cancer, prostate cancer and breast cancer.^^^ Its tribenzoyl ester 181 showed similar activity profile as 180, but was considerably more active probably because of better cell permeation.^^^ HoN n2iN H n RO- N-..V=N OH 179 180R = H 181 R = Bz OR OR OH 182 Figure 70. "Fat" nucleosides. Neplanocin A (183, Figure 71) is a natural unsaturated nucleoside endowed with antiviral and antitumor properties.^^ Structure-activity relationships of neplanocin A analogs revealed interesting antiviral activity. Particularly, the D-cytosine and 5-F-cytosine derivatives 184 and 185 show anti-HIV activity (EC^Q 0.06 and 5.34 |iM, respectively) and are also the first nucleosides active against West Nile virus (EC^^ 0.2 and 15 |iM, respectively). However, their severe toxicity will prevent their development as antiviral agents.^^^ 53 Recent Advances in Antiviral Nucleosides NHp NH HO N-^N*^ OH HO ^ OH 183(neplanocinA) ^N'**0 ^ OH OH 184X = H 185X = F Figure 71. Neplanocin A and its D-cytidine analogs. Aristeromycin (186, Figure 72) is a natural carbocyclic ribonucleoside endowed with antitumor properties.^^^ Its 5'-nor analog 187 is active against HCMV,^^^'^^'^ vaccinia virus and measles, while its (+)-enantiomer 188 is active against HBV. ^^^'^^^ The guanine analog 189 has anti-EBV activity.^^^ X NHo 1 ^ N N - ^ N ^ Y HO OH OH OH 186 (aristeromycin) NH2 1 OH 1 8 7 X = NH2, Y = H 0::> N^^^N OH OH OH 188 1 8 9 X = OH, Y = NH2 Figure 72. Aristeromycin and its 5'-nor analogs. 1.10. References 1. 2. Kinchington, D. Recent advances in antiviral therapy. /. Clin. Pathol. 1999, 52, 89-94. (a) Agrofoglio, L.; Suhas, E.; Fares, A.; Condom, R.; Challand, S. R.; Earl, R. A. Synthesis of carbocyclic nucleosides. Tetrahedron 1994, 50, 10611-10670. (b) Nair, V.; Jahnke, T. S. Antiviral activities of isomeric dideoxynucleosides of D- and L-related stereochemistry. Antimicrob. Agents Chemother. 1995, 39, 1017-1029. (c) Marquez, V. E. Carbocyclic nucleosides. Adv. Antiviral Drug Design 1996, 2, 89-145. (d) Freeman, S.; Gardiner, J. M. Acyclic nucleosides as antiviral compounds. Mol. Biotechnol. 1996, 5, 125-137. (e) Naesens, L.; Snoeck, R.; Andrei, G.; Balzarini, J.; Neyts, J.; De Clercq, E. HPMPC (cidofovir), PMEA (adefovir) and related acyclic nucleoside phosphonate analogs: a review of their 54 G. Gumina, Y. Choi and C. K. Chu pharmacology and clinical potential in the treatment of viral infections. Antiviral Chem. Chemther. 1997, 8, 1-23. (f) Wang, P.; Hong, J. H.; Cooperwood, J. S.; Chu, C. K. Recent advances in L-nucleosides: chemistry and biology. Antiviral Res. 1998, 40, 19-44. (g) Crimmins, M. New development in the enantioselective synthesis of cyclopentyl carbocyclic nucleosides. Tetrahedron 1998, 54, 9229-9272. (h) Zhu, X.-F. The latest progress in the synthesis of carbocyclic nucleosides. Nucleosides Nucleotides 2000,19, 651-690. (i) Gumina, G.; Song, G. -Y.; Chu, C. K. L-Nucleosides as chemotherapeutic agents. 2001, 202, 9-15. Cj) Cooperwood, J. S.; Gumina, G.; Boudinot, F. D.; Chu, C. K. Nucleoside and nucleotide prodrugs. In Recent Advances in Nucleosides: Chemistry and Chemotherapy. C. K. Chu Ed. 3. 2002, 91-147. (a) Perigaud, C ; Gosselin, G.; Imbach, J. -L. Nucleoside analogs as chemotherapeutic agents: a review. Nucleosides Nucleotides 1992, 11, 903-945. (b) Mansour, T. S.; Storer, R. Antiviral nucleosides. Curr. Pharm. Design 1997, 3, 227-264. 4. 5. Huryn, D. M.; Okabe, M. AIDS-driven nucleoside chemistry. Chem. Rev. 1992, 92, 1745-1768. (a) De Clercq, E. HIV inhibitors targeted at the reverse transcriptase. AIDS Res. Hum. Retroviruses 1992, 8, 119-134. (b) Schinazi, R. F.; Mead, J. R.; Feorino, P. M. Insight into HIV chemotherapy. AIDS Res. Hum. Retroviruses 1992, 8, 963-990. (c) De Clercq, E. Antiviral activity spectrum and target of action of different classes of nucleoside analogs. Nucleosides Nucleotides 1994,13, 1271-1295. (d) De Clercq, E. In search of a selective antiviral chemotherapy. Clin. Microbiol. Rev. 1997,10, 674-693. (e) Hong, J. H.; Choi, Y.; Chun, B. K.; Lee, K.; Chu, C. K. Current status of anti-HBV chemotherapy. Arch. Pharm. Res. 1998, 21, 89-105. (f) Tan, X.; Chu, C. K.; Boudinot, F. D. Development and optimization of anti-HIV nucleoside analogs and prodrugs: a review of their cellular pharmacology, structure-activity relationships and pharmacokinetics. Adv. Drug Delivery Reviews 1999, J9, 117-151. (g) Zemlicka, J. Enantioselectivity of the antiviral effects of nucleoside analogs. Pharmacol. Ther. 2000, 85, 251-266. (h) Gumina, G.; Song, G. -Y.; Chu, C. K. Advances in antiviral agents for hepatitis B virus. Antiviral Chem. Chemother. 2001, 72,89-112. 6. Challand, R.; Young, R. J. Antiviral Chemotherapy Mann, J. Ed.; Biochemical & Medicinal Chemistry Series; Spektrum: UK, 1997. (a) Crick, F. Life Itself, Simon and Shuster: New York, 1981. (b) Spadari, S.; Maga, G.; Verri, A.; Bendiscioh, A.; Tondelli, L.; Capobianco, M.; Colonna, F.; Garbesi, A.; Focher, F. Lack of stereospecificity of some cellular and viral enzymes involved in the synthesis of deoxyribonucleotides and DNA: molecular basis for the antiviral activity of unnatural L-p-nucleosides. Biochimie 1995, 77, 861-867. 7. 8. 9. Focher, F.; Maga, G.; Bendiscioli, A.; Capobianco, M.; Colonna, F.; Garbesi, A.; Spadari, S. Stereospecificity of human DNA polymerases a, y, 6 and £, HIV-reverse transcriptase, HSV-1 DNA polymerase, calf thymus transferase and Escherichia coli DNA polymerase I in recognizing D- and L-thymidine 5'-triphosphate as substrate. Nucleic Acids Research 1995, 23, 2840-2847. Spadari, S.; Ciarrocchi, G.; Focher, F.; Verri, A.; Maga, G.; Arcamone, F.; lafrate, E.; Manzini, S.; Garbesi, A.; Tondelli, L. 5-Iodo-2'-deoxy-L-uridine and (£)-5-(2-bromovinyl)-2'-deoxy-L-uridine: selective phosphorylation by herpes simplex virus type 1 thymidine kinase, antiherpetic activity, and cytotoxicity studies. Mol. Pharmacol. 1995, 47, 1231-1238. 10. Faraj, A.; Loi, A. G.; Xie, M. Y.; Bryant, M.; Bridges, E. G.; Juodawlkis, A.; Pierra, C ; Dukhan, D.; Gosselin, G.; Imbach, J.-L.; Schinazi, R. F.; Pai, S. B.; Korba, B.; Marion, P.; Sommadossi, J.-P. P-L-Thymidine and P-L-2'-deoxycytidine are potent, selective and specific anti-hepatitis B virus agents. Abstracts of Papers, 3rd International Conference on Therapies for Viral Hepatitis, Maui, USA; International Society for Antiviral Therapy: 1999; Abstract 119. Recent Advances in Antiviral Nucleosides 55 11. De Clercq, E. Vaccinia virus inhibitors as a paradigm for the chemotherapy of poxvirus infections. Clin. Microb. Rev. 2001,14, 382-397. 12. Chu, C. K.; Ma, T.; Shanmuganathan, K.; Wang, C ; Xiang, Y.; Pai, S. B.; Tao, G.-Q.; Sommadossi, J. -P.; Cheng, Y. -C. Use of 2'-fluoro-5-methyl-p-L-arabinofuranosyluracil as a novel antiviral agent for hepatitis B virus and Epstein-Barr virus. Antimicrob. Agents Chemother. 1995, 39, 979-981. 13. (a) Prusoff, W. H. Synthesis and biological activities of iododeoxyuridine, an analog of thymidine. Biochim. Biophys. Acta 1959, 32, 295-296. (b) Kaufman, H. E.; Martola, E.; Heidelberger, C. Therapeutic antiviral action of 5-trifluoromethyl-2-deoxyuridine in herpes simplex keratitis. Science 1964, 745, 585-586. (c) De Clercq, E. Synthetic pyrimidine nucleoside analogs. In Approaches to Antiviral Agents, Hamden, M. R., Ed.; Macmillan: London, 1985, pp. 57-99. (d) De Clercq, E. In Advances in Drug Research, Testa, B., Ed.; Academic Press Ltd.: London, 1988, Vol. 17, pp. 1-59. 14. De Clercq, E.; Descamps, J.; De Somer, P.; Barr, P. J.; Jones, A. S.; Walker, R. T. (£r)-5-(2-Bromovinyl)2'-deoxyuridine: a potent and selective anti-herpes agent. Proc. Natl. Acad. Sci. USA 1979, 76, 29472951. 15. Desgranges, C ; Razaka, G.; Rabaud, M.; Bricaud, H.; Balzarini, J.; De Clercq, E. Phosphorolysis of (F)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) and other 5-substituted-2'-deoxyuridines by purified human thymidine phosphorylase and intact blood-platelets. Biochem. Pharmacol. 1983, 32, 3583-3590. 16. Harris, S. A.; McGuigan, C ; Andrei, G.; Snoeck, R.; De Clercq, E.; Balzarini, J. Synthesis and antiviral evaluation of phsphoramidate derivatives of (F)-5-(2-bromovinyl)-2'-deoxyuridine. Antiviral Chem. Chemother. 2001, 72, 293-300. 17. De Clercq, E.; Descamps, J.; Balzarini, J.; Giziewicz, J.; Barr, P. J.; Robins, M. J. Nucleic acid related compounds. 40. Synthesis and biological activities of 5-alkynyluracil nucleosides. J. Med. Chem. 1983, 26, 661-666. 18. Wigerinck, P.; Snoeck, R.; Claes, P.; De Clercq, E.; Herdewijn, P. Synthesis and antiviral activity of 5-heteroaryl-substituted 2'-deoxyuridines. J. Med. Chem. 1991, 34, 1161-1712. 19. (a) Wigerinck, P.; Pannecouque, C ; Snoeck, R.; Claes, P.; De Clercq, E.; Herdewijn, P. 5-(5-Bromothien2-yl)-2'-deoxyuridine and 5-(5-chlorothien-2-yl)-2'-deoxyuridine are equipotent to (F)-5-(2-bromovinyl)2'-deoxyuridine in the inhibition of herpes simplex virus type I replication. J. Med. Chem. 1991,34,23832389. (b) Wigerinck, P.; Kerremans, L.; Claes, P.; Snoeck, R.; Maudgal, P.; De Clercq, E.; Herdewijn, P. Synthesis and antiviral activity of 5-thien-2-yl-2'-deoxyuridines. J. Med. Chem. 1993, 36, 538-543. 20. Choi, Y.; Li, L.; Grill, S.; GuUen, E.; Lee, C. S.; Gumina, G.; Cheng, Y.-C; Chu, C. K. Structure-activity relationships of (F)-5-(2-bromovinyl)uracil nucleosides and related analogs as anti-herpesvirus agents. /. Med. Chem. 2000, 43, 2538-2546. 21. Bryant, M. L.; Bridges, E. G.; Placidi, L.; Faraj. A.; Loi, A. G.; Pierra, C ; Dukhan, D.; Gosselin, G.; Imbach, J. -L.; Hernandez, B.; Juodawlkis, A.; Tennant, B.; Korba, B, Cote, P.; Marion, P.; Cretton-Scott, E.; Schinazi, R. F.; Sonmiadossi, J. -P. Antiviral L-nucleosides specific for hepatitis B virus infection. Antimicrob. Agents Chemother. 2001, 45, 229-235. 22. Pharmacology of p-L-thymidine and P-L-2'-deoxycytidine in HepG2 cells and primary human hepatocytes: relevance to chemotherapeutic efficacy against hepatitis B virus. Antimicrob. Agents Chemother. 2002, 46, 1728-1733. 23. McGuigan, C ; Yamold, C. J.; Jones, G.; Velazquez, S.; Barucki, H.; Brancale, A.; Andrei, G.; Snoeck, R.; De Clercq, E.; Balzarini, J. Potent and selective inhibition of varicella-zoster virus (VZV) by nucleoside analogs with an unusual bicyclic base. /. Med. Chem. 1999, 42, 4479-4484. 24. Dyson, M. R.; Coe, P. L.; Walker, R. T. The synthesis and antiviral properties of £'-5-(2-bromovinyl)-4'thio-2'-deoxyuridine. J. Chem. Soc, Chem. Commun. 1991, 741-742. 56 G. Gumina, Y. Choi and C. K. Chu 25. Rahim, S. G.; Trivedi, N.; Bogunovic-Batchelor, M. V.; Hardy, G. W.; Mills, G.; Selway, J. W. T.; Snowden, W.; Littler, E.; Coe, P. L.; Basnak, I.; Whale, R. F. Walker, R. T. Synthesis and anti-herpes virus activity of 2'-deoxy-4'-thiopyrimidine nucleosides. J. Med. Chem. 1996, 39, 789-795. 26. Basnak, I.; Sun, M.; Coe, P. L.; Walker, R. T. The synthesis of some 5-alkyl (cycloalkyl) substituted 2'-deoxy-4'-thiouridines. Nucleosides Nucleotides 1996, 75, 121-134. 27. Draanen, N. A.; Freeman, G. A.; Short, S. A.; Harvey, R.; Jansen, R.; Szczech, G.; Koszalka, G. W. Synthesis and antiviral activity of 2'-deoxy-4'-thio purine nucleosides. /. Med. Chem. 1996, 39, 538-542. 28. Uenishi, J.; Takahashi, K.; Motoyama, M.; Akashi, H.; Sasaki, T. Syntheses and antitumor activities of D- and L-2'-deoxy-4'-thio pyrimidine nucleosides. Nucleosides Nucleotides 1994,13, 1347-1361. 29. Herdewijn, P.; De Clercq, E.; Balzarini, J.; Vanderhaeghe, H. Synthesis and antiviral activity of the carbocyclic analogs of (F)-5-(2-halovinyl)-2'-deoxyuridine and (F)-5-(2-halovinyl)-2'-deoxycytidines. J. Med. Chem. 1985, 28, 550-555. 30. Balzarini, J.; Baumgartner, H.; Bodenteich, M.; De Clercq, E.; Griengl, H. Synthesis and antiviral activity of the enantiomeric forms of carba-5-iodo-2'-deoxyuridine and carba-(F)-5-(2-bromovinyl)-2'deoxyuridine. /. Med. Chem. 1989, 32, 1861-1865. 31. Shealy, Y. F.; O'Dell, C. A.; Shannon, W. M.; Amett, G. Synthesis and antiviral activity of carbocyclic analogs of 2'-deoxyribofuranosides of 2-amino-6-substituted-purines and of 2-amino-6-substituted-8azapurines. J. Med. Chem. 1984, 27, 1416-1421. 32. Secrist III, J. A.; Montgomery, J. A.; Shealy, Y. F.; O'Dell, C. A.; Clayton, S. J. Resolution of racemic carbocyclic analogs of purine nucleosides through the action of adenosine deaminase. Antiviral activity of the carbocyclic 2'-deoxyguanosine enantiomers. /. Med. Chem. 1987, 30, 746-749. 33. (a) Bennett Jr., L. L.; Shealy, Y. F.; Allan, P. W.; Rose, L. M.; Shannon, W. M.; Amett, G. Phosphorylation of the carbocyclic analog of 2'-deoxyguanosine in cells infected with herpes viruses. Biochem. Pharmacol. 1990, 40, 1515-1522. (b) Bennett Jr., L. L.; Parker, W. B.; Allan, P. W.; Rose, L. M.; Shealy, Y. F.; Secrist III, J. A.; Montgomery, J. A.; Amett, G.; Kirkman, R. L.; Shannon, W. M. Phosphorylation of the enantiomers of the carbocyclic analog of 2'-deoxyguanosine in cells infected with herpes simplex virus type 1 and in uninfected cells. Lack of enantiomeric selectivity with viral thymidine kinase. Mol. Pharmacol. 1993,44, 1258-1266. 34. Bennett Jr., L. L.; Allan, P. W.; Amett, G.; Shealy, Y. F.; Shewach, D. S.; Mason, W. S.; Fourel, I.; Parker, W. B. Metabolism in human cells of the D and L enantiomers of the carbocyclic analog of 2'-deoxyguanosine: substrate activity with deoxycytidine kinase, mitochondrial deoxyguanosine kinase, and 5'-nucleotidase. Antimicrob. Agents Chemother. 1998, 42, 1045-1051. 35. Price, P. M.; Banjit, R.; Acs, G. Inhibition of the replication of hepatitis B vims by the carbocyclic analog of 2'-deoxyguanosine. Proc. Natl. Acad. Sci. USA 1989, 86, 8541-8544. 36. Price, P. M.; Banerjee, R.; Jeffrey, A. M.; Acs, G. The mechanism of inhibition of hepatitis B virus repHcation by the carbocyclic analog of 2'-deoxyguanine. Hepatology 1992,16, 8-12. 37. Jansen, R. W.; Johnson, L. C ; Averett, D. R. High capacity in vitro assessment of anti-hepatitis B vims compound selectivity by a virion-specific polymerase chain reaction assay. Antimicrob. Agents Chemother. 1993, 37, 441-447. 38. (a) Slusarchyk, W. A.; Field, A. K.; Greytok, J. A.; Taunk, P.; Tuomari, A. V.; Young, M.G.; Zahler, R. 4-Hydroxy-3-(hydroxymethyl)-2-methylenecyclopentyl purines and pyrimidines, a new class of antiherpesvims agents. Antiviral Res. 1992, 17, suppl.l, 98. (b) Bisacchi, G. S.; Chao, S. T.; Bachard, C ; Daris, J. P.; Innaimo, S.; Jacobs, G. A.; Kocy, O.; Lapointe, P.; Martel, A.; Merchant, Z.; Slusarchyk, W. A.; Sundeen, J. E.; Young, M. G.; Colonno, R.; Zahler, R. BMS-200475, A novel carbocyclic 2'-deoxyguanosine analog with potent and selective anti-hepatitis B vims activity in vitro. Bioorg. Med. Recent Advances in Antiviral Nucleosides 57 Chem. Lett. 1997, 7, 127-132. (c) Innaimo, S. F.; Seifer, M.; Bisacchi, G. S.; Standring, D. N.; Zahler, R,; Colonno, R. J. Identification of BMS-200475 as a potent and selective inhibitor of hepatitis B virus. Antimicrob. Agents Chemother. 1997, 41, 1444-1448. 39. Genovesi, E. V.; Lamb, L.; Medina, I.; Taylor, D.; Seifer, M.; Innaimo, S.; Colonno, R. J.; Standring, D. N.; Clark, J. M. Efficacy of the carbocyclic 2'-deoxyguanosine nucleoside BMS-200475 in the woodchuck model of hepatitis B virus infection. Antimicrob. Agents Chemother. 1998, 42, 3209-3217. 40. Yamanaka, G.; Wilson, T.; Innaimo, S.; Bisacchi, G. S.; Egh, P.; Rinehart, J. K.; Zahler, R.; Colonno, R. J. Metabolic studies on BMS-200475, a new antiviral compound active against hepatitis B virus. Antimicrob. Agents Chemother. 1999, 43, 190-193. 41. (a) Shannon, W. M. In Antiviral Agents and Viral diseases of Man; Galasso, G. J., Ed.; Raven Press: New York, 1984; Chapter 3, pp. 55-121. (b) Robins, R. K.; Revenkar, G. R. In Antiviral Drug Development. A Multidisciplinary Approach; De Clercq, E; Walker, R. T., Eds.; Nato Advanced Institutes Series, Series A: Life Sciences; Plenum Press: New York, 1987; Vol. 143, pp. 11-36. 42. Jacyna, M. R.; Thomas, H. C. Antiviral therapy: hepatitis B virus. Br. Med. Bull. 1990, 46, 368-382. 43. Machida, H.; Sakata, S.; Kumnaka, A.; Yoshino, H. Anti-herpesviral and anti-cellular effects of 1-p-Darabinofuranosyl-£-5-(2-halogenovinyl)uracils. Antimicrob. Agents Chemother. 1981, 20, 47-52. 44. (a) Miyauchi, S.; Imaoka, T.; Okada, T.; Motoyama, M.; Kawaguchi, T.; Akiyama, H.; Odomi, M. Leukopenia-inducing effect of a combination of a new 5-fluorouracil (5-FU)-derived drug, BOF-A2 (emitefur), with other 5-FU-derived drugs or BV-araU (sorivudine) in rats. Jpn. J. Pharmacol. 1996, 70,139-148. (b) Watabe, T.; Okuda, H.; Ogura, K. Lethal drug interaction of the new antiviral, sorivudine, with anticancer prodrugs of 5-fluorouracil. Yakugaku Zasshi 1997,117, 910-921. 45. Lin, T.-S.; Luo, M.-Z.; Liu, M.-C. Synthesis of several pyrimidine L-nucleoside analogs as potential antiviral agents. Tetrahedron 1995, 57, 1055-1068. 46. Machida, H.; Sakata, S. 5-Substituted arabinofuranosyluracil nucleosides as antiherpesvims agents: from araT to BV-araU. In Nucleosides and Nucleotides as Antitumor and Antiviral Agents; Chu, C. K.; Baker, D. C , Eds.; Plenum Press: New York, 1993; pp. 245-264. 47. (a) Rahim, S. G.; Trevidi, N.; Selway, J.; Darby, G.; Collins, P.; Powell, K. L.; Purifoy, D. J. M. 5-Alkynyl pyrimidine nucleosides as potent selective inhibitors of Varicella-Zoster Virus. Antiviral Chem. Chemother. 1992, 3, 293-297. (b) Purifoy, D. J. M.; Beauchamp, L. M.; de Miranda, P.; Ertl, P.; Lacey, S.; Roberts, G.; Rahim, S. G.; Darby, G.; Krenitsky, T. A.; Powell, K. L. Review of research leading to new anti-herpesvirus agents in clinical and 882C, a specific agent for varicella zoster virus. /. Med. Virol. 1993, suppl. 1, 139-145. 48. (a) Machida, H.; Ashida, N.; Miura, S.; Endo, M.; Yamada, K.; Kitano, K.; Yoshimura, Y.; Sakata, S.; Ijichi, O.; Eizuru, Y. Anti-herpesvirus activity profile of 4'-thioarabinofuranosyl purine and uracil nucleosides and activity of l-p-D-2'-fluoro-4'-thioarabinofuranosyl guanine and 2,6-diaminopurine against clinical isolates of human cytomegalovirus. Antiviral Res. 1998, 39, 129-137. (b) Satoh, H.; Yoshimura, Y.; Sakata, S.; Miura, S.; Machida, H. Synthesis of L-enantiomers of 4'-thioarabinofuranosyl pyrimidine nucleosides. Bioorg. Med. Chem. Lett. 1998, 8, 989-992. 49. (a) Watanabe, K. A.; Reichman, U.; Hirota, K.; Lopez, C ; Fox, J. J. Nucleosides. 110. Synthesis and antiherpes virus activity of some 2'-fluoro-2'-deoxy-arabinofuranosyl pyrimidine nucleosides. /. Med. Chem. 1979, 22, 21-24. (b) Watanabe, K. A.; Su, T.-L.; Klein, R. S.; Chu, C. K.; Matsuda, A.; Chun, M. W.; Lopez, C ; Fox, J. J. Nucleosides. 123. Synthesis of antiviral nucleosides, 5-substituted l-(2-deoxy-2halogeno-p-D-arabinofuranosyl) cytosines and uracils, some structure-activity relationships. J. Med. Chem. 1983, 26, 152-156. (c) Watanabe, K. A.; Su, T. -L.; Reichman, U.; Greenberg, N.; Lopez, C ; Fox, J. J. Nucleosides. 129. Synthesis of antiviral nucleosides, 5-alkenyl-l-(2-deoxy-2-fluoro-p-D- 58 G. Gumina, Y. Choi and C. K. Chu arabinofuranosyl)uracils. /. Med. Chem. 1984, 27, 91-94. 50. Kreis, W.; Damin, L.; Colacino, J.; Lopez, C. In vitro metabolism of 1-P-D-arabinofuranosylcytosine and l-P-D-2'-fluoroarabino-5-iodocytosine in normal and herpes simplex type 1 infected cells. Biochem. Pharmacol. 1982, 31, 767-773. 51. Abbruzzese, J. L.; Schmidt, S.; Raber, M. N.; Levy, J. K.; Castellanos, A. M.; Legha, S. S.; Krakoff, I. H. Phase I trial of l-(2-deoxy-2-fluoro-|3-D-arabinofuranosyl)-5-methyluracil (FMAU) terminated by severe neurologic toxicity. Invest. New Drugs 1989, 7, 195-201. 52. (a) Macilwain, C. NIH, FDA seek lessens from the hepatitis B drug trial deaths. Nature 1993,364,275-275. (b) Touchette, N. HBV-drug deaths prompt restudy of similar antivirals. /. NIH Res. 1993, 5, 33-36. 53. (a) Parker, W. B.; Cheng, Y. -C. Mitochondrial toxicity of antiviral nucleoside analogs. /. NIH Res. 1994, 6, 57-61. (b) Cui, L.; Yoon, S.; Schinazi, R. F.; Sommadossi, J.-P. Cellular and molecular events leading to mitochondrial toxicity of l-(2-deoxy-2-fluoro-l-P-D-arabinofuranosyl)-5-iodouracil in human liver cells. /. Clin. Invest. 1995, 95, 555-563. 54. Chu, C. K.; Boudinot, F. D.; Peek, S. F.; Hong, J. H.; Choi, Y. S.; Korba, B. E.; Gerin, J. L.; Cote, P. J.; Tennant, B. C ; Cheng, Y. -C. Preclinical investigation of L-FMAU as an anti-hepatitis B vims agent. In Therapies for viral Hepatitis; Schinazi, R. F.; Sommadossi, J.-P.; Thomas, H. C. Eds,; International Medical Press: UK, 1999; Chapter 32, pp 303-312. 55. Jeong, L. S.; Moon, H. R.; Yoo, S. J.; Lee, S. N.; Chun, M. W.; Lim, Y. -H. Ring contraction and rearrangement of 4-thiofuranose deravatives observed during DAST fluorination in the synthesis of L-2'"up"-fluoro-4'-thiothymidine. Tetrahedron Lett. 1998, 39, 5201-5204. 56. Borthwick, A. D.; Evans, D. N.; Kirk, B. E.; Biggadike, K.; Exall, A. M.; Youds, P.; Roberts, S. M.; Knight, D. J.; Coates, J. A. V. Fluoro carbocyclic nucleosides: synthesis and antiviral activity of 2'- and 6'-fluoro carbocyclic pyrimidine nucleosides including carbocyclic l-(2-deoxy-2-fluoro-P-D-arabinofuranosyl)5-methyluracil and carbocyclic l-(2-deoxy-2-fluoro-p-D-arabinofuranosyl)-5-iodouracil. J. Med. Chem. 1990,53, 179-186. 57. Borthwick, A. D.; Butt, S.; Biggadike, K.; Exall, A. M.; Roberts, A. M.; Youds, P. M.; Kirk, B. E.; Booth, B. R.; Cameron, J. M.; Cox, S. W.; Marr, C. L. P.; Shill, M. D. Synthesis and enzymatic resolution of carbocyclic 2'-ara-fluoro-guanosine: a potent new anti-herpetic agent. /. Chem. Soc., Chem. Commun. 1988, 657-658. 58. Biggadike, K.; Borthwick, A. D.; Exall, A. M.; Kirk, B. E.; Ward, R. A. 2'-Deoxy-2'-fluoro-araaristeromycin, a new anti-herpes agent: the first direct introduction of a 2'-fluoro substituent into a carbocyclic nucleoside. J. Chem. Soc, Chem. Commun. 1988, 898-900. 59. Hertel, L. W.; Kroin, J. S.; Misner, J. W.; Tustin, J. M. Synthesis of 2-deoxy-2,2-difluoro-D-ribose and 2-deoxy-2,2-difluoro-D-ribofuranosyl nucleosides. J. Org. Chem. 1988, 53, 2406-2409. 60. Plunkett, W.; Huang, P.; Ganghi, V. Gemcitabine: actions and interactions. Nucleosides Nucleotides 1997, 16, 1261-1270. 61. Gandhi, V.; Mineishi, S.; Huang, P.; Chapman, A. J.; Young, Y.; Chen, F.; Nowak, B.; Chubb, S.; Hertel, L. W.; Plunkett, W. Cytotoxicity, metabolism, and mechanism of action of 2',2'-difluorodeoxyguanosine in Chinese hamster ovary cells. Cancer Res. 1995, 55, 1517-1524. 62. Kotra, L. P.; Xiang, Y.; Newton, M. G.; Schinazi, R. F.; Cheng, Y. - C ; Chu, C. K. Structure-activity relationships of 2'-deoxy-2',2'-difluoro-L-ery^/zrc?pentofuranosyl nucleosides. /. Med. Chem. 1997, 40, 3635-3644. 63. Yoshimura, Y.; Kitano, K.; Yamada, K.; Satoh, H.; Watanabe, M.; Miura, S.; Sakata, S.; Sasaki, T.; Matsuda, A. A novel synthesis of 2'-modified 2'-deoxy-4'-thiocytidines from D-glucose. J. Org. Chem. 1997,62,3140-3152. Recent Advances in Antiviral Nucleosides 59 64. Jeong, L. S.; Moon, H. R.; Choi, Y. J.; Chun, M. W.; Kim, H. O. A short and efficient synthesis of L-thioarabinose derivative: a versatile synthon for the synthesis of L-2'-deoxy-2',2'-disubstituted-4'thionucleosides. J. Org. Chem. 1998, 63, 4821-4825. 65. Takenuki, K.; Matsuda, A.; Ueda, T.; Sasaki, T.; Fujii, A.; Yamagami, K. Design, synthesis, and antineoplastic activity of 2'-deoxy-2'-methyHdenecytidine (DMDC). /. Med. Chem. 1988,31, 1063-1064. 66. Yamagami,K,;Fujii,A.;Arita,M.;Okumoto,T.;Sakata,S.;Matsuda,A.;Ueda,T.;Sasaki,T.Antitumoractivity of 2'-deoxy-2'-methylidenecytidine, anew 2'-deoxycytidine derivative. Cancer Res. 1991,57,2319-2323. 67. Baker, C. H.; Banzon, J.; Bollinger, J. M.; Stubbe, J.; Samano, V.; Robins, M. J.; Lippert, B.; Jarvi, E.; Resvick, R. 2'-Deoxy-2'-methylidenecytidine and 2'-deoxy-2',2'-difluorocytidine 5'-diphosphates: potent mechanism-based inhibitors of ribonucleotide reductase. /. Med. Chem. 1991, 34, 1879-1884. 68. Machida, H.; Sakata, S.; Ashida, N.; Takenuki, K.; Matsuda, A. In vitro anti-herpesvirus activities of 5substituted 2'-deoxy-2'-methylidene pyrimidine nucleosides. Antiviral Chem. Chemother. 1993,4, 11-17. 69. Satoh, H.; Yoshimura, Y.; Watanabe, M.; Ashida, N.; Ijichi, K.; Sakata, S.; Machida, H. Synthesis and antiviral activities of 5-substituted l-(2-deoxy-2-C-methylene-4-thio-p-D-ery?/ir(?-pentofuranosyl)uracils. Nucleosides Nucleotides 1998,17, 65-79. 70. Maag, H.; Rydzewski, R. M.; McRoberts, M. J.; Crawford-Ruth, D.; Verheyden, J. P. H.; Prisbe, E. J. Synthesis and anti-HIV activity of 4'-azido- and4'-methoxynucleosides. /. Med. Chem. 1992,35,1440-1451. 71. Chen, M. S.; Suttmann, R. T.; Wu, J. C ; Prisbe, E. J. Metabolism of 4'-azidothymidine. J. Biol Chem. 1992, 267, 257-260. 72. Chen, M. S.; Suttmann, R. T.; Papp, E.; Cannon, P. D.; McRoberts, M. J.; Bach, C ; Copeland, W. C ; Wang, T. S.-F. Selective action of 4'-azidothymidine triphosphate on reverse transcriptase of human immunodeficiency virus type 1 and human DNA polymerases a and p. Biochemistry 1993,32,6002-6010. 73. Nomura, M.; Shuto, S.; Tanaka, M.; Sasaki, T.; Mori, S.; Shigeta, S.; Matsuda, A. Nucleosides and nucleotides. 185. Synthesis and biological activities of 4'a-C-branched-chain sugar pyrimidine nucleosides. J. Med. Chem. 1999, 42, 2901-2908. 74. Kitano, K.; Machida, H.; Miura, S.; Ohrui, H. Synthesis of novel 4'-C-methyl-pyrimidine nucleosides and their biological activities. Bioorg. Med. Chem. Lett. 1999, 9, 827-830. 75. Borthwick, A. D.; Biggadike, K. Eur. Pat. Appl. 0345 016, 1989. 76. Ohrui, H.; Kohgo, S.; Kitano, K.; Sakata, S.; Kodama, E.; Yoshimura, K.; Matsuoka, M.; Shigeta, S.; Mitsuya, H. Syntheses of 4'-C-ethynyl-P-D-arfl^mo- and 4'-C-ethynyl-2'-deoxy-P-D-n^(9-pentofuranosy Ipyrimidines and -purines and evaluation of their anti-HIV activity. /. Med. Chem. 2000, 43, 4516-4525. 77. Marquez, V. E.; Siddiqui, M. A.; Ezzitouni, A.; Russ, P.; Wang, J.; Wagner, R. W.; Matteucci, M. D. Nucleosides with a twist. Can fixed forms of sugar ring pucker influence biological activity in nucleosides and oligonucleotides? /. Med. Chem. 1996, 39, 3739-3747. 78. Huheihel, M.; Salah, L.; Konson, A.; Manor, E.; Ford Jr., H.; Marquez, V. E.; Johns, D. G.; Agbaria, R. Intracellular phosphorylation of A^-methanocarbathymidine, a novel antiherpetic agent. Antiviral Res. 2000, 53, A55-A55. 79. Chun, B. K.; Olgen, S.; Hong, J. H.; Newton, M. G.; Chu, C. K. Enantiomeric syntheses of conformationally restricted D- and L-2',3'-dideoxy-2',3'-en<io-methylene nucleosides from carbohydrate chiral templates. /. Org. Chem. 2000, 65, 685-693. 80. Herdewijn, P.; De Clercq, E. The cyclohexene ring as bioisostere of a furanose ring: synthesis and antiviral activity of cyclohexenyl nucleosides. Bioorg. Med. Chem. Lett. 2001,11, 1591-1597. 81. Wang, J.; Froeyen, M.; Hendrix, C ; Andrei, C ; Snoeck, R.; Lescrinier, E.; De Clercq, E.; Herdewijn, P. (D)- And (L)-cyclohexenyl-G, a new class of antiviral agents: synthesis, conformational analysis, molecular modehng, and biological activity. Nucleosides Nucleotides 2001, 20, 727-730. 60 G. Gumina, Y. Choi and C K. Chu 82. Mitsuya, H.; Broder, S. Inhibition of the in vitro infectivity and cytopathic effect of human T-lymphotropic virus type Ill/lymphadenopathy-associated virus (HTLV-III/LAV) by 2',3'-dideoxynucleosides. Proc. Natl Acad. Sci. USA 1986, 83, 1911-1915. 83. (a) Mitsuya, H.; Weinhold, K. J.; Furman, P. A.; St Clair, M. H.; Lehman, S. N.; Gallo, R. C ; Bolognesi, D.; Barry, D. W.; Broder, S. 3'-Azido-3'-deoxythymidine (BW A509U): an antiviral agent that inhibits the infectivity and cytopathic effect of human T-lymphotropic virus type Ill/lymphadenopathy-associated virus in vitro. Proc. Natl. Acad. Sci. USA 1985, 82, 7096-7100. (b) Pemo, C.-F.; Yarchoan, R.; Cooney, D. A.; Hartman, N. R.; Gartner, S.; Popovic, M.; Hao, Z.; Gerrard, T. L.; Wilson, Y. A. Johns, D. G.; Broder, S. Inhibition of human immunodeficiency virus (HIV-1/HTLV-IIIg^ J replication in fresh and cultured human peripheral blood monocytes/macrophages by azidothymidine and related 2',3'-dideoxynucleosides. J. Exp. Med. 1988,168, 1111-1125. 84. (a) Lin, T.-S.; Schinazi, R. F.; Prusoff, W. H. Potent and selective in vitro activity of 3'-deoxythymidine2'-ene (3'-deoxy-2',3'-didehydrothymine) against human immunodeficiency virus. Biochem. Pharmacol. 1987, 36, 2713-2718. (b) Baba, M.; Pauwels, R.; Herdewijn, P.; De Clercq, E.; Desmyter, J.; Vandeputte. Both 2',3'-dideoxythymidine and its 2',3'-unsaturated derivative (2',3'-dideoxythymidinene) are potent and selective inhibitors of human immunodeficiency virus replication in vitro. Biochem. Biophys. Res. Commun. 1987, 142, 128-134. (c) Hamamoto, Y.; Nakashima, H.; Matsuda, A.; Ueda, T.; Yamamoto, N. Inhibitory effect of 2',3'-didehydro-2',3'-dideoxynucleosides on infectivity, cytopathic effects, and replication of human immunodeficiency virus. Antimicrob. Agents Chemother. 1987, 31, 907-910. 85. Soudeyns, H.; Yao, X. J.; Gao, Q. Anti-human immunodeficiency virus type 1 activity and in vitro toxicity of 2'-deoxy-3'-thiacytidine (BCH-189), a novel heterocyclic nucleoside analog. Antimicrob. Agents Chemother. 1991, 35, 1386-1390. 86. Daluge, S. M.; Good, S. S.; Faletto, M. B.; Miller, W. H.; St Clair, M. H.; Boone, L. R.; Tisdale, M.; Parry, N. R.; Reardon, J. E.; Domsife, R. E.; Averett, D. R.; Krenitsky, T. A. 1592U89, a novel carbocyclic nucleoside analog with potent, selective anti-human immunodeficiency virus activity. Antimicrob. Agents Chemother. 1997, 41, 1082-1093. 87. Balzarini, J.; Aquaro, S.; Pemo, C. -F.; Witvrouw, M.; Holy, A.; De Clercq, E. Activity of the {R)enantiomers of 9-(2-phosphonylmethoxypropyl)adenine and 9-(2-phosphonylmethoxypropyl)-2,6diaminopurine against human immunodeficiency virus in different human cell systems. Biochem. Biophys. Res. Comm. 1996, 219, 337-341. 88. Balzarini, J.; Holy, A.; Jindrich, J.; Naesens, L.; Snoeck, R.; Schols, D.; De Clercq E. Differential antiherpesvirus and antiretrovirus effects of the {S) and {R) enantiomers of acyclic nucleoside phosphonates: potent and selective in vitro and in vivo antiretrovirus activities of (/?)-9-(2-phosphonylmethoxypropyl)2,6-diaminopurine. Antimicrob. Agents and Chemother. 1993, 37, 332-338. 89. First nucleotide analog for treatment of HIV-1 infection; dosed once daily. Formulary 2001, 36, 826-827. 90. Coleman, C ; Ross, J.; Reddy, P. Tenofovir disoproxil fumarate. The first nucleotide reverse transcriptase inhibitor for treatment of patients with HIV-1 infection. Formulary 2002, 37, 15-22. 91. Norbeck, D. W.; Spanton, S.; Broder, S.; Mitsuya, H. (±)-Dioxolane-T ((±)-l-[(2p,4P)-2-(hydroxymethyl4-dioxolanyl]thymine). A new 2',3'-dideoxy nucleoside prototype with in vitro activity against HIV. Tetrahedron Lett. 1989, 30, 6263-6266. (b) Belleau, B., Dixit, D.M., Ba, N.N., Brown, W.L., Gulini, U., Lafleur, D., Cameron, J.M. Synthesis and anti-HIV activity of (±)-l,3-dioxolane nucleoside analogs and their enzymatic resolution. Abstr. Papers Am. Chem. Soc. 1992, MEDI 138. 92. Storer, R.; Clemens, I. R.; Lamont, B.; Noble, S. A.; Williamson, C ; Belleau, B. The resolution and absolute stereochemistry of enantiomers of cw-l,2-hydroxymethyl-l,3-oxathiolan-5-yl-cytosine BCH189 equipotent anti-HIV agents. Nucleosides Nucleotides 1993,12, 225-236. Recent Advances in Antiviral Nucleosides 61 93. (a) Johnson, M. A.; Fridland, A. Phosphorylation of 2',3'-dideoxyinosine by cytosoHc 5'-nucleotidase of human lymphoid cells. Mol Pharmacol. 1989, 36, 291-295. (b) Ahluwalia, G.; Cooney, D. A.; Mitsuya, H.; Fridland, A.; Flora, K. P.; Hao, Z.; Dalai, M.; Broder, S.; Johns, D. G. Initial studies on the cellular pharmacology of 2',3'-dideoxyinosine, an inhibitor or HIV infectivity. Biochem. Pharmacol. 1987, 36,3797-3800. (c) Johnson, M. A.; Ahluwalia, G.; Connelly, M. C ; Cooney, D. A.; Broder, S.; Johns, D. G.; Fridland, A. Metabolic pathways for the activation of the antiretroviral agent 2',3'-dideoxyadenosine in human lymphoid cells. /. Biol. Chem. 1988, 263, 15354-15357. 94. AhluwaUa, G.; Johnson, M. A.; Fridland, A.; Cooney, D. A.; Broder, S.; Johns, D. G. Cellular pharmacology of the anti-HIV agent 2',3'-dideoxyadenosine. Proc. Am. Acad. Cancer Res. 1988, 29, 349. 95. (a) Stames, M. C ; Cheng, Y. -C. Cellular metaboHsm of 2',3'-dideoxycytidine, a compound against human immunodeficiency virus. /. Biol. Chem. 1987, 262, 988-991. (b) Chen, C. -H.; Cheng, Y. -C. Delayed cytotoxicity and selective loss of mitochondrial DNA in cells treated with the anti-human immunodeficiency virus compound 2',3'-dideoxycytidine. /. Biol. Chem. 1989, 264, 11934-11937. 96. Lewis, L. D.; Hamzeh, F. M.; Lietman, P. S. Ultrastructure changes associated with reduced mitochonrial DNA and impaired mitochondrial function in the presence of 2',3'-dideoxycytidine. Antimicroh. Agents Chemother. 1992, 36, 2061-2065. 97. Lin, T. -S.; Luo, M. -Z.; Liu, M. - C ; Pai, S. B.; Dutschman, G. E.; Cheng, Y. -C. Synthesis and biological evaluation of 2',3'-dideoxy-L-pyrimidine nucleosides as potential antiviral agents against human immunodeficiency virus (HIV) and hepatitis B virus (HBV). /. Med. Chem. 1994, 37, 798-803. 98. Pelicano, H.; Pierra, C ; Eriksson, S.; Gosselin, G.; Imbach, J. -L.; Maury, G. Enzymatic properties of the unnatural p-L-enantiomers of 2',3'-dideoxyadenosine and 2',3'-didehydro-2',3'-dideoxyadenosine. /. Med. Chem. 1997, 40, 3969-3973. 99. (a) Secrist III, J. A.; Riggs, R. M.; Tiwari, K. N.; Montgomery, J. A. Synthesis and anti-HIV activity of 4'-thio-2',3'-dideoxynucleosides. /. Med. Chem. 1992, 35, 533-538. (b) Young, R. J.; Shaw-Ponter, S.; Thomson, J. B.; Miller, J. A.; Gumming, J. G.; Pugh, A. W.; Rider, P. Synthesis and antiviral evaluation of enantiomeric 2',3'-dideoxy- and 2',3'-didehydro-2',3'-dideoxy-4'-thionucleosides. Bioorg. Med. Chem. Lett. 1995, 5, 2599-2604. (c) Beres, J.; Sagi, G.; Tomoskozi, I.; Gruber, L.; Gulacsi, E.; Otvos, L. Stereospecific synthesis of (-)-carbocyclic 2',3'-dideoxythymidine, a potential anti-AIDS agent. Tetrahedron Lett. 1988, 29, 2681-2684. (d) Wolffkugel, D.; Halazy, S. Studies towards the synthesis of the saturated and unsaturated carbocyclic methylene phosphonate analogs of dideoxyadenosine. Nucleosides Nucleotides 1993, 12, 279-294. (e) Wang, P.; GuUen, B.; Newton, M. G.; Cheng, Y. - C ; Schinazi, R. F.; Chu, C. K. Asymmetric synthesis and antiviral activities of L-carbocychc 2',3'didehydro-2',3'-dideoxy and 2',3'-dideoxy nucleosides. /. Med. Chem. 1999, 42, 3390-3399. (d) Rassu, G.; Zanardi, F.; Battistini, L.; Gaetani, E.; Casiraghi, G. Expeditious syntheses of sugar-modified nucleosides and collections thereof exploiting furan-, pyrrole-, and thiophene-based siloxy dienes. /. Med. Chem. 1997, 40, 168-180. 100. Lin, T. -S.; Guo, J. -Y.; Schinazi, R. F.; Chu, C. K.; Xiang, J. -N.; Prusoff, W. H. Synthesis and antiviral activity of various 3'-azido analogs of pyrimidine deoxyribonucleosides against human immunodeficiency virus (HIV-1, HTLV/LAV). /. Med Chem. 1988, 31, 336-340. 101. Baba, M.; Pauwels, R.; Balzarini, J.; Herdewijn, P.; De Clercq, E. Selective inhibition of human immunodeficiency virus (HIV) by 3'-azido-2',3'-dideoxyguanosine in vitro. Biochem. Biophys. Res. Commun. 1987, 145, 1080-1086. 102. Balzarini, J.; Baba, M.; Pauwels, R.; Herdewijn, P.; Wood, S. G.; Robins, M. J.; De Clercq, E. Potent and selective activity of 3'-azido-2,6-diaminopurine-2',3'-dideoxyriboside, 3'-fluoro-2,6-diaminopurine2',3'-dideoxyriboside, and 3'-fluoro-2',3'dideoxyguanine against human immunodeficiency virus. Mol. 62 G. Gumina, Y. Choi and C. K. Chu Pharmacol 1988, 33, 243-249. 103. Furman, P. A.; Fyfe, J. A.; St Clair, M. H.; Weihold, K.; Rideut, J. L.; Freeman, G. A.; Lehrman, S. N.; Bolognesi, D. P.; Broder, S.; Mitsuya, H.; Barry, D. W. Phosphorylation of 3'-azido-3'-deoxythymidine and selective interaction of the 5'-triphosphate with human immunodeficiency virus reverse transcriptase. Proc. Natl. Acad. Sci. USA 1986, 83, 8333-8337. 104. St Clair, M. H.; Richard, C. A.; Spector, T.; Weinhold, K.; Miller, W. H.; Langloir, A. J.; Furman, P. A. 3'Azido-3'-deoxythymidine triphosphate as an inhibitor and substrate of purified human immunodeficiency virus reverse transcriptase. Antimicrob. Agents Chemother. 1987, 31, 1972-1977. 105. Ono, K.; Nakane, H.; Herdewijn, P.; Balzarini, J.; De Clercq. Differential inhibitory effects of several pyrimidine 2',3'-dideoxynucleoside 5'-triphosphates on the activities of reverse transcriptase and various cellular DNA polymerases. Mol. Pharmacol. 1989, 35, 578-583. 106. Sommadossi, J.-P.; Carlisle, R.; Zhou, Z. Cellular pharmacology of 3'-azido-3'-deoxythymidine with evidence of incorporation into DNA of human bone marrow cells. Mol. Pharmacol. 1989, 36, 9-14. 107. Larder, B. A.; Darby, G.; Richman, D. D. HIV with reduced sensitivity to zidovudine (AZT) isolates during prolonged therapy. Science 1989, 243, 1731-1734. 108. Richman, D. D. Subsceptibility to nucleoside analogs of zidovudine-resistant isolates of humanimmunodeficiency-virus. Am. J. Med. 1990, 88, 8-10. 109. Wengel, J.; Lau, J.; Pederson, E. B.; Nielson, C. M. Synthesis of L-3'-azido-3'-deoxythymidine and its stereoisomers. /. Org. Chem. 1991, 56, 3591-3594. 110. Gosselin, G.; Boudou, V.; Griffon, J.-F.; Pavia, G.; Pierra, C ; Imbach, J.-L.; Aubertin, A.-M.; Schinazi, R. F.; Faraj, A.; Sommadossi, J.-P. New unnatural L-nucleoside enantiomers: from their stereospecific synthesis to their biological activities. Nucleosides Nucleotides 1997,16, 1389-1398. 111. Eriksson, B. F. H.; Chu, C. K.; Schinazi, R. F. Phosphorylation of 3'-azido-2',3'-dideoxyuridine and preferential inhibition of human and simian inmiunodeficiency virus reverse transcriptases by its 5'-triphosphate. Antimicrob. Agents Chemother. 1989, 33, 1729-1734. 112. Chu, C. K.; Schinazi, R. F.; Ahn, M. K.; UUas, G. V.; Gu, Z. P. Structure-activity relationships of pyrimidine nucleosides as antiviral agents for human immunodeficiency virus type 1 in peripheral blood mononuclear cells. /. Med. Chem. 1989, 32, 612-617. 113. Schinazi, R. F.; Chu, C. K.; Ahn, M. K.; Sommadossi, J. -P. Selective in vitro inhibition of human immunodeficiency virus (HIV) replication by 3'-azido-2',3'-dideoxyuridine (CS-87). /. Cell Biochem. 1987,11 (suppl. D), 405. 114. Zhu, Z.; Schinazi, R. F.; Chu, C. K.; Wilhams, G. J.; Colby, C. B.; Sommadossi, J. -P. Cellular metabolism of 3'-azido-2',3'-dideoxyuridine with formation of 5'-0-diphosphohexose derivative by previously unrecognized metabolic pathways for 2'-deoxyuridine analogs. Mol. Pharmacol. 1990, 38, 929-938. 115. Schinazi, R. F.; Chu, C. K.; Eriksson, B. F.; Sommadossi, J. -P.; Doshi, K. J.; Boudinot, F. D.; Oswald, B.; McClure, H. M. Antiretroviral activity, biochemistry, and pharmacokinetics of 3'-azido-2',3'-dideoxy-5'methyIcytidine. Ann. New York Acad. Sci. 1990, 616, 385-397. 116. Watanabe, K. A.; Harada, K.; Zeidler, J.; Matulic-Adamic, J.; Takahashi, K.; Ren, W. -Y.; Cheng, L. - C ; Fox, J. J.; Chou, T. - C ; Zhu, Q. -Y.; Polsky, B.; Gold, J. W. M.; Armstrong, D. Synthesis and antiHIV activity of 2'-"up"-fluoro and analogs of active anti-AIDS nucleosides 3'-azido-3'-deoxythymidine (AZT) and 2',3'-dideoxycytidine (DDC). J. Med. Chem. 1990, 33, 2145-2150. 117. Tber, B.; Fahmi, N.-E.; Ronco, G.; Villa, P.; Ewing, D. F.; Mackenzie, G. An alternative strategy for the synthesis of 3'-azido-2',3'-dideoxy-4'-thionucleosides starting from D-xylose. Carbohydr. Res. 1995, 267, 203-215. 118. Bodenteich, M.; Griengl, H. Synthesis of enantiomerically pure carbocyclic 3'-azido-2',3'- Recent Advances in Antiviral Nucleosides 63 dideoxythymidine, a potential anti-AIDS drug. Tetrahedron Lett. 1978, 28, 5311-5312. 119. Marquez, V. E.; Ezzitouni, A.; Russ, P.; Siddiqui, M. A.; Ford, Jr., H.; Feldman, R. J.; Mitsuya, H.; George, C ; Barchi, Jr., J. J. HIV-1 reverse transcriptase can discriminate between two conformationally locked carbocyclic AZT triphosphate analogs. /. Am. Chem. Soc. 1998, 120, 2780-2789. 120. Herdewijn, P.; Balzarini, J.; De Clercq, E.; Pauwels, R.; Baba, M.; Broder, S.; Vanderhaeghe, H. 3'Substituted 2',3'-dideoxynucleoside analogs as potential anti-HIV (HTLV-III/LAV) agents. J. Med. Chem. 1987, 30, 1270-1278. 121. Balzarini, J.; Baba, M. Pauwels, R.; Herdewijn, J.; De Clercq, E. Anti-retrovirus activity of 3'-fluoroand 3'-azido-substituted pyrimidine 2',3'-dideoxynucleoside analogs. Biochem. Pharmacol. 1988, 37, 2847-2856 122. Hartmann, H.; Vogt, M. W.; Dumo, A. G.; Hirsch, M. S.; Hunsmann, G.; Eckstein, F. Enhanced in vitro inhibition of HIV-1 replication by 3'-fluoro-3'-deoxythymidine compared to several other nucleoside analogs. AIDS Res. Human Retroviruses 1988, 4, 457-466. 123. Matthes, E.; Lehmann, Ch.; Scholz, D.; Rosenthal, H. A.; Langen, P. Phosphorylation, anti-HIV activity and cytotoxicity of 3'-fluorothymidine. Biochem. Biophys. Res. Commun. 1988,153, 825-831. 124. Matthes, E.; Lehmann, C. H.; Scholz, D.; von Janta-Lipinski, M.; Gaertner, K.; Rosenthal, H. A.; Langen, P. Inhibition of HIV-associated reverse transcriptase by sugar-modified derivatives of thymidine 5'-triphosphate in comparison to cellular DNA a and p. Biochem. Biophys. Res. Commun. 1987, 148, 78-85. 125. Mansuri, M. M.; Hitchcock, M. J. M.; Buroker, R. A.; Bregman, C. L.; Ghazzouli, I.; Desiderio, J. V.; Starrett, J. E.; Sterzycki, R. Z.; Martin, J. C. Comparison of in vitro biological properties and mouse toxicities of three thymidine analogs active against human immunodeficiency virus. Antimicrob. Agents Chemother. 1990, 34, 637-641. 126. Lietamn, P. S. Lessons learned from pharmacodynamic studies of anti-HIV drugs. Antiviral Res. 1993, 20 (Suppl. 1), 22. 127. Balzarini, J.; Aerschot, A.; Pauwels, R.; Baba, M.; Scholz, D.; Herdewijn, P.; De Clercq, E. 5-Halogeno3'-fluoro-2',3'-dideoxyuridines as inhibitors of human immunodeficiency virus (HIV): potent and selctive anti-HIV activity of 3'-fluoro-2',3'-dideoxy-5-chlorouridine. Mol. Phamacol. 1989, 35, 571-577. 128. Daluge, S.; Purifoy, D. J. M.; Savina, P. M.; St Clair, M. H.; Parry, N. R.; Dev, I. K.; Novak, P.; Ayers, K. M.; Reardon, J. E.; Roberts, G. B.; Fyfe, J. A.; Blum, M. B.; Averett, D. R.; Domsife, R.E.; Domin, B. A.; Ferone, R.; Lewis, D. A.; Krenitsky, T. A. 5-Chloro-2',3'-dideoxy-3'-fluorouridine (93U83), a selective anti-human immunodeficiency virus agent with an improved metabolic and toxicological profile. Antimicrob. Agents Chemother. 1994, 38, 1590-1603. 129. Chun, B. K.; Schinazi, R. F.; Cheng, Y. - C ; Chu, C. K. Synthesis of L-2',3'-dideoxy-3'-fluoro-Lribonucleosides as potential antiviral agents from D-sorbitol. Carbohydr. Res. 2000, 328, 49-59. 130. Baumgarten, H.; Bodenteich, M.; Griengl, H. Chemo-enzymatic synthesis of (-)-carba-2',3'dideoxythymidine and (-)-carba-2',3'-dideoxy-3'-fluorothymidine. Tetrahedron Lett. 1988,29,5745-5746. 131. Nakayama, T.; Matsumura, Y.; Morizawa, Y.; Yasuda, A.; Uchida, K.; Takase, H.; Murakami, Y.; Atarashi, S.; Ikeuchi, T.; Osada, Y. Regio- and stereoselective synthesis of carbocyclic 2',3'-dideoxy-3'fluoro nucleosides as potential antiviral agents. Chem. Pharm. Bull. 1989, 42, 183-187. 132. Marquez, V. E.; Tseng, C. K. -H.; Mitsuya, H.; Aoki, S.; Kelly, J. A.; Ford Jr., H.; Roth, J. S.; Broder, S.; Johns, D. G.; Driscoll, J. S. Acid-stable 2'-fluoro purine dideoxynucleosides as active agents against HIV. /. Med. Chem. 1990, 33, 978-985. 133. Masood, R.; Ahluwalia, G. S.; Cooney, D. A.; Fridland, A.; Marquez, V. E.; Driscoll, J. S.; Hao, Z.; Mitsuya, H.; Pemo, C.-F.; Broder, S. Johns, D. G. 2'-Fluoro-2',3'-dideoxyarabinosyladenine: a metabolic 64 G. Gumina, Y. Choi and C. K. Chu stable analog of the antiretroviral agent 2',3'dideoxyadenosine. Mol. Pharmacol. 1990, 37, 590-596. 134. Ahluwalia, G. S.; Cooney, D. A.; Shirasaka, T.; Mitsuya, H.; Driscoll, J. S.; Johns, D. G. Enhancement by 2'-deoxycoformycin of the 5'-phosphorylation and anti-human immunodeficiency virus activity of 2',3'-dideoxyadenosine and 2'-P-fluoro-2',3'-dideoxyadenosine. Mol. Pharmacol. 1994, 46, 1002-1008. 135. Sterzycki, R. Z.; Ghazzouli, I.; Brankovan, V.; Martin, J. C ; Mansuri, M. M. Synthesis and anti-HIV activity of several 2'-fluoro-containing pyrimidine nucleosides. J. Med. Chem. 1990, 33, 2150-2157. 136. Van Aerschot, A.; Herdewijn, P.; Balzarini, J.; Pauwels, R.; De Clercq, E. 3'-Fluoro-2',3'-dideoxy5-chlorouridine: most selective anti-HIV-1 agent among a series of new 2'- and 3'-fluorinated 2',3'-dideoxynucleoside analogs. /. Med. Chem. 1989, 32, 1743-1749. 137. (a) Huang, J.-T.; Chen, L.-C; Wang, L.; Kim, M.-H.; Warshaw, J. A.; Armstrong, D.; Zhu, Q.-Y.; Chou, T.-C; Watanabe, K. A.; Matulic-Adamic, J.; Su, T.-L.; Fox, J. J.; Polsky, B.; Baron, P. A.; Gold, J. W. M.; Hardy, W. D.; Zuckerman, E. Fluorinated sugar analogs of potential anti-HIV nucleosides. /. Med. Chem. 1991, 34, 1640-1646. (b) McAtee, J. J.; Schinazi, R. F.; Liotta, D. C. A completely diastereoselective electrophilic fluorination of a chiral, noncarbohydrate sugar ring precusor: application to the synthesis of several novel 2'-fluoronucleosides. J. Org. Chem. 1998, 63, 2161-2167. 138. Cavalcanti, S. C. H.; Xiang, Y.; Newton, M. G.; Schinazi, R. F.; Cheng, Y. - C ; Chu, C. K. Synthesis of 2',3'-dideoxy-2'-fluoro-L-//zr£o-pentofuranosyl nucleosides as potential antiviral agents. Nucleosides Nucleotides 1999, 18, 2233-2252. 139. Kotra, L. P.; Newton, M. G.; Chu, C. K. Synthesis of 2,3-dideoxy-2,2-difluoro-L-^/>'c^r6>-pentofuranosyl nucleosides. Carbohydr. Res. 1998, 306, 69-80. 140. Marquez, V. E.; Jeong, L. S.; Nicklaus, M. C ; George, C. Synthesis and biological activity of sugarfluorinated 2',3'-dideoxy-4'-thioribofuranosyl nucleosides. Nucleosides Nucleotides 1995,14, 555-558. 141. Acton, E. M.; Goener, R. N.; Uh, H. S.; Ryan, K. J.; Henry, D. W. Improved antitumor effects in 3'-branched homologues of 2'-deoxythioguanosine. Synthesis and evaluation of thioguanosine nucleosides of 2,3-dideoxy-3-(hydroxymethyl)-D-^ry''/2rc>-pentofuranose. /. Med. Chem. 1979, 22, 518-525. 142. (a) Svansson, L.; Kvamstrom, I.; Classon, B.; Samuelsson, B. Synthesis of 2',3'-dideoxy-3'-Chydroxymethyl nucleosides as potential inhibitors of HIV. /. Org. Chem. 1991, 56, 2993-2997. (b) Lee-Ruff, E.; Ostrowski, M.; Ladha, A.; Stynes, D. V.; Vemik, I.; Jiang, J.-L.; Wan, W.-Q.; Ding, S.-F.; Joshi, S. Synthesis and HIV inhibition activity of 2',3'-dideoxy-3'-C-hydroxymethyl nucleosides. J. Med. Chem. 1996, 39, 5276-5280. 143. (a) Branalt, J.; Kvamstrom, I.; Niklasson, G.; Svensson, S. C. T. Classon, B.; Samuelsson, B. Synthesis of 2',3'-dideoxy-3'-C-(hydroxymethyl)-4'-thionucleosides as potential inhibitors of HIV. /. Org. Chem. 1994, 59, 1783-1788. (b) Janson, M.; Svansson, L.; Svensson, S. C. T.; Kvamstrom, I.; Classon, B.; Samuelsson, B. Synthesis of some purine carbocyclic isosters of 2',3'-dideoxy-3'-C-hydroxymethyl nucleosides as potential inhibitors of HIV. Nucleosides Nucleotides 1992, 11, 1739-1747. 144. Lin, T. -S.; Zhu, J. -L.; Dutschman, G. F.; Cheng, Y. - C ; Prusoff, W. H. Syntheses and biological evaluations of 3'-deoxy-3'-C-branched-substituted nucleosides. J. Med. Chem. 1993, 36, 353-362. 145. Chun, M. W.; Lee, K.; Choi, Y.; Lee, J.; Kim, J. H.; Lee, C. K.; Choi, B. G.; Xu, Y. C. Synthesis and antiviral activity of fluoro sugar nucleosides 2: synthesis and biological evaluations of 2',3'-dideoxy-2'fluoro-3'-C-hydroxymethyl-p-D-arabinofuranosyl nucleosides. Arch. Pharm. Res. 1996,19, 243-245. 146. Wachtmeister, J.; Classon, B.; Samuelsson, B. Synthesis of carbocyclic 2',3'-dideoxy-2'-fluoro-3'-Chydroxy methyl nucleoside analogs as potential inhibitors of HIV and HS V. Tetrahedron 1997,55,1861 -1872. 147. (a) Ho, H. -T.; Hitchcock, M. J. M. Cellular pharmacology of 2',3'-dideoxy-2',3'-didehydrothymidine, a nucleoside analog active against human immunodeficiency. Antimicrob. Agents Chemother. 1987, 33, 844-849. (b) Balzarini, J.; Herdewijn, P.; De Clercq, E. Differential patterns of intracellular metabolism Recent Advances in Antiviral Nucleosides 65 of 2',3'-didehydro-2',3'-dideoxythymidine, two potent anti-human immunodeficiency virus compounds. J. Biol. Chem. 1989, 264, 6127-6133. 148. Huang, P.; Farquhar, D.; Plunkett, W. Selective action of 2',3'-didehydro-2',3'-dideoxythymidine triphosphate on human immunodeficiency virus reverse transcriptase and human DNA polymerases. J. Biol. Chem. 1992, 267, 2817-2822. 149. Mansuri, M. M.; Strarrett Jr., J. E.; Ghazzouli, I.; Hitchcock, M. J. M.; Sterzycki, R. Z.; Brankovan, V.; Lin, T.-S.; August, E. M.; Prusoff, W. H.; Sommadossi, J.-P.; Martin, J. C. (l-(2,3-Dideoxy-p-D-g/3;c^rapent-2-enofuranosyl)thymine. A potent and selective anti-HIV agent. /. Med. Chem. 1989, 32, 461-466. 150. Zhu, Z.; Hitchcock, M. J. M.; Sommadossi, J. -P. MetaboHsm and DNA interaction of 2',3'-didehydro2',3'-dideoxythymidine in human bone marrow cells. MoL Pharmacol. 1991, 40, 838-845. 151. Chen, C. -H.; Vazquez-Padua, M.; Cheng, Y. -C. Effect of anti-human immunodeficiency virus nucleoside analogs on mitochondrial DNA and its implication for delayed toxicity. Mol. Pharmacol. 1991,39,625-628. 152. Lin, T. -S.; Luo, M. -Z.; Liu, M. - C ; Zhu, Y. -L.; GuUen, E.; Dutschman, G. E.; Cheng, Y. -C. Design and synthesis of 2',3'-dideoxy-2',3'-didehydro-p-L-cytidine (P-L-d4C) and of 2',3'-dideoxy-2',3'didehydro-p-L-5-fluorocytidine (p-L-Fd4C), two exceptionally potent inhibitors of human hepatitis B virus (HB V) and potent inhibitors of human immunodeficiency virus (HIV) in vitro. J. Med. Chem. 1996, 39,1757-1759. 153. (a) Faraj, A.; Schinazi, R. F.; Joudawlkis, A.; Lesnikowski, Z.; McMillan, A.; Morrow, C. D.; Sommadossi, J. -P. Effects of p-D-2',3'-didehydro-2',3'-dideoxy-5-fluorocytidine 5'-triphosphate (p-D-D4FCTP) and its p-L-enantiomer 5'-triphosphate (p-L-D4FCTP) on viral DNA polymerases. Antiviral Res. 1997, 34, A66. (b) Ma, L.; Hurwitz, S. J.; Shi, J.; McAtee, J. J.; Liotta, D. C ; McClure, H. M.; Schinazi, R. F. Pharmacokinetics of the antiviral agent p-D-2',3'-didehydro-2',3'-dideoxy-5-fluorocytidine in rhesus monkeys, Antimicrob. Agents Chemother. 1999, 43, 381-384. 154. Shi, J.; McAtee, J. J.; Schlueter Wirts, S.; Thamish, P.; Juodawlkis, A.; Liotta, D. C ; Schinazi, R. F. Synthesis and biological evaluation of 2',3'-didehydro-2',3'-dideoxy-5-fluorocytidine (D4FC) analogs: discovery of carbocyclic nucleoside triphosphates with potent inhibitory activity against HIV-1 reverse transcriptase. J. Med. Chem. 1999, 42, 859-867. 155. Chen, S. -H.; Lin, S.; King, I.; Spinka, T.; Dutschman, G. F.; GuUen, E. A.; Cheng, Y. - C ; Doyle, T. W. Synthesis and comparative evaluation of two antiviral agents: p-L-Fd4C and p-D-Fd4C. Bioorg. Med. Chem. Lett. 1998, 8, 3245-3250. 156. Schinazi, R. F.; Mellors, J.; Bazmi, H.; Diamond, S.; Garber, S.; Gallagher, K.; Geleziunas, R.; Klabe, R.; Pierce, M.; Rayner, M.; Wu, J. -T.; Zhang, H.; Hammond, J.; Bacheler, L.; Manion, D. J.; Otto, M. J.; Stuyver, L.; Trainor, G.; Liotta, D. C ; Erickson-Viitanen, S. DPC 817: a cytidine nucleoside analog with activity against zidovudine- and lamivudine-resistant viral variants. Antimicrob. Agents Chemother. 2002,46, 1394-1401. 157. Bolon, P. J.; Wang, P.; Chu, C. K.; Gosselin, G.; Boudou, V.; Pierra, C ; Mathe, C ; Imbach, J. -L.; Faraj, A.; Alaoui, M. A.; Sommadossi, J. -P.; Pai, S. B.; Zhu, Y. -L.; Lin, J. -S.; Cheng, Y. - C ; Schinazi, R. F. Anti-human immunodeficiency and anti-hepatitis B virus activities of p-L-2',3'-dideoxy purine nucleosides. Bioorg. Med. Chem. Lett. 1996, 6, 1657-1662. 158. Chu, C. K.; Schinazi, R. F.; Arnold, B. H.; Cannon, D. L.; Doboszewski, B.; Bhadti, V. B.; Gu, Z. Comparative activity of 2',3'-saturated and unsaturated pyrimidine and purine nucleosides against human immunodeficiency virus type 1 in peripheral blood mononuclear cells. Biochem. Pharmacol. 1988, 37, 3543-3548. 159. Ray, A. S.; Yang, Z.; Chu, C. K.; Anderson, K. S. Novel use of a guanosine prodrug approach to convert 2',3'-didehydro-2',3'-dideoxyguanosine into a viable antiviral agent. Antimicrob. Agents Chemother. 66 G. Gumina, Y. Choi and C. K. Chu 2002,^(5,887-891. 160. Lee, K.; Choi, Y.; Schinazi, R. F.; Cheng, Y. - C ; Chu, C. K. Structure-activity relationships of 2'-fluoro2',3'-unsaturated D-nucleosides as anti-HIV agents. Abstracts of Papers, 218th American Chemical Society National Meeting, 1999, Part 1, MEDI 9. 161. Lee, K.; Choi, Y.; Gumina, G.;Zhou, W.; Schinazi, R. F.; Chu, C. K. Structure-activity relationships of 2'-fluoro-2',3'-unsaturated D-nucleosides as anti-HIV agents. /. Med.Chem. 2002, 45, 1313-1320. 162. (a) Choi, Y.; Lee, K.; Hong, J. H.; Schinazi, R. F.; Chu, C. K. Synthesis and anti-HIV activity of L-2'fluoro-2',3'-unsaturated purine nucleosides. Tetrahedron Lett. 1998, 39, 4437-4440. (b) Lee, K.; Choi, Y.; GuUen, E.; Schlueter-Wirtz, S.; Schinazi, R. F.; Cheng, Y.-C; Chu, C. K. Synthesis and anti-HIV and anti-HBV activities of 2'-fluoro-2',3'-unsaturated L-nucleosides. J. Med. Chem. 1999, 42, 1320-1328. 163. Pai, S. B.; Liotta, D. C ; Chu, C. K.; Schinazi, R. F. Inhibition of hepatitis B virus by novel 2'-fluoro-2',3'unsaturated D- and L-nucleosides. Abstrats of Papers, 3rd International Conference on Therapies for Viral Hepatitis, Maui, USA; International Society for Antiviral Therapy: 1999; Abstract 125. 164. Koshida, R.; Cox, S.; Harmenberg, J.; Gilljam, G.; Wahren, B. Structure-activity relationships of fluorinated nucleoside analogs and their synergistic effect in combination with phosphonoformate against human immunodeficiency virus type 1. Antimicrob. Agents Chemother. 1989, 33, 2083-2088. 165. Chu, C. K.; Gumina, G.; Zhou, W.; Schinazi, R. F. Synthesis and anti-HIV activity of D- and L-3'-fluoro2',3'-unsaturated cytidines. Antiviral Res. 2002, 53, A41-A41. 166. Gumina, G.; Schinazi, R. F.; Chu, C. K. Synthesis and potent anti-HIV activity of L-3'-fluoro unsaturated cytidine. Org. Lett. 2001, 3, 4177-4180. 167. Choi, Y.; Choo, H.; Chong, Y.; Lee, S.; Olgen, S.; Schinazi, R. F.; Chu, C. K. Synthesis and potent anti-HIV activity of L-2',3'-didehydro-2',3'-dideoxy-2'-fluoro-4'-thiocytidine. Org. Lett. 2002,4,305-307. 168. Choi, Y.; Choo, H.; Chong, Y.; Lee, S.; Schinazi, R. F.; Chu, C. K. Synthesis and antiviral activitiesof L-2',3'didehydro-2',3'-dideoxy-2'-fluo^o-4'-thionucleosides. AZ75rr. Papers Am. Chem. Soc. 2001, CARB 029 169. Chu, C. K.; Choi, Y.; Choo, H.; Chong, Y.; Lee, S.; Zhu, W.; Schinazi, R. F. Synthesis and anti-HIV activity of D- and L-2',3'didehydro-2',3'-dideoxy-2'-fluoro-4'-thiocytidines. Antiviral Res. 2002, 53, A36-A36. 170. Vince, R.; Hua, M.; Brownell, J.; Daluge, S.; Lee, F.; Shannon, W. M.; Lavelle, G. C ; Quails, J.; Weislow, O. S.; Kiser, R.; Canonico, P. G.; Schitz, R. H.; Narayanan, V. L.; Mayo, J. G.; Shoemaker, R. H.; Boyd, M. R. Potent and selective activity of a new carbocyclic nucleoside analog (carbovir: NSC 614846) against human immunodeficiency virus in vitro. Biochem. Biophys. Res. Commun. 1988, 156, 1046-1053. 171. White, E. L.; Parker, W. B.; Macy, L. J.; Shaddis, S. C ; McCaleb, G.; Secrist III, J. A.; Vince, R.; Shannon, W. M. Comparison of the effect of carbovir, AZT, and dideoxynucleoside triphosphates on the activity of human immunodeficiency virus reverse transcriptase and selected human polymerases. Biochem. Biophys. Res. Commun. 1989, 161, 393-398. 172. Bondoc Jr., L. L.; Shannon, W. M.; Secrist III, J. A.; Vince, R.; Fridland, A. Metabolism of the carbocyclic nucleoside analog carbovir, an inhibitor of human immunodeficiency virus, in human lymphoid cells. Biochemistry 1990, 29, 9839-9843. 173. Miller, W. A.; Daluge, S.; Garvey, E. P.; Hopkins, S.; Reardon, J. E.; Boyd, F. L.; Miller, R. L. Phosphorylation of carbovir enantiomers by cellular enzymes determines the stereoselectivity of antiviral activity. J. Biol. Chem. 1992, 267, 21220-21224. 174. Parker, W. B.; White, E. L.; Shaddix, S. C ; Rose, L. J.; Buckheit Jr., R. W.; Germay, J. M.; Secrist III, J. A.; Vince, R.; Shannon, W. M. Mechanism of inhibition of human immunodeficiency virus type 1 reverse transcriptase a, P, and yby the 5'-triphosphates of carbovir, 3'-azido-3'-deoxythymidine, 2',3'- Recent Advances in Antiviral Nucleosides 67 dideoxyguanosine, and 3'-deoxythymidine. J. Biol Chem. 1991, 266, 1754-1762. 175. Orr, D. C ; Figueiredo, H. T.; Mo, C. -L.; Penn, C. R.; Cameron, J. M. DNA chain termination activity and inhibition of human immunodeficiency virus reverse transcriptase by carbocyclic 2',3'-didehydro-2',3'dideoxyguanosine triphosphate. J. Biol. Chem. 1992, 267, 4177-4182. 176. Parker, W. B.; Shaddix, S.; Bowdon, B. J.; Rose, L. M.; Vince, R.; Shannon, W. M. Bennett Jr., L. L. Metabolism of carbovir, a potent inhibitor of human immunodeficiency virus type 1, and its effects on cellular metabolism. Antimicrob. Agents Chemother. 1993, 37, 1004-1009. 177. Parker, W. B.; Shaddix, S.; Vince, R.; Bennett Jr., L. L. Lack of mitochondrial toxicity in CEM cells treated with carbovir. Antiviral Res. 1997, 34, 131-136. 178. Faletto, M. B.; Miller, W. H.; Garvey, E. P.; St Clair, M. H.; Daluge, S. M.; Good, S. S. Unique intracellular activation of the potent anti-human immunodeficiency virus agent 1592U89. Antimicrob. Agents Chemother. 1997, 41, 1099-1107. 179. Dobkin, J. F. Two new agents for HIV: Kaletra and Trizivir. Infect. Med. 2000,17,111-111. 180. Trizivir - Abacavir sulfate/lamivudine/zidovudine tablets - Glaxo Wellcome - Fixed-dose combination of 3 nucleoside analogs for twice-daily Tx of HIV. Formulary 2001, 36, 13-13. 181. Katagiri, N.; Nomura, M.; Sato, H.; Kaneko, C ; Tsuruo, T. Synthesis of nucleosides and related compounds - Synthesis and anti-HIV activity of 9-(c-4,t-5-bis[hydroxymethyl]cyclopent-2-en-R-l-yl)9//-adenine. /. Med. Chem. 1992, 35, 1882-1886. 182. Katagiri, N.; Shiraishi, T.; Sato, H.; Toyota, A.; Kaneko, C ; Yusa, K.; Ohhara, T.; Tsuruo, T. Resolution of 9-(c-4,t-5-Bis[hydroxymethyl]cyclopent-2-en-r-l-yl)-9//-adenine and selective inhibition of human immunodeficiency virus by the (-)-enantiomer. Biochem. Biophys. Res. Commun. 1992,184, 154-159. 183. (a) Valette, G.; Pompon, A.; Giradet, J. -L.; Gosselin, G.; Perigaud, C ; Gosselin, G.; Imbach, J. -L. Decomposition pathways and in vitro HIV inhibitory effects of isoddA pronucleotides: toward a rational approach for intracellular delivery of nucleoside 5'-monophosphates. J. Med. Chem. 1996,39,1981-1990. (b) Siddiqui, A. Q.; Ballatore, C ; McGuigan, C ; De Clercq, E.; Balzarini, J. The presence of substituents on the aryl moiety of the aryl phosphoramidate derivative of d4T enhances anti-HIV efficacy in cell culture: a structure-activity relationship. /. Med. Chem. 1999,42, 393-399. (c) McGuigan, C ; Cahard, D.; Sheeka, H. M.; De Clercq, E.; Balzarini, J. Aryl phosphoramidate derivatives of d4T have improved anti-HIV efficacy in tissue culture and may act by the generation of a novel intracellular metabohte. /. Med. Chem. 1996, 39, 1748-1753. (d) Balzarini, J.; Karlsson, A.; Aquaro, S.; Pemo, C. -F. Cahard, D.; Naesens, L.; De Clercq, E.; McGuigan, C. Mechanism of anti-HIV action of masked alaninyl d4T-MP derivatives. Proc, Natl. Acad. Sci. USA 1996,93,7295-7299. (e) Balzarini, J.; Egberink, H.; Hartmann, K.; Cahard, D.; Vahlenkamp, T.; Thormar, H.; De Clercq, E.; McGuigan, C. Antiretrovirus specificity and intracellular metaboUsm of 2',3'-didehydro-2',3'-dideoxythymidine (stavudine) and its 5'-monophosphate triester prodrug So324. Mol. Pharmacol. 1996, 50, 1207-1213. (f) Beltran, T.; Egron, D.; Lefebvre, L; Perigaud, C ; Pompon, A.; Gosselin, G.; Aubertin, A. -M.; Imbach, J. -L. Anti-HIV pronucleotides: SATE versus phenyl as a protecting group of AZT phosphoramidate derivatives. Nucleosides Nucleotides 1999, 4-5, 973-975. (g) McGuigan, C ; Bidois, L.; Hiouni, A.; Ballatore, C ; De Clercq, E.; Balzarini, J. Phosphoramidate derivatives of stavudine as inhibitors of HIV: unnatural amino acids may substitute for alanine. Antiviral Chem. Chemother. 2000,11, 111-116. 184. Balzarini, J.; Wedgwood, O.; Kruining, J.; Pelemans, H.; Heijtink, R.; De Clercq, E.; McGuigan, C. Anti-HIV and anti-HBV activity and resistance profile of 2', 3'-dideoxy-3'-thiacytidine (3TC) and its arylphosphoramidate derivative CF 1109. Biochem. Biophys. Res. Comm. 1996, 225, 363-369. 185. McGuigan, C ; Pathirana, R. N.; Balzarini, J.; De Clercq, E. Intracellular delivery of bioactive AZT nucleotides by aryl phosphate derivatives of AZT. /. Med. Chem. 1993, 36, 1048-1052. 68 G. Gumina, Y. Choi and C. K. Chu 186. Balzarini, J.; Kruining, J.; Wedgwood, O.; Pannecouque, C ; Aquaro, S.; Pemo, C. - R ; Naessens, L.; Witvrouw, M.; Heijtink, R.; De Clercq, E.; McGuigan, C. Conversion of 2', 3'-dideoxyadenosine (ddA) and 2', 3'-didehydro-2', 3'-dideoxyadenosine (d4A) to their corresponding aryloxyphosphoramidate derivatives markedly potentiates their activity against human immunodeficiency virus and hepatitis B virus. FEES Letters 1997, 410, 324-328. 187. McGuigan, C ; Wedgwood, O. M.; De Clercq, E.; Balzarini, J. Phosphoramidate derivatives of 2',3'didehydro-2',3'-dideoxyadenosine [d4A] have markedly improved anti-HIV potency and selectivity. Bioorg. Med. Chem. Lett. 1996, 6, 2359-2362. 188. Meier, C. 4H-1.3.2.-Benzodioxaphosphorin-2-nucleosyl-2-oxide: a new concept for lipophilic, potential prodrugs of biologically active nucleoside monophosphates. Angew. Chem. 1996,108, 77-79. 189. Meier, C ; Knispel, T.; Lorey, M.; Balzarini, J. Cyc/oSal-pronucleotides: the design and biological evaluation of a new class of hpophilic nucleotide prodrugs. Int. Antiviral News 1997, 5, 183-186. 190. Meier, C. Pro-nucleotides - recent advances in the design of efficient tools for the delivery of biologically active nucleoside monophosphate. Synlett 1997, 233-242. 191. Meier, C ; Lorey, M.; De Clercq, E.; Balzarini, J. Cyclic saUgenyl phosphotriesters of 2',3'-dideoxy-2',3'didehydrothymidine (d4T) - a new pro-nucleotide approach. Bioorg. Med. Chem. Lett. 1997, 7, 99-104. 192. Meier, C ; Lorey, M.; De Clercq, E.; Balzarini, J. C>'c/c>Sal-2',3'-dideoxy-2',3'-didehydrothymidine monophosphate (c>'<:/cSal-d4TMP): synthesis and antiviral evaluation of a new d4TMP delivery System. J. Med. Chem. 1998, 41, 1417-1427. 193. Balzarini, J.; Aquaro, S.; Knispel, T.; Rampazzo, C ; Bianchi, V.; Pemo, C. -P.; De Clercq, E. Meier, C. Cyclosaligenyl-2',3'-didehydro-2',3'-dideoxythymidine monophosphate: efficient intracellular delivery of d4TMP. Mol. Pharm. 2000, 58, 928-935. 194. Lorey, M.; Meier, C ; De Clercq, E.; Balzarini, J. New synthesis and antitumor activity of cyclo Salderivatives of 5-fluoro-2'-deoxyuridine monophosphate. Nucleosides Nucleotides 1997,16, 789-792. 195. Meier, C ; De Clercq, E.; Balzarini, J. Cyclosaligenyl-3'-azido-2',3'-dideoxythymidine monophosphate (cycloSal-AZTMP): a new pro-nucleotide approach. Nucleosides Nucleotides 1997, 16, 793-796. 196. Meier, C ; De Clercq, E.; Balzarini, J. Nucleotide delivery from cyclosaligenyl-3'-azido-3'deoxythymidine monophosphates (c};c/oSal-AZTMP). Eur. J. Org. Chem. 1998, 837-846. 197. Balzarini, J.; Naesens, L.; Aquaro, S.; Knispel, T.; Pemo, C. -F.; De Clercq, E.; Meier, C. Intracellular metabolism of cyclosaligenyl 3'-azido-2',3'-dideoxythimidine monophosphate, a prodrug of 3'-azido2',3'-dideoxythymidine (zidovudine). Mol. Pharmacol. 1999, 56, 1354-1361. 198. Meier, C ; Knispel, T.; De Clercq, E.; Balzarini, J. ADA-bypass by lipophilic cyc/oSal-ddAMP pronucleotides: a second example of the efficiency of the c>'c/oSal concept. Bioorg. Med. Chem. Lett. 1997, 7, 1577-1582. 199. Meier, C ; Knispel, T.; De Clercq, E.; Balzarini, J. Cyc/oSal pronucleotides of 2',3'-dideoxyadenosine and 2',3'-dideoxy-2',3'-didehydroadenosine: synthesis and antiviral evaluation of a highly efficient nucleotide delivery system. J. Med. Chem. 1999, 42, 1604-1614. 200. Meier, C.; Knispel, T.; Marquez, V. E.; Siddiqui, M. A.; De Clercq, E.; Balzarini, J. Cyclo^dX pronucleotides of 2'-fluoro-flra- and 2'-fluoro-n77o-2',3'-dideoxyadenosine as a strategy to bypass a metabolic blockade. /. Med. Chem. 1999, 42, 1615-1624. 201. Schinazi, R. P.; McMillan, A.; Cannon, D.; Mathis, R.; Lloyd, R. M.; Peck, A.; Sommadossi, J. -P.; St Clair, M.; Wilson, J.; Furman, P. A.; Painter, G.; Choi, W. B.; Liotta, D. C. Selective inhibition of human immunodeficiency vimses by racemates and enantiomers of c/5-5-fluoro-l-[2-(hydroxymethyl)l,3-oxathiolan-5-yl]cytosine. Antimicrob. Agents Chemother. 1992, 36, 2423-2431. 202. (a) Chu, C. K.; Ahn, S. K.; Kim, H. O.; Beach, J. W.; Alves, A. J.; Jeong, L. S.; Islam, Q.; Van Roey, P.; Recent Advances in Antiviral Nucleosides 69 Schinazi, R. F. Asymmetric synthesis of enantiomerically pure (-)-(rR,4'R)-dioxolane-thymine and its anti-HIV activity. Tetrahedron Lett. 1991,32,3791-3794. (b) Kim, H. O.; Shanmuganathan, K. Alves, A. J.; Shanmuganathan, K.; Cannon, D. L.; Alves, A. J.; Jeong, L. S.; Beach, J. W.; Chu, C. K. Potent anti-HIV and anti-HBV activities of (-)-L-p-dioxolane-C and (+)-L-p-dioxolane-T and their asymmetric synthesis. Tetrahedron Lett. 1992,33,6899-6902. (c) Kim, H. O.; Schinazi, R. F.; NampaUi, S.; Shanmuganathan, K.; Cannon, D. L.; Alves, A. J.; Jeong, L. S.; Beach, J. W.; Chu, C. K. 1,3-Dioxolanylpurine nucleosides (2R,4R) and (2R,4S) with selective anti-HIV-1 activity in human lymphocytes. J. Med. Chem. 1993, 36, 30-37. (d) Kim, H. O.; Schinazi, R. F.; Shanmuganathan, K.; Jeong, L. S.; Beach, J. W.; NampaUi, S.; Cannon, D. L.; Chu, C. K. L-p-(2S,4S)- and L-a-(2S,4R)-Dioxolanyl nucleosides as potential anti-HIV agents: asymmetric synthesis and structure-activity relationships. /. Med. Chem. 1993, 36, 519-528. 203. Doong, S. -L.; Tsai, C. H.; Schinazi, R. F.; Liotta, D. C ; Cheng, Y. -C. Inhibition of the replication of hepatitis B virus in vitro by 2'-deoxy-3'-thiacytidine and related analogs. Proc. Natl. Acad. Sci. USA 1991, 88, 8495-8499. 204. (a) Mahmoudian, M.; Bains, B. S.; Drake, C. S.; Hale, R. S.; Jones, P.; Piercery, J. E.; Montgomery, D. S.; Purvis, I. J.; Storer, R.; Dawson, M. J.; Lawrence, G. C. Enzymatic production of optically pure (2'/?-cw)-2'-deoxy-3'-thiacytidine (3TC, lamivudine): a potent anti-HIV agent. Enzym. Microb. Technol. 1993, 75, 749-755. (b) Choi, W. B.; Wilson, L. J.; Yeola, S.; Liotta, D. C. In situ complexation directs the stereochemistry of iV-glycosylation in the synthesis of oxathiolanyl and dioxolanyl nucleoside analogs. /. Am. Chem. Sac. 1991, 113, 9377-9379. 205. (a)Coates,J. A. V.; Cammack,N.; Jenkinson,H. J.;Mutton,I. M.;Pearson,B. A.; Storer,R.; Cameron,J.M.; Penn, C. R. The separated enantiomers of 2'-deoxy-3'-thiacytidine (BCH-189) both inhibit human immunodeficiency virus in vitro. Antimicrob. Agents Chemother. 1992, 36, 202-205. (b) Schinazi, R. F.; Chu, C. K.; Peck, A.; McMillan, A.; Mathis, S.; Cannon, D.; Jeong, L. S.; Beach, J. W.; Choi, W. B.; Yeola, S.; Liotta, D. C. Activities of the four optical isomers of 2',3'-dideoxy-3'-thiacytidine (BCH-189) against human immunodeficiency virus type I in human lymphocytes. Antimicrob. Agents Chemother. 1992, 36, 672-676. 206. (a) Chu, C. K.; Beach, J. W.; Jeong, L. S.; Choi, B. G.; Comer, F. I.; Alves, A. J.; Schinazi, R. F. Enantiomeric synthesis of (+)-BCH-l89 [(+)-(2S,5R)-l-[2-(hydroxymethyl)-l,3-oxathiolan-5-yl]cytosine] from D-mannose and its anti-HIV activity. J. Org. Chem. 1991, 56, 6503-6505. (b) Beach, J. W.; Jeong, L. S.; Alves, A. J.; Pohl, D.; Kim, H. O.; Chang, C. -N.; Doong, S. -L.; Schinazi, R. F.; Cheng, Y. - C ; Chu, C. K. Synthesis of enantiomerically pure 2'/?,5'iS-(-)-[2-(hydroxymethyl)-oxathiolan-5-yl]cytosine as a potent antiviral agent against hepatitis B virus (HBV) and human immunodeficiency virus (HIV). J. Org. Chem. 1992, 57, 2217-2219. (c) Jeong, L. S.; Schinazi, R. F.; Beach, J. W.; Kim, H. O.; Shanmuganathan, K.; Nampalli, S.; Chun, M. W.; Chung, W. K.; Choi, B. G.; Chu, C. K. Structureactivity relationships of j3-D-(25',57?)- and a-D-(25,55)-l,3-oxathiolanyl nucleosides as potential anti-HIV agents. /. Med. Chem. 1993, 36, 2627-2638. (d) Jeong, L. S.; Schinazi, R. F.; Beach, J. W.; Kim, H. O.; NampaUi, S.; Shanmuganathan, K.; Alves, A. J.; McMiUan, A.; Chu, C. K.; Mathis, R. Asymmetric synthesis and biological evaluation of P-L-(2/?,55)- and a-L-(27?,5/?)-l,3 oxathiolane-pyrimidine and purine nucleosides as potential anti-HIV agents. J. Med. Chem. 1993, 36, 181-195. 207. Chang, C. -N.; Doong, S. -L.; Zhou, J. H.; Beach, J. W.; Jeong, L. S.; Chu, C. K.; Tsai, C. H.; Cheng, Y. -C. Deoxycytidine deamination-resistant stereoisomer is the active form of (-)-2',3'-dideoxy-3'-thiacytidine in the inhibition of hepatitis B virus replication. /. Biol. Chem. 1992, 267, 13938-13942. 208. Severini, A.; Liu, X.-Y.; WUson, J. S.; Tyrrell, D. L. J. Mechanism of inhibition of duck hepatitis B virus polymerase by (-)-p-L-2',3'-dideoxy-3'-thiacytidine. Antimicrob. Agents Chemother. 1995,39,1430-1435. 209. Shewach, D. S.; Liotta, D. C ; Schinazi, R. F. Affinity of the antiviral enantiomers of oxathiolane cytosine 70 G. Gumina, Y. Choi and C. K. Chu nucleosides for human 2'-deoxycytidine kinase. Biochem. Pharmacol. 1993, 45, 1540-1543. 210. Hart, G. J.; Orr, D. C ; Penn, C. R.; Figueiredo, H. T.; Gray, N. M.; Boehme, R. E.; Cameron, J. M. Effects of (-)-2'-deoxy-3'-thiacytidine (3TC) 5'-triphosphate on human immunodeficiency virus reverse transcriptase and mammalian DNA polymerase a, p, and y. Antimicrob. Agents Chemother. 1992, 36, 1688-1694. 211. Gish, R. G.; Leung, N. W. Y.; Wright, T. L.; Trinh, H.; Lang, W.; Kessler, H. A.; Fang, L.; Wang, L. H.; Delehanty, J.; Rigney, A.; Mondou, E.; Snow, A.; Rousseau, F. Dose range study of pharmacokinetics, safety, and preliminary antiviral activity of emtricitabine in adults with hepatitis B virus infection. Antimicrob. Agents Chemother. 2002, 46, 1734-1740. 212. Furman, P. A.; Davis, M.; Liotta, D. C ; Paff, M.; Frick, L. W.; Nelson, D. J.; Domsife, R. E.; Wyrster, J. A.; Wilson, L. J.; Fyfe, J. A.; Tuttle, J. V.; Miller, W. H.; Condreay, L.; Averett, D. R.; Schinazi, R. F.; Painter, G. R. The anti-hepatitis B virus activities, cytotoxicities, and anabolic profiles of the (-) and (+) enantiomers of c/5-5-fluoro-l-[2-(hydroxymethyl)-l,3-oxathiolan-5-yl]cytosine. Antimicrob. Agents Chemother. 1992, 36, 2686-2692. 213. (a) Schinazi, R. F.; Boudinot, F. D.; Ibrahim, S. S.; Manning, C ; McClure, H. M.; Liotta, D. C. Pharmacokinetics and metabolism of racemic 2',3'-dideoxy-5-fluoro-3'-thiacytidine in rhesus monkeys. Antimicrob. Agents Chemother. 1992, 36, 2432-2438. (b) Schinazi, R. F.; Lloyd, R. M.; Nguyen, M. H.; Cannon, D. L.; McMillan, Ilksoy, N.; Chu, C. K.; Liotta, D. C ; Bazmi, H. Z.; Mellors, J. W. Characterization of human immunodeficiency viruses resistant to oxathiolane-cytosine nucleosides. Antimicrob. Agents Chemother. 1993, 38, 875-881. 214. (a) Grove, K. L.; Guo, X.; Liu, S.-H.; Gao, Z.; Chu, C. K.; Cheng, Y. -C. Anticancer activity of p-Ldioxolane-cytidine, a novel nucleoside analog with the unnatural L-configuration. Cancer Res. 1995, 55, 3008-3011. (b) Grove, K. L.; Cheng, Y. -C. Uptake and metabolism of the new anticancer compound p-L-(-)-dioxolane-cytidine in human prostate carcinoma DU-145 cells. Cancer Res. 1996, 56, 4187-4191. 215. Kukhanova, M.; Liu, S.-H.; Mozzherin, D.; Lin, T. -S.; Chu, C. K.; Cheng, Y. -C. L- and D-enantiomers of 2',3'-dideoxycytidine 5'-triphosphate analogs as substrates for human DNA polymerases. /. Biol. Chem. 1995, 270, 23055-23059. 216. (a) Rajagopalan, P.; Boudinot, F. D.; Chu, C. K.; Tennant, B. C ; Baldwin, B. H.; Schinazi, R. F. Pharmacokinetics of (-)-p-D-2,6-diaminopurine dioxolane and its metabolite, dioxolane guanosine, in woodchucks (Marmota monax). Antiviral Chem. Chemother. 1996, 7,65-70. (b) Chen, H.; Boudinot, F. D.; Chu, C. K.; McClure, H. M.; Schinazi, R. F. Pharmacokinetics of (-)-p-D-2-aminopurine dioxolane and (-)-p-D-2-amino-6-chloropurine dioxolane and their antiviral metabolite (-)-P-D-dioxolane guanine in rhesus monkeys. Antimicrob. Agents Chemother. 1996, 40, 2332-2336. 217. Bednarski, K.; Dixit, D. M.; Wang, W.; Evans, C. A.; Jin, H.; Yuen, L.; Mansour, T. S. Inhibitory activities of herpes simplex viruses type 1 and 2 and human cytomegalovirus by stereoisomers of 2'-deoxy-3'-oxa-5-(£)-(2-bromovinyl)uridines and their 4'-thioanalogs. Bioorg. Med. Chem. Lett. 1994, 4, 2667-2672. 218. (a) Li, L.; Dutschman, G. E.; Gullen, E. A.; Grill, S. P.; Choi, Y.; Chu, C. K.; Cheng, Y.-C. Inhibition of the growth of varicella-zoster virus by L-p-5-bromovinyl-(2-hydroxymehtyl)-(l,3-dioxolanyl)uracil. Abstracts of Papers, 91th Annual Meeting of the American Assciation for Cancer Research, San Francisco, CA; American Association for Cancer Research: 2000; A22. (b) Li, L.; Dutschman, G. E.; Gullen, E. A.; Tsujii, E.; Grill, S. P.; Choi, Y.; Chu, C. K.; Cheng, Y. -C. MetaboUsm and mode of inhibition of varicella-zoster virus by L-5-bromovinyl-(2-hydroxymethyl)-(l,3-dioxolanyl)uracil is dependent on viral thymidine kinase. Mol. Pharmacol. 2000, 58, 1109-1114. 219. Lin, J. -S.; Kira, T.; Gullen, E.; Choi, Y.; Qu, F.; Chu, C. K.; Cheng, Y. -C. Structure-activity relationships Recent Advances in Antiviral Nucleosides 71 of L-dioxolane uracil nucleosides as anti-Epstein Barr virus agents. /. Med. Chem. 1999, 42, 2212-2217. 220. Kira, T.; Grill, S. P.; Dutschman, G. E.; Lin J. -S.; Qu, F.; Choi, Y.; Chu, C. K.; Cheng, Y. -C. AntiEpstein-Barr virus (EBV) activity of -L-5-iododioxolane uracil is dependent on EBV thymidine kinase. Antimicrob. Agents Chemother. 2000, 44, 3278-3284. 221. Mansour, T. S.; Jin, H.; Wang, W.; Hooker, E. U.; Ashman, C ; Cammack, N.; Salomon, H.; Belmonte, A. R.; Wainberg, M. A. Anti-human immunodeficiency virus and anti-hepatitis B virus activities and toxicities of the enantiomers of 2'-deoxy-3'-oxa-4'-thiocytidine and their 5-fluoro analogs in vitro. J. Med. Chem. 1995, 38, 1-4. 222. De Muys, J. -M.; Gourdeau, H.; Nguyen-Ba, N.; Taylor, D. L.; Ahmed, P. S.; Mansour, T.; Locas, C ; Richard, N.; Wainberg, M. A.; Rando, R. F. Anti-human immunodeficiency virus type 1 activity, intracellular metabolism, and pharmacokinetic evaluation of 2'-deoxy-3'-oxa-4'-thiocytidine. Antimicrob. Agents Chemother. 1999, 43, 1835-1844. 223. Du, J.; Surzhykov, S.; Lin, J. S.; Newton, M. G.; Cheng, Y. - C ; Schinazi, R. F.; Chu, C. K. Synthesis, antihuman immunodeficiency virus and anti-hepatitis B virus activities of novel oxaselenolane nucleosides. /. Med.Chem. 1997, 40, 2991-2993. 224. Chu, C. K.; Pierra, C ; Ma, L.; Olgen, S.; Du, J.; Gumina, G.; Gullen, E.; Cheng, Y. - C ; Schinazi, R. F. Synthesis and antiviral activities of oxaselenolane nucleosides. J. Med.Chem. 2000, 43, 3906-3912. 225. Du, J.; Chu, C. K. Asymmetric synthesis of oxazolidine nucleosides and related chemistry. Nucleosides Nucleotides 1998, 17, 1-13. 226. Nair, V.; Jahnke, T. Antiviral activities of isomeric dideoxnucleosidesof D- and L-related stereochemistry. Antimicrob. Agents Chemother. 1995, 39, 1017-1029. 227. Huryn, D. M.; Sluboski, B. C ; Tam, S. Y. Weigele, M.; Sim, L; Anderson, B. D.; Mitsuya, H.; Broder, S. Synthesis and anti-HIV activity of isonucleosides. /. Med. Chem. 1992, 35,1'iAl-l^^A. 228. Nair, V.; St Clair, M. H.; Reardon, J. E.; Krasny, H. C ; Hazen, R. J.; Paff, M. T.; Boone, L. R.; Tisdale, M.; Najera, L; Domsife, R. E.; Averett, D. R.; Borroto-Esoda, K.; Yale, J. L.; Zimmerman, T. P.; Rideout, J. L. Antiviral, metabolic, and pharmacokinetic properties of the isomeric dideoxynucleoside 4(5')-(6-amino-9H-purin-9-yl)tetrahydro-2(*S)-furanmethanol. Antimicrob. Agents Chemother. 1995, 39, 1993-1999. 229. Frank, K. B.; Council, E. V.; Holman, M. J.; Huryn, D. M.; Sluboski, B. C ; Tam, S. Y.; Todaro, L. J.; Weigele, M.; Richman, D. D.; Mitsuya, H.; Broder, S.; Sim, L S. Anabolism and mechnism of action of Ro24-5098, an isomer of 2',3'-dideoxyadenosine (ddA) with anti-HIV activity. Ann. N. Y. Acad. Sci. 1990, 616, 408-414. 230. (a) Ng, K. M. E.; Orgel, L. E. Replacement of the 3'-CH group by nitrogen in the carbocyclic analog of thymidine. /. Med. Chem. 1989, 32, 1754-1757. (b) Peterson, M. L.; Vince, R. Synthesis and biological evaluation of 4-purinylpyrrolidine nucleosides. /. Med. Chem. 1991, 34, 2787-2797. 231. (a) Jones, M. F.; Nobel, S. A.; Robertson, C. A.; Storer, R. Tetrahydrothiophenenucleosides as potential anti-HIV agents. Tetrahedron Lett. 1991, 32, 247-250. (b) Jones, M. F.; Nobel, S. A.; Robertson, C. A.; Storer, R.; Highcock, R. M.; Lamont, R. B. Enantiospecific synthesis of 3'-hetero-dideoxy nucleoside analogs as potential anti-HIV agents. /. Chem. Soc. Perkin Trans. 11992, 1427-1436. 232. Pal, S.; Nair, V. Phosphorylation of the anti-HIV compound (5,5)-isodideoxyadenosine by human recombinant deoxycytidine kinase. Biochem. Pharmacol. 2000, 60, 1505-1508. 233. Sells, T. B.; Nair, V. Synthetic approaches novel cis and trans dideoxynucleosides of the apiose family. Tetrahedron 1994, 50, 117-138. 234. (a) Hammerschmidt, F.; Ohler, E.; Polsterer, J.-P.; Zbiral, E.; Balzarini, J.; De Clercq, E. A convenient route to D-apio-p-D-furanosyl- and 2'-deoxyapio-p-D-furanosyl nucleosides. Liebigs Ann. 1995, 551-558. 72 G. Gumina, Y. Choi and C. K. Chu (b) Hammerschmidt, F.; Ohler, E.; Polsterer, J.-P.; Zbiral, E.; Balzarini, J.; De Clercq, E. A convenient route to 3'-deoxy-a-L- and 3'-deoxy-|3-D-apionucleosides. Liebigs Ann. 1995, 559-565. 235. Hong, J. H.; Lee, K.; Choi, Y.; Chu, C. K. Enantiomeric synthesis of 3'-fluoro-apionucleosides using Claisen rearrangement. Tetrahedron Lett. 1998, 39, 3443-3446. 236. Jeong, L. S.; Lee, Y. A.; Moon, H. R.; Chun, M. W. Syntheses and antiviral activity of apio dideoxy nucleosides with azido or amino substituent. Nucleosides Nucleotides 1998,17, 1473-1487. 237. Tino, J. A.; Clark, J. M.; Kirk Field, A.; Jacobs, G. A.; Lis, K. A.; Michalik, T. L.; McGeever-Rubin, B.; Slusarchyk, W. A.; Spergel, S. H.; Sundeen, J. E.; Tuomari, V.; Weaver, E. R.; Young, M. G.; Zahler, R. Synthesis and antiviral activity of novel isonucleoside analogs. J. Med. Chem. 1993, 36, 1221-1229. 238. (a) Nair, V.; Purdy, D. F. Synthetic approaches to new regioisomers of AZT and AZU. Heterocycles 1993, 36,421-425. (b) Purdy, D. F.; Zintek, L. B.; Nair, V. Synthesis of isonucleosides related to AZT and AZU. Nucleosides Nucleotides 1994,13, 109-126. (c) Zintek, L. B.; Jeon, G. S.; Nair, V. The synthesis of (5) and {R) enantiomers of novel hydroxymethylated isodideoxynucleosides. Heterocycles 1994, 37, 1853-1864. 239. Shimada, N.; Hasegawa, N.; Harada, T.; Tomisawa, T.; Fujii, A.; Takita, T. Oxetanocin, a novel nucleoside from bacteria. J. Antibiot. 1986, 39, 1623-1625. 240. Seki, J. -L; Shimada, N.; Takahashi, K.; Takita, T.; Takeuchi, T.; Hoshino, H. Inhibition of infectivity of human immunodeficiency virus by a novel nucleoside, oxetanocin, and related compounds. Antimicrob. Agents Chemother. 1989, 33,113-115. 241. Nishiyama, Y.; Yamamoto, N.; Takahashi, K.; Shimada, N. Selective inhibition of human cytomegalovirus replication by a novel nucleoside, oxetanocin G. Antimicrob. Agents Chemother. 1988, 32, 1053-1056. 242. Alder, J.; Mitten, M.; Norbeck, D.; Marsh, K.; Kern, E. R.; Clement, J. Efficacy of A-73209, a potent orally active agent against VZV and HSV infections. Antiviral Res. 1994, 23, 93-105. 243. Gumina, G.; Chu, C. K. Synthesis of L-Oxetanocin. Org. Lett. 2002, 4, 1147-1149. 244. Hayashi, S.; Norbeck, D. W.; Rosenbrook, W.; Fine, R. L.; Matskura, M.; Plattner, J. J.; Broder, S.; Mitsuya, H. Cyclobut-A and cyclobut-G, carbocyclic oxetanocin analogs that inhibit the replication of human immunodeficiency virus in T cells and monocytes and macrophages in vitro. Antimicrob. Agents Chemother. 1990, 34, 287-294. 245. Field, A. K.; Tuomari, A. V.; McGreever-Rubin, B.; Terry, B. J.; Mazina, K. E.; Haffey, M. L.; Hagen, M. E.; Clark, J. M.; Braitman, A.; Slusatchyk, W. A.; Young, M. G.; Zahler, R. (±)-(la,2p,3oc)9-[2,3-S/5(hydroxymethyl)-cyclobutyl]guanine [(±)-BHCG or SQ 33054]: a potent and selective inhibitor of herpesvirus. Antiviral Res. 1990, 13, 41-52. 246. Norbeck, D. W.; Kern, E.; Hayashi, S.; Rosenbrook, W.; Sham, H.; Herrin, T.; Plattner, J. J.; Erickson, J.; Clement, J.; Swanson, R.; Shipkowitz, N.; Hardy, D.; Marsh, K.; Amett, G.; Shannon, W.; Broder, S.; Mitsuya, H. Cyclobut-A and cyclobut-G: broad-spectrum antiviral agents with potential utility for the therapy of AIDS. J. Med. Chem. 1990, 33, 1281-1285. 247. Tenney, D. J.; Yamanaka, G.; Voss, S. M.; Cianci, C. W.; Tuomari, A. V.; Sheaffer, A. K.; Alam, M.; Colonno, R. J. Lobucavir is phosphorylated in human cytomegalovirus infected and -uninfected cells and inhibits the viral DNA polymerase. Antimicrob. Agents Chemother. 1997, 41, 2680-2685. 248. Sato, Y.; Maruyama, T. Synthesis and antiviral activity of 3'-fluorocarbocyclic oxetanocin A. Chem. Pharm. Bull. 1995, 43, 91-95. 249. Ashton, W. T.; Meurer, L. C ; Cantone. C. L.; Field, K.; Hannah, J.; Karkas, J. D.; Liou, R.; Patel, G. F.; Perry, H. C ; Wagner, A. F.; Walton, E.; Tolman, R. L. Synthesis and antiherpetic activity of (±)-9-[[(Z)-2(hydroxymethyl)cyclopropyl]methyl]guanine and related compounds. J. Med. Chem. 1988,31,2304-2315. 250. Pierra, C ; Olgen, S.; Cavalcanti, S. C. H.; Cheng, Y. - C ; Schinazi, R. F.; Chu, C. K. Synthesis and antiviral activities of enantiomeric l-[2-(hydroxymethyl)cyclopropyl]methyl nucleosides. Nucleosides Recent Advances in Antiviral Nucleosides 73 Nucleotides 2000,19, 253-268. 251. Sekiyama, T.; Hatsuya, S.; Tanaka, Y.; Uchiyama, M.; Ono, N.; Iwayama, S.; Oikawa, M.; Suzuki, K.; Okunishi, M.; Tsuji, T. Synthesis and antiviral activity of novel acyclic nucleosides: discovery of cyclopropyl nucleoside with potent inhibitory activity against herpesviruses. /. Med. Chem. 1998, 41, 1284-1298. 252. Iwayama, S.; Ono,N.; Ohmura, Y.; Suzuki, K.; Aoki, M.;Nakazawa, H.; Oikawa,M.; Kato,T.; Okunishi, M.; Mishiyama, Y.; Yamanishi, K. Antiherpesvirus activities of (rS,2'R)-9-{[r,2'-bis(hydroxymethyl)cyclo prop-r-yl]methyl}guanine (A-5021) in cell culture. Antimicrob. Agents Chemother. 1998,42,1666-1670. 253. Onoshi, T.; Mukai, C ; Nakagawa, R.; Sekiyama, T.; Aoki, M.; Suzuki, K.; Nakazawa, H.; Ono, N.; Ohmura, Y.; Iwayama, S.; Okunishi, M.; Tsuji, T. Synthesis and antiviral activity of novel anti-VZV 5-substituted uracil nucleosides with a cyclopropane sugar moiety. /. Med. Chem. 2000, 43, 278-282. 254. (a) Csuk, R.; Scholz, Y. Synthesis of cyclopropyl carbocyclic nucleosides. Tetrahedron 1994, 50, 1043110442. (b) Zhao, Y. F.; Yang, T. -F.; Lee, M. G.; Lee, D. W.; Newton, M. G.; Chu, C. K. Asymmetric synthesis of (r5,2'/?)-cyclopropyl carbocyclic nucleosides. J. Org. Chem. 1995, 60, 5236-5242. (c) Lee, M. G.; Du, J. F.; Chun, M. W.; Chu, C. K. Enantiomeric synthesis of L- (or l'R,2'S)-carbocyclic cyclopropyl nucleosides. /. Org. Chem. 1997, 62, 1991-1995. (d) Yang, T. -F.; Kim, H.; Kotra, L. P.; Chu, C. K. Design and synthesis of 2'-hydroxyethylcyclopropyl carbocyclic nucleosides. Tetrahedron Lett. 1996, 37, 8849-8852. 255. (a) Qiu, Y. -L.; Ksebati, M. B.; Ptak, R. G.; Fan, B. Y.; Breitenbach, J. M.; Lin, J. -S.; Cheng, Y. - C ; Kern, E. R.; Drach, J. C ; Zemlicka, J. (Z)- and (F)-2-((Hydroxymethyl)cyclopropylidene)methyladenine and -guanine. New nucleoside analogs with a broad-spectrum antiviral activity. J. Med. Chem. 1998, 41, 10-23. (b) Qiu, Y. -L.; Hempel, A.; Camerman, N.; Camerman, A.; Geiser, F.; Ptak, R. G.; Breitenbach, J. M.; Kira, T.; Li, L.; GuUen, E.; Cheng, Y. - C ; Drach, J. C ; Zemlicka, J. (/?)-(-)- And {S)(+)-synadenol: synthesis, absolute configuration, and enantioselectivity of antiviral effect. /. Med. Chem. 1998, 41, 5257-5264. (c) Rybak, R. J.; Zemlicka, J.; Qiu, Y. -L.; Hartline, C. B.; Kern, E. R. Effective treatment of murine cytomegalovirus infections with methylenecyclopropane analogs of nucleosides. Antiviral Res. 1999, 43, 175-188. 256. Uchida, H.; Komada, E. N.; Yoshimura, K.; Maeda, Y.; Kosalaraksa, P.; Maroun, V.; Qiu, Y. -L.; Zemlicka, J.; Mitsuya, H. In vitro anti-human immunodeficiency virus activities of Z- and ^-methylenecyclopropane nucleoside analogs and their phosphoro-L-alaninate diesters. Antimicrob. Agents Chemother. 1999, 43, 1487-1490. 257. Rybak, R. J.; Hartline, C. B.; Qiu, Y. -L.; Zemlicka, J.; Harden, E.; Marshall, G.; Sommadossi, J. -P.; Kern, E. R. In vitro activities of methylenecylopropane analogs of nucleosides and their phosphoralaninate prodrugs against cytomegalovirus and other herpesvirus infections. Antimicrob. Agents Chemother. 2000, 44, 1506-1511. 258. Guan, H. -P.; Ksebati, M. B.; Cheng, Y. - C ; Drach, J. C ; Kern, E. R.; Zemlicka, J. Spiropentane mimics of nucleosides: analogs of 2'-deoxyadenosine and 2'-deoxyguanosine. Synthesis of all stereoisomers, isomeric assignment, and biological activity. /. Org. Chem. 2000, 65, 1280-1290. 259. Whitley, R. J.; Gnann, J. W. Acyclovir: a Decade Later. N. Engl. J. Med. 1992, 327, 782-788. 260. Schaeffer, H. J.; Beauchamp, L.; De Miranda, P.; Elion, G. B.; Bauer, D. J.; Collins, P. 9-(2-Hydroxyeth oxymethyl)guanine activity against viruses of the herpes group. Nature 1978, 272, 583-585. 261. Lewis, L. D.; Fowle, A. S. E.; Bittiner, S. B. Human gastrointestinal absorption of acyclovir from tablet, duodenal infusion and sipped solution. Br. J. Clin. Pharmacol. 1986, 21, 459-462. 262. Laskin, O. L.; Cederberg, D. M.; Mills, J.; Eron, L. J.; Mildvan, D.; Spector, S. S. Ganciclovir for the treatment and suppression of serious infections caused by cytomegaloviruses. Am. J. Med. 1987,83,201-207. 74 G. Gumina, Y. Choi and C. K. Chu 263. Buhles, W. C ; Mastre, B. J. Jr.; Tinker, A. J. Ganciclovir treatment of life or sight-threatening cytomegalovirus infection: experience in 314 immunocompromised patients. Rev. Infect. Dis. 1988, 10 (Suppl. 3), S495-506. 264. Drew, W. L; Ives, D.; Lalezari, J. P.; Crumpacker, C ; Follansbee, S. E.; Spector, S. A.; Benson, C. A.; Friedberg, D. N.; Hubbard, L. Oral ganciclovir as maintenance treatment for cytomegalovirus retinitis in patients with AIDS. N. Engl. J. Med. 1995, 333, 615-620. 265. Spector, S. A.; Busch, D. P.; Pollansbee, S.; Squires, K.; Lalezari, J. P.; Jacobson, M. A.; Connor, J. D.; Jung, D.; Shadman, A.; Mastre, B.; Buhles, W.; Drew, W. L. Pharmacokinetics, safety, and antiviral profiles of oral ganciclovir in persons infected with human immunodeficiency virus: a phase II study. J. Infect. Dis. 1995, 777, 1431-1437. 266. Harden, M. R.; Jarvest, R. L.; Boyd, M. R.; Sutton, D.; Vere Hodge, R. A. Prodrugs of selective antiherpesvirus agent 9-[4-hydroxy-3-(hydroxymethyl)but-l-yl]guanine (BRL 39123) with improved gastrointestinal absorption properties. J. Med. Chem. 1989, 32, 1738-1743. 267. Boyd, M. R.; Bacon, T. H.; Sutton, D. Comparative activity of penciclovir and acyclovir in mice infected intraperitoneally with herpes simplex virus type 1SC 16. Antimicroh. Agents Chemother. 1993,37,642-645. 268. Boyd, M. R.; Bacon, T. H.; Sutton, D. Antiherpesvirus activity of 9-(4-hydroxy-3-hydroxymethylbut-lyl)guanine (BRL 39123) in animals. Antimicroh. Agents Chemother. 1988, 32, 358-363. 269. Vere Hodge, R. A.; Cheng, Y. -C. The mode of action of penciclovir. Antiviral Chem. Chemother. 1993, 4, 13-24. 270. Rolan, P. Pharmacokinetics of new antiherpetic agents. Clin. Pharmacokinet. 1995, 29, 333-340. 271. Pue, M. A.; Benet, L. Z. Pharmacokinetics of famciclovir in man. Antiviral Chem. Chemother. 1993, 4 (Suppl. 1), 47-55. 272. Parang, K.; Knaus, E. E.; Wiebe, L. I.; Sardari, S.; Daneshtalab, M.; Csizmadia, F. Synthesis and antifungal activities of myristic acid analogs. Arch. Pharm.-Pharm. Med. Chem. 1996, 329, 475-482. 273. Naesens, L.; Balzarini, J.; De Clercq, E. Therapeutic potential of PMEA as an antiviral drug. Med. Virol. 1994, 4, 147-159. 274. De Clercq, E. Broad-spectrum anti-DNA virus and anti-retrovirus activity of phosphonylmethoxyalkylpu rine and pyrimidines. Biochem. Pharmacol. 1991, 42, 963-972. 275. Walker, R. E.; Vogel, S. E.; Jaffe, H.S.; PoHs, M. A.; Kovacs, J.; Markowitz, N.; Masur, H.; Lane, H. C. A phase I/II study of PMEA in HIV infected patients. Abstracts of Papers; P^ National Conference on Human Retroviruses and Related Infections, 1993, Washington DC. 276. Starrett, J. E., Jr.; Tortolani, D. R.; Russel, J.; Hitchcock, M. J. M.; Whiterock, V.; Martin, J. C ; Mansuri, M. M. Synthesis, oral bioavailability determination, and in vitro evaluation of prodrugs of the antiviral agent 9-[2-(phosphonomethoxy)-ethyl]adenine (PMEA). /. Med. Chem. 1994, 37, 1857-1864. 277. De Clercq, E. Acyclic nucleoside phosphonates in the chemotherapy of DNA virus and retrovirus infections. Intervirology 1997, 40, 295-303. 278. Srinivas, R. V.; Robbins, B. L.; Connelly, M. C ; Gong, Y. -F., Bischofberger, N.; Fridland, A. Metabolism and in vitro antiretroviral activities of bis(pivaloyloxymethyl) prodrugs of acyclic nucleoside phosphonates. Antimicroh. Agents Chemother. 1993, 37, 2247-2250. 279. Starrett, J. E. Jr.; Tortolani, D. R.; Hitchcock, M. J. M.; Martin, J. C ; Mansuri, M. M. Synthesis and in vitro evaluation of a phosphonate prodrug: bis(pivaloyloxymethyl) 9-(2-phosphonylmethoxyethyl)ade nine. Antiviral Res. 1992, 19, 267-273. 280. Delaugerre, C ; Marcelin, A. -G.; Thibalut, V.; Peytavin, G.; Bombled, T.; Bochet, M. -V.; Katlama, C ; Benhamou, Y.; Calvez, V. Human immunodeficiency virus (HIV) type 1 reverse transcriptase resistance mutations in hepatitis B virus (HBV)-HIV-coinfected patients treated for HBV chronic infecion once Recent Advances in Antiviral Nucleosides 75 daily with 10 milligrams of adefovir dipivoxil combined with lamivudine. Antimicrob. Agents Chemother. 2002, 46, 1586-1588. 281. Ying, C ; De Clercq, E.; Nicholson, W.; Furman, P.; Neyts, J. Inhibition of the replication of the DNA polymerase M550V mutation variant of human hepatitis B virus by adefovir, tenofovir, L-FMAU, DAPD, penciclovir and lobucavir. J. Viral Hepat. 2000, 7, 161-165. 282. (a) Srinivas, R. V.; Fridland, A. Antiviral activities of 9-R-2-phosphonomethoxypropyl adenine (PMPA) and bis(isopropyloxymethylcarbonyl)PMPA against various drug-resistant human immunodeficiency virus strains. Antimicrob. Agents Chemother. 1998,42,1484-1487. (b) Palmer, S.; Buckheit, R. W.; Gilbert, H.; Shaw, N.; Miller, M. D. Anti-HIV-1 activity of PMEA, PMPA (9-(2-phosphonylmethoxypropyl)adenine) and AZT against HIV-1 Subtypes A, B, C, D, E, F, G and O. Antiviral Res. 2000, 46, A42-A42. 283. ArimiUi, M. N.; Kim, C. U.; Dougherty, J.; Mulato, A.; Oliyai, R.; Shaw, J. P.; Gundy, K. C ; Bischofberger, N. Synthesis, in vitro biological evaluation and oral bioavailability of 9-[2-(phosphonome thoxy)propyl]adenine (PMPA) prodrugs. Antiviral Chem. Chemother. 1997, 8, 557-564. 284. Cihlar, T.; Birkus, G.; Greenwalt, D. E.; Hitchcock, M. J. M. Tenofovir exhibits low cytotoxicity in various human cell types: comparison with other nucleoside reverse transcriptase inhibitors. Antiviral Res. 2002, 54, 37-45. 285. Naeger, L. K.; Margot, N. A.; Miller, M. D. Tenofovir (PMPA) is less susceptible to pyrophosphorolysis and nucleotide-dependent chain-terminator removal than zidovudine or stavudine. Nucleosides Nucleotides 2001, 20, 635-639. 286. Fridland, A.; Robbins, B. L.; Srinivas, R. V. Antiretroviral activity and metabolism of bis(POC)PMPA, an oral bioavailable prodrug of PMPA. Antiviral Res. 1997, 34, A49. 287. De Clercq, E.; Sakuma, T.; Baba, M.; Pauwels, R.; Balzarini, J.; Rosenberg, I.; Holy, A. Antiviral activity of phosphylmethoxyalkyl derivatives of purines and pyrimidines. Antiviral. Res. 1987, 8, 261-272. 288. Hitchcock, M. J. M.; Jaffe, H. S.; Martin, J. C.; Stagg, R. J. Cidofovir - a new agent with potent antiherpesvirus activity. Antiviral Chem. Chemother. 1996, 7, 115-127. 289. (a) Yokota, T.; Konno, K.; Chonan, E.; Mochizuki, S.; Kojima, K.; Shigeta, S.; De Clercq, E. Comparative activities of several nucleoside analogs against duck hepatitis B virus in vitro. Antimicrob. Agents Chemother. 1990,34, 1326-1330. (b) Yokota, T.; Mochizuki, S.; Konno, K.; Mori, S.; Shigeta, S.; De Clercq, E. Inhibitory effects of selected antiviral compounds on human hepatitis B virus DNA synthesis. Antimicrob. Agents Chemother. 1991, 35, 394-397. 290. Mendel, D. B.; Cihlar, T.; Moon, K.; Chen, M. S. Conversion of l-[((5)-2-hydroxy-2-oxo-l,4,2dioxaphosphorinan-5-yl)methyl]cytosine to cidofovir by an intracellular cyclic CMP phosphodiesterase. Antimicrob. Agents Chemother. 1997, 41, 641-646. 291. Weller, S.; Blum, M. R.; Doucette, M. Pharmacokinetics of the acyclovir pro-drug valaciclovir after escalating single and mutiple-dose administration to normal volunteers. Clin. Pharmacol. Ther. 1993, 54, 595-605. 292. Hostetler, K. Y.; Beadle, J. R.; Hombuckle, W. E.; Bellezza, C. A.; Tochkov, I. A.; Cote, P. J.; Gerin, J. L.; Korba, B. E.; Tennant, B. C. Antiviral activities of oral l-O-hexadecylpropanediol-3phosphoacyclovir and acyclovir in woodchucks with chronic woodchuck hepatitis virus infection. Antimicrob. Agents Chemother. 2000, 44, 1964-1969. 293. Cheng, L. Y.; Hostetler, K. Y.; Ozerdem, U.; Gardner, M. F.; Mach-Hofacre, B.; Freeman, W. R. Intravitreal toxicology and treatment efficacy of a long acting anti-viral lipid prodrug of ganciclovir (HDP-GCV) in liposome formulation. Invest. Ophtalmol. Vis. Sci. 1999, 40, S872-S872. 294. Cheng, L.; Hostetler, K. Y.; Chaidhawangul, S.; Gardner, M. F.; Beadle, J. R.; Keefe, K. S.; BergeronLynn, G.; Severson, G. M.; Soules, K. A.; Mueller, A. J.; Freeman, W. Intravitreal toxicology and 76 G. Gumina, Y. Choi and C. K. Chu duration of efficacy of a novel antiviral lipid prodrug of ganciclovir in liposome formulation. Invest. Ophtalmol Vis. Sci. 2000, 41, 1523-1532. 295. (a) Beadle, J. R.; Rodriguez, N.; Aldem, K. A.; Hostetler, K. Y.; Hartline, C ; Kern, E. R. Antiviral activity of alkoxyalkyl esters of cidofovir against drug-resistant strains of cytomegalovirus. Antiviral Res. 2002, 53, A60-A60. (b) Williams, S. L.; Kushner, N. L.; Hartline, C. B.; Harden, E. A.; Beadle, J. R.; Hostetler, K. Y.; Kern, E. R. Enhanced activity of alkoxyalkyl esters of cidofovir and cyclic cidoforiv against replication of herpesviruses in vitro. Antiviral Res. 2002, 53, A61-A61. (c) Aldem, K. A.; Ciesla, S. L.; Winegarden, K. L.; Hostetler, K. Y. l-0-Hexadecyloxypropyl-[^'^C]cidofovir: cellular uptake and metabolism in MRC-5 human lung fibroblasts, in vitro. Antiviral Res. 2002, 53, A62-A62. (d) Huggins, J. W.; Baker, R. O.; Beadle, J. R.; Hosteder, K. Y. Orally active ether lipid prodrugs of cidofovir for the treatment of smallpox. Antiviral Res. 2002, 53, A66-A66. (e) Winegarden, K. L.; Ciesla, S. L.; Aldem, K. A.; Beadle, J. R.; Hostetler, K. Y. Antiviral Res. 2002, 53, A67-A67. 296. Kumar, R.; Nath, M.; Tyrrell, D. L. J. Design and synthesis of novel 5-substituted acyclic pyrimidine nucleosides as potent and selective inhibitors of hepatitis B vims. J. Med. Chem. 2002, 45, 2032-2040. 297. Pockros,P. J. Developments in the treatment of chronic hepatitis C.£';c/7. Opin. Inv. Drugs 20Q2,77,515-528. 298. Sood, R. K.; Bhadti, V. S.; Fattom, A. I.; Naso, R. B.; Korba, B. E.; Kern, E. R.; Chen, H. -M.; Hosmane, R. S. Novel ring-expanded nucleoside analogs exhibit potent and selective inhibition of hepatitis B vims replication in cultured human hepatoblastoma cells. Antiviral Res. 2002, 53, 159-164. 299. Gill, J. K.; Wang, L.; Bretner, M.; Newman, R.; Kyprianou, N.; Hosmane, R. S. Potent in vitro anticancer activities of ring-expanded ("fat") nucleosides containing the imidazo[4,5-^][l,3]diazepine ring system. Nucleosides Nucleotides 2001, 20, 1043-1047. 300. De Clerq, E. Antiviral and antimetabolic activities of neplanocins. Antimicrob. Agents. Chemother. 1985, 28, 84-89. 301. Song, G. -Y.; Paul, V.; Choo, H.; Morrey, J.; Sidwell, R. W.; Schinazi, R. R; Chu, C. K. Enantiomeric synthesis of D- and L-cyclopentenyl nucleosides and their antiviral activity against HIV and West Nile vims. J. Med. Chem. 2001, 44, 3985-3993. 302. Kusaka, T.; Yamamoto, H.; Shibata, M.; Muroi, M.; Kishi, T.; Mizuno, K. Streptomyces citricolor nov. sp. and a new antibiotic, aristeromycin. J. Antibiot. 1968, 27, 255-263. 303. Rajappan, V.; Schneller, S. W.; Williams, S. L.; Kem, E. R. The enantiomers of carbocyclic 5'-norguanosine: activity towards Epstein-Barr virus. Bioorg. Med. Chem. Lett. 2002,10, 883-886. 304. Siddiqi, S. M.; Chen, X.; Schneller, S. W. Antiviral enantiomeric preference for 5'-noraristeromycin. J. Med. Chem. 1994, 37, 551-554. 305. Seley, K. L.; Schneller, S. W.; Korba, B. A 5'-noraristeromycin enantiomer with activity towards hepatitis B vims. Nucleosides Nucleotides 1997, 16, 2095-2099.