Wiley · 2020. 2. 6. · CHAPTER 1 RECENT ADVANCES IN CARBOCYCLIC NUCLEOSIDES: SYNTHESIS AND...

CHAPTER 1

RECENT ADVANCES IN CARBOCYCLICNUCLEOSIDES: SYNTHESIS ANDBIOLOGICAL ACTIVITY

JIANING WANG, RAVINDRA K. RAWAL, and CHUNG K. CHUThe University of Georgia, College of Pharmacy, Athens, GA

1.1. INTRODUCTION

As fundamental building blocks of nucleic acids, nucleosides are essential to theprocess of replication and transcription of genetic information in living organ-isms [1]. Therefore, a nucleoside analog is able to interfere with the replicationof pathogenic agents or with the proliferation of cancer cells by competingwith their natural counterparts, and this conception has attracted considerableattention in the field of chemotherapy. Indeed, the past decades have witnessedthe emergence of numerous therapeutically important nucleosides. In antivi-ral chemotherapy, eight nucleosides/nucleotides are currently licensed for thetreatment of human immunodeficiency virus (HIV) infection, and five nucleo-sides/nucleotides have been approved for anti-hepatitis B virus (HBV) therapy.A number of other nucleoside analogs are widely used against herpes simplexvirus (HSV), varicella-zoster virus (VZV), cytomegalovirus (CMV), influenzavirus, respiratory syncytial virus (RSV), and hepatitis C virus (HCV) [2]. Incancer chemotherapy, several nucleoside analogs have also demonstrated theirclinical application [3].

Carbocyclic nucleosides are analogs of natural nucleosides in which a methy-lene group replaces the oxygen atom in the carbohydrate ring. This modifica-tion results in the loss of the labile glycosidic bond and thus increases theirmetabolic stability toward phosphorylase and/or hydrolase [4]. Although carbo-cyclic nucleosides were first conceived and synthesized by medicinal chemists

Medicinal Chemistry of Nucleic Acids, First Edition. Edited by L.H. Zhang, Z. Xi and J. Chattopadhyaya.© 2011 John Wiley & Sons, Inc. Published 2011 by John Wiley & Sons, Inc.

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2 RECENT ADVANCES IN CARBOCYCLIC NUCLEOSIDES: SYNTHESIS AND BIOLOGICAL ACTIVITY

[5], aristeromycin 1 and neplanocin A 3, two naturally occurring antibiotics [6,7],prompted extensive investigations in carbocyclic nucleosides. Although synthe-sis of carbocyclic nucleosides have been mainly focused on the five-memberedring system, three-, four-, and six-membered carbocyclic nucleosides have alsobeen synthesized and discussed in this review as well. Thus, a large number ofnovel carbocyclic nucleosides have been prepared, and many of these compoundsare endowed with interesting biological activities. Particularly, this strategy hassuccessfully led to the discoveries of abacavir (a pro-drug of carbovir) and ente-cavir as clinically useful anti-HIV and anti-HBV agents, respectively. Despite thesubstantial progress that has been achieved [8–13], the effort to discover novelchemotherapeutic agents with enhanced biological activity and reduced toxicitycontinues, in order to treat emerging infectious organisms. This chapter coversthe most recent advances in carbocylic nucleosides up to September 2010.

1.2. FIVE-MEMBERED CARBOCYCLIC NUCLEOSIDES

1.2.1. Aristeromycin and neplanocin analogs

Although the majority of carbocyclic nucleosides are of synthetic origin, naturehas provided two of the most interesting compounds, aristeromycin (1) andneplanocin A (3) (Figure 1.1). The d-(-)-aristeromycin was first isolated fromStreptomyces citricolor in 1968 [7], although the racemic form was chemicallysynthesized in 1966 [5]. The discovery of neplanocin A, another carbocylicnucleoside, was achieved later in 1981 [6]. These two carbocyclic furanose nucle-osides exhibit significant antitumor as well as antiviral activity. In particular, thebroad-spectrum antiviral activity of these agents has been correlated with potentinhibitory effect of S -adenosyl-L-homocysteine (SAH) hydrolase [14–19].

It is well known that SAH hydrolase is one of the key enzymes in regulatingthe methylation reactions, which are essential to a number of important biolog-ical processes [20–22]. For instance, methylation of mRNA (i.e., 5′-capping)is required for mRNA maturation in many viruses. As shown in Figure 1.2,after the methyltransfer reaction, S -adenosyl-L-methionine (SAM) is convertedto S -adenosyl-L-homocysteine (SAH), which is a powerful feedback inhibitor of

Figure 1.1 Aristeromycin and neplanocin A and their 3-deaza analogs.

FIVE-MEMBERED CARBOCYCLIC NUCLEOSIDES 3

Figure 1.2 Inhibition of SAH hydrolase by aristeromycin and neplanocin A [13].

this cycle. SAH hydrolase efficiently removes SAH by cleaving it to adenosineand homocysteine, which maintains the balance of SAM and SAH. Inhibition ofSAH hydrolase (tight binding) results in increased level of SAH and consequentinhibition of viral mRNA methylation [13].

There are two types of mechanisms by which SAH hydrolase acts. The first onewas elucidated by Palmer and Abeles [20]. The reaction proceeds with oxidation,deprotonation, elimination, Michael-type addition, and reduction, with all stepsreversible. Methionine-bound aristeromycin and neplanocin A appear to act asanalogs of SAH and are oxidized by SAH hydrolase at the 3′-position. Thisprocess leads to a depletion of enzyme-bound NAD, and SAH hydrolase can nolonger initiate the catalysis cycle [19,23,25]. Such inhibition of SAH hydrolasecan be reversed after incubation with NAD+ or dialysis. On the other hand, thesecond type of inhibition is caused not only by the NAD+ depletion, but also bythe covalent binding of the inhibitor to SAH hydrolase, which cannot be rescuedby the addition of NAD+ [26,30].

The inhibition of SAH by aristeromycin and neplanocin A, as well as their 3-deaza analogs (2 and 4), has been investigated (Table 1.1). All these compounds,especially 3-deazaneplanocin A (4) (Ki 0.05 nM), are potent SAH inhibitors [31].De Clercq and coworkers have also demonstrated that vaccinia virus (VV) andvesicular stomatitis virus (VSV) are markedly senstitive to these carbocyclic

TABLE 1.1 Inhibition of SAH and antiviral activity of compounds 1–4 [18,31]

Inhibitory constantsagainst SAH Antiviral potency

EC50 (μg/ml)

Compounds Source Ki (nM) Cell VV VSV CC50 (μg/ml)

Aristeromycin (1) Beef liver 5 PRK N/A N/A N/A3-Deazaaristeromycin (2) Beef liver 4–13 PRK 0.8 0.2 >400Neplanocin A (3) Beef liver 2–8.4 PRK 0.03 0.01 403-Deazaneplanocin A (4) Hamster liver 0.05 PRK 0.07 0.07 >400


nucleosides. Presumably, the mRNA, which needs to be heavily methylated, isindispensable to the life cycle of VV and VSV. Shuting down the methylationpathway of mRNA, therefore, results in the inhibition of viral replication [31].

In addition to VV (belonging to poxvirus) and VSV (belonging to rhab-dovirus), a number of species of viruses, encompassing paramyxoviruses, her-pesviruses, reoviruses, arenavirus, and retroviruses, have been shown to be sus-ceptible to the SAH inhibitors [31].

In view of the interesting mechanism of action, as well as the significant bio-logical activity, syntheses of SAH hydrolase inhibitors, particularly aristeromycinand neplanocin A, as well as their analogs, has been the subject of a number ofinvestigations.

1.2.1.1. Aristeromycin (1) A number of approaches have been used to syn-thesize aristeromycin since Shealy’s original work [5]. Several published methodstook advantage of aminotriol 5 as an important intermediate for building up thetarget nucleosides, whereas others focused on utilizing alcohol 6 (or appropriatederivatives therefrom) as a pseudosugar source to couple with the base moiety(Figure 1.3).

In general, the aminotriol routes always started from achiral materials and gen-erated racemic compounds [5,32–37]. Although in other instances, the chiralitycan be resolved by using enzymatic resolutions [38], asymmetric cycloaddition[39–42], or palladium-catalyzed reactions [43–45], the synthesis of opticallypure aristeromycin 1 is unsatisfactory due to lengthy sequences and low yields,as well as scale-up difficulties. On the contrary, the pathway via alcohol 6 hasbeen more fruitful. Borchardt and coworkers [46,47] developed a more directmethod starting from d-ribonic acid γ-lactone 7, via an enone intermediate 8,which was treated with lithium di-(tert-butoxymethylene)cuprate followed byDIBAL-H to provide the desired key intermediate 10. Condensation of triflate11 with adenine salt followed by deprotection afforded the chiral aristeromycin(Scheme 1.1).

Chu and coworkers made modifications of this scheme in which d-ribose wasconverted to the key intermediate 14 as well as its enantiomer 16 in eight stepsin large scale (>40 g) in good yield (d-series >54%; l-series >45%, Scheme1.2) [48,49]. A series of l-aristeromycin analogs have been prepared using 16 asa key intermediate [50,51].

Figure 1.3 General approaches of the synthesis of aristeromycin.


Scheme 1.1 Synthesis of aristeromycin by Borchardt and coworkers [46]. Reagentsand conditions: (a) (i) cyclohexanoe, FeCl3, NaIO4, NaOH; (ii) 2-propanol, PPTS; (iii)CH3PO(OMe)2, nBuLi, THF; (b) (tBuOCH2)2CuLi; (c) DIBAL-H; (d) Tf2O/Py; and (e)(i) adenine, NaH, (ii) TFA/H2O.

Scheme 1.2 Modified sequences of the synthesis of aristeromycin by Chu and coworkers[48,49]. Reagents and conditions: (a) (i) 2,2-dimethoxypropane, p-TSA; (ii) TBDMSCl,Im; (b) (i) vinylmagnesium bromide; (ii) TBAF; (iii) NaIO4; (iv) NaH, DMSO, Ph3PMeBr;(c) (i) NaH, DMSO, Ph3PMeBr; (ii) DCC, DMSO, Py, TFA; (iii) vinylmagnesium bro-mide; (iv) TBAF; (v) NaIO4; (vi) NaBH4, CeCl3 · 7H2O; (d) (i) Grubbs’ catalyst; (ii)PDC, 4 A MS, AcOH.

Recently, Schneller and coworkers also described a hybrid sequence of thepreviously reported methods to produce aristeromycin (Scheme 1.3) [52]. Thekey step of this scheme is the 1,4-addition using a vinyl anion instead of tert-butoxymethylene. By this modification, the deprotection step of tert-butyl groupis avoided, which requires very harsh conditions, resulting in incompatibility ofsome protecting groups.

1.2.1.2. Neplanocin (3) An endocyclic double bond in the carbocyclic ringdistinguishes neplanocin A from aristeromycin. Therefore, the allylic alcohol 19,which is more reactive than the saturated alcohol 6, has been employed frequentlyas a precursor for the synthesis of neplanocin A (Figure 1.4).

A representative sequence for synthesis was developed by Lim and Marquez(Scheme 1.4) [53], in which neplanocin A was synthesized from ribonolactone20, which in turn was available in two steps from d-ribonic acid γ-lactone 7 [54].Treatment of lactone 20 with lithium dimethyl methylphosphonate followed by


Scheme 1.3 Synthesis of aristeromycin by Schneller and coworkers [52]. Reagents andconditions: (a) (i) (MeO)2CMe2, MeOH; (ii) Ph3P, I2, Im; (iii) Zn; (iv) vinylmagnesiumbromide; (v) Grubbs’ catalyst; (vi) PCC; (b) (i) vinylmagnesium bromide, TMSCl, HMPA,CuBr • Me2S; (ii) LiAlH4; (c) Ph3P, DIAD, 6-chloropurine; (d) (i) NaIO4, OsO4; (ii)NaBH4; (iii) NH3/MeOH; (iv) HCl/MeOH.

Figure 1.4 General synthesis approach via allylic alcohol 19.

Scheme 1.4 Synthesis of neplanocin A by Lim and Marquez [53]. Reagents and condi-tions: (a) (i) LiCH2P(O)(OCH3)2; (ii) NaOMe; (b) CrO3, Py; (c) K2CO3, 18-crown-6; (d)NaBH4, CeCl3; (e) (i) p-CH3PhSO2Cl; (ii) 6-chloropurine, NaH; (iii) NH3/MeOH; (iv)BCl3.

sodium methoxide in methanol afforded keto phosphonate 21. Oxidation of 21with modified Collins reagent produced ketone 22, which underwent intramolecu-lar cyclization under basic condition to generate key intermediate 23. Regioselec-tive reduction of 23 to allylic alcohol 24, followed by condensation condensationwith 6-chloropurine, eventually afforded the chiral neplanocin A.


Optimization of this sequence was carried out by Johnson and coworkers[55], who utilized the enone 16 as a precursor, which was converted to acetate26 via a sequence of 1,2-addition/acetylation/1,3-σ rearrangement (Scheme 1.5).Deprotection of 26 provided known compound 24, which was converted to theneplanocin A 3 by the known chemistry.

In view of the interesting biological activity as well as the unique structureof neplanocin A, Chu and coworkers conducted the SAR study of d- andl-neplanocin analogs and observed interesting antiviral activity (vida infra ,Table 1.11). It is noteworthy that the Mitsunobu reaction was performed toconstruct the nucleosides instead of classic SN2 coupling reaction (Scheme 1.6)[56].

More recently, Strazewski and Michel reported a short pathway to neplanocinA in good overall yield (Scheme 1.7) [57]. This approach used allylic alcohol36 as the key intermediate, which was prepared from d-ribose in eight steps.Mitsunobu reaction coupled the carbocyclic moiety with di -Boc protected ade-nine moiety to give the desired nucleoside 37, which was deblocked to provideneplanocin A (3).

Scheme 1.5 Synthesis of neplanocin A by Johnson and coworkers [55]. Reagents and con-ditions: (a) (i) n-Bu3SnCH2OBn, n-BuLi; (ii) Ac2O, Et3N, DMAP; (b) PdCl2(CH3CN)2,benzoquinone; (c) (i) K2CO3; (ii) MsCl, Et3N; (iii) adenine, K2CO3, 18-crown-6; (iv)Pd(OH)2, cyclohexene; (d) HCl/MeOH.

Scheme 1.6 Synthesis of d- and l-neplanocin analogs by Chu and coworkers [56].Reagents and conditions: (a) (i) (CH3)3COCH3,

t BuOK, sec-BuLi; (ii) Ac2O, Et3N,DMAP; (b) (i) PdCl2(CH3CN)2, benzoquinone; (ii) K2CO3; (c) Mitsunobu conditions,proper base moieties.


Scheme 1.7 Synthesis of neplanocin A by Strazewski and Michel [57]. Reagents andconditions: (a) (i) acetone, H+; (ii) TBDPSCl, Et3N, DMAP; (b) (i) Ph3PMeBr,t BuOK;(ii) (COCl)2, DMSO, Et3N; (c) vinylmagnesium bromide; (d) Neolyst dichloride; (e) (i)PDC, 4A MS; (ii) NaBH4, CeCl3; (f) (i) PPh3, DIAD, base; (ii) TBAF; (g) TFA.

1.2.2. Aristeromycin and neplanocin A analogs

Although aristeromycin and neplanocin A are potent SAH inhibitors, their thera-peutic utility has been limited due to their significant toxicity, which was shownto be mediated through phosphorylation by adenosine kinase and subsequentconversion to the corresponding cytotoxic nucleotides (Figure 1.5) [58–61].

Therefore, modifications based on the prototypes of these natural productshave generated some of the carbocyclic analogs that retain the inhibitory activitytoward SAH hydrolase, but are devoid of toxicity (Figure 1.6).

1.2.2.1. Aristeromycin analogs 5′-Fluoro-5′-deoxyaristeromycin 38 hasbeen prepared via a Mitsunobu coupling of (1S,2S,3R,4S)-2,3-(cyclopentylid-

Figure 1.5 Metabolites of aristeromycin and neplanocin A.


Figure 1.6 Biologically active aristeromycin analogs.

enedioxy)-4-fluoromethylcyclopentan-1-ol (78) with N6-bis-boc protectedadenine (Scheme 1.8) [62]. This procedure is adaptable to preparing a numberof 5′-fluoro-5′-deoxycarbocyclic nucleoside analogs with diversity in theheterocyclic base. To gain a glimpse into the biological potential of 38, it wassubjected to antiviral evaluation against herpes simplex-1, herpes simplex-2,


Scheme 1.8 Synthesis of 5′-fluoro-5′-deoxyaristeromycin by Schneller and coworkers[62]. Reagents and conditions: (a) NaIO4, OsO4, MeOH/H2O, then NaBH4, MeOH, 53%;(b) DAST, Pyridine, CH2Cl2/H2O, 60%; (c) DDQ, CH2Cl2/H2O, 60%; (d) DIAD,TPP,Ad(Boc)2; (e) 3N HCl/MeOH, 58% for two steps.

herpes simplex-1 (TK−), vaccinia, cowpox, vesicular stomatitis, coxsackieB4, respiratory syncytial, parainfluenza 3, reovirus-1, Sindbis, Punta Toro,rhinovirus, adenovirus, hepatitis C, virus, WestNile virus, and feline coronavirus.No activity was found against these viruses. However, antiviral test foundcompound 38 (Figure 1.6) showed activity against measles (MO6) withEC50′s 2.8 μM in neutral red assay and 13 μM in visual assay. No cytotoxicitywas observed in the cell lines.

2-Modified aristeromycin derivatives 39 and the related analog 40 were synthe-sized to investigate their inhibitory activity against human and Plasmodium fal-ciparum S -adenosyl-L-homocysteine hydrolase (PfSAHH) as shown in Scheme1.9 [63]. The 2-fluorinated compound 39 and 2-amino compound 40 showedstrong inhibitory activities against PfSAHH with IC50 value of 1.98 and 4.51 andselective index 24 and 20, respectively (Table 1.2), and a complete resistance toadenosine deaminase.

Given the fact that the cytotoxicity of aristeromycin is attributed to themetabolism to its 5′-phosphates, Schneller and coworkers addressed thissituation by preparing (±) 5′-noraristeromycin 41 (Figure 1.6) to avoid the

Scheme 1.9 Synthesis of 2-modified aristeromycins and their analogs [63]. Reagentsand conditions: (a) 2-fluoroadenine or 2-amino-6-chloropurine, NaH, DMSO, then(Ph3P)4Pd, Ph3P, THF, 55◦C, 73% (for 81) or 46% (for 82); (b) NH3/MeOH, 0◦C, 99%(for 83) or 120◦C, 71% (for 84); (c) OsO4, NMO, THF–H2O, rt, 45%, 52%, 35%, and40% (for 39, 40, and 2′,3′-epimer).


TABLE 1.2 Inhibitory activities of aristeromycin and noraristeromycin carbocyclicnucleosides analogs against human and P. falciparum SAH hydrolases [63–65]

IC50 (μM)

Compound HsSAHH PfSAHH SIa

1 4.85 57 0.08539 47.2 1.98 2440 90.7 4.51 2042 1.1 3.1 0.3543 200 NDb <0.244 NDb NDb –45 NDb NDb –46 NDb 220 >4.547 63 13 4.848 79 7.6 9.650 9 18 0.5

aSI: mean of IC50 values for HsSAH/mean of IC50 value for PfSAHH.bNo inhibitory activity showed at 500 μM.

phosphorylation step by displacing the 5′-phosphate-accepting hydroxyl groupfrom its original place [66]. Surprisingly, nucleoside 41 was found to benontoxic to host cells but still active against a variety of viruses (Table 1.3).

Subsequently, the enantiomerically pure d-5′-noraristeromycin and itsl-isomer were synthesized using chiral precursors 85 and 88 as startingmaterials. The Pd-catalyzed reaction was conducted to couple the base and sugarto provide the target nucleosides (Scheme 1.10) [67].

As shown in Table 1.3, d-form 42 was, on the average, 10-fold more potentthan its l-enantiomer 91 in inhibiting virus replication. The 4′-epimer of 5′-

TABLE 1.3 Antiviral Activity of (–)- and (+)-5′-noraristeromycin (41 and 42)

EC50 (μg/mL)

Compd 41 Compd 42 Compd 91 NeplanocinVirus Cell (racemic) (d-form) (l-form) A

Vaccinia virus E6SM 0.3 0.04 0.7 0.2a/0.2b

Vesicular stomatitis virus E6SM 0.07 0.1 2.0 0.2a/2.0b

Parainfluenza-3 HeLa 0.4 0.07 0.2 0.4a/0.2b

Reovirus-1 Vero 0.07 0.7 7.0 0.4a/0.7b

Cytomegalovirus HEL 0.4 0.01–0.05 5–20 0.4a/0.2–0.5b

Measles Vero 0.4 / / 0.4a

Respiratory syncytial virus Hela 2.0 / / 0.2a

Tacaribe Vero 1.0 8 50 0.4a/0.4b

aPositive control for compound 41.bPositive control for compounds 42 and 91.


Scheme 1.10 Synthesis of d and l-noraristeromycin by Siddiqi and coworkers [67].Reagents and conditions: (a) (EtO)2P(=O)Cl, Py; (b) (i) NH3/MeOH; (ii) Pd(PPh3)4, Ph3P,Base; (c) (i) OsO4, NMO; (ii) NH4OH/MeOH.

noraristeromycin 48 [68] (Figure 1.6) and 5′-homoaristeromycin 56 [69](Figure 1.6) were also prepared. Compound 48 inhibited the replicationof various DNA and RNA viruses at concentrations similar to those forneplanocin A, but were significantly less cytotoxic. Interestingly, an extensionof 5′-hydroxylmethyl chain (56) also retained antiviral activity against vaccinia(EC50 1.2 μg/mL), cowpox (EC50 0.12 μg/mL), and moneypox (EC50 0.12μg/mL) viruses without cytotoxicity up to 100 μg/mL.

Kitade and coworkers [64] synthesized 4′-fluorinated analogue of 9-[(1′R,2′S,3′R)-2′,3′-dihydroxy-cyclopentan-1′-yl]adenine (DHCaA) and theirrelated analogues under the Mitsunobu and palladium(0) coupling conditionsfollowed by fluorination with inversion of the configuration by using diethy-laminosulfur trifluoride as shown in Scheme 1.11. The 4′-β-Fluoro DHCaA(43 and 44) and 2-amino-4′-α-fluoro DHCaA (45 and 46) demonstrated slightinhibitory activity against human and P. falciparum S -adenosyl-L-homocysteinehydrolase (Table 1.2), respectively. A favorable antiviral activity of compound43 was observed toward measles (IC50 of 1.2 μg/mL by the neutral red assayand 14 μg/mL by the visual assay), but this was accompanied by the cytotoxicityin the CV-1 host cells (21–36 μg/mL) [70].

The same group also synthesized 4′-modified noraristeromycin (NAM)analogs [65], 2-fluoronoraristeromycin 47, 5′-α-noraristeromycin 48, 4′-sulfo-,4′-sulfamoy, 4′-azido, and 4′-amino-NAM. The inhibitory activities of theseanalogs and related compounds against P. falciparum and human S -adenosyl-L-homocysteine hydrolase were investigated (Table 1.2), but none of themdemonstrated good activity.

Borchardt and coworkers also described several other aristeromycin analogswithout a 5′-OH [71,72]. Among these compounds, nucleoside 50 (Figure 1.6)was one of the most potent SAH hydrolase inhibitors. A number of otheraristeromycin analogs with modifications at the 5′-position have been reported[73–77]. However, no significant antiviral activity was observed for those


Scheme 1.11 Synthesis of 4′-fluorinated carbocyclic nucleoside [64]. Reagents and con-ditions: (i) adenine (for 93) or 2-amino-6-chloropurine (for 94), Ph3P, DEAD, THF, rt; (ii)NH3, MeOH, rt (for 95), 55◦C (for 100), 100◦C (for 96 and 102); (iii) DAST, CH2Cl2,0◦C; (iv) OsO4, NMO, THF–H2O, rt; (v) N6-benzoyladenine (for 99) or 2-amino-6-chloropurine (for 100), NaH, (Ph3P)4Pd, Ph3P, DMSO, THF, 55◦C.

molecules with one exception of nucleoside 57 (Figure 1.6), which showedpotent antiviral activity against yellow fever (EC50 0.32 μg/mL). Seley-Radtkeand coworkers synthesized 5′-deoxy pyrimidine analogues 52 and 53 [78] andevaluated against SAH, but they were inactive.

The replacement of the 2′-hydrogen of natural ribonucleosides with a methylgroup yields compounds with excellent RNA chain-terminating properties as anti-HCV agents. Among them, 2′-C -methyladenosine [79] and 2′-C -methylcytidine[80] were discovered as potent anti-HCV agents and have undergone clinicaltrials. To explorer the effect of substitution in the carbocyclic moiety of theaforementioned antiviral agents, Hong and coworkers decided to synthesize thenucleoside analogues 54 and 55 containing a novel 2′,3′-dimethylcarbasugar [81].These compounds were evaluated as inhibitors of HCV in Huh-7 cell in vitro.But these nucleosides failed to inhibit HCV RNA replication in the cell-basedreplication assay.

Modification of aristeromycin was carried out on its 6′-position as well. Nucle-oside 58 (Figure 1.6) is a racemic compound with a fluorine atom on the 6′-β face[82]. The synthesis of 58 was accomplished, starting from the epoxide precursor105, which was subjected to nucleophilic attack by an azide anion to open up theepoxide ring. Fluorination and azide reduction provided the amine intermediate107, which was used to construct the target nucleoside 58 (Scheme 1.12).

Although 58 and its α-epimer 59 are good SAH hydrolase inhibitors (IC50 8and 80 nM for β and α epimers, respectively), its analog 60 is not active (IC50

28,000 nM). As shown in Scheme 1.13, the mechanism of this type of SAHhydrolase inhibitors has been proposed. The intermediate 115, generated from6′-fluoroaristeromycin, is the same one produced by the action of SAH hydrolase


Scheme 1.12 Synthesis of 6′-modified aristeromycin analogs [82]. Reagents andconditions: (a) (i) NaN3; (ii) 2,2-dimethoxypropane, H+; (b) (i) Tf2O, Py; (ii)TASF; (iii) H2/Lindlar catalyst; (c) (i) 5-amino-4,6-dichloropyrimidine, Et3N; (ii)diethoxymethyl acetate; (iii) NH3/MeOH; (iv) cyclohexene/Pd(OH)2/C; (v) HCl; (d)p-anisylchlorodiphenylmethane, Py; (e) Tf2O, 2,6-di-tert-butyl-4-methylpyridine; (iii)lithium benzonate; (iv) NH3/MeOH; (f) i) Tf2O, 2,6-di-tert-butyl-4-methylpyridine; (ii)TBAF; (g) adenine, K2CO3; (h) (i) formic acid; (ii) cyclohexene/Pd(OH)2/C.

Scheme 1.13 Proposed mechanism of 6′-F-neplanocin analogs as SAH hydrolaseinhibitors [82].


on neplanocin A and can irreversibly bind with the enzyme and consequentlyinhibits the SAH hydrolase. Therefore, the poor activity of 60 may be explainedby the fact that the 6′-hydroxyl group would be more difficult to eliminate than 6′-fluorine to generate active intermediate 115. On the basis of this information, thed-enantiomer of 6′-β-fluoroaristeromycin (61) was synthesized [83]. However,no biological data have been reported. Installing exocyclic double bond on the6′-position of aristeromycin produced novel nucleoside 436 (Figure 1.15) whichwill be discussed in detail in the next section (vide infra).

One of important discoveries of selective SAH hydrolase nucleoside inhibitorsis the replacement of adenine moiety with a 3-deazaadenine moiety. This modi-fication provides the reduced metabolic susceptibility of the nucleoside to theadenosine deaminase as well as adenosine kinase [84,85], which may resultin increased antiviral potency and/or reduced toxicity profile (Figure 1.7). Forinstance, 3-deazaaristeromycin 2 exhibited potent antiviral activity against VVand VSV in vitro (vida supra Table 1.1). In vivo, it decreased mortality rate innewborn mice infected with VSV at doses of 20, 100, and 500 μg/day [85].The 3-Deazanoraristermycin analog 49 (Figure 1.6) showed inhibition of VVand VSV with EC50s of 0.4 and 0.7 μg/mL, respectively, and was nontoxic inE6SM, Hela, Vero, and MDCK cells at concentrations up to 200 μg/mL [86]. Theother interesting 3-deaza analog is compound 51 (Figure 1.6), which does nothave 5′-substitution. This nucleoside inhibited L929 cell SAH with IC50 values of14.3 nM and was not a substrate or inhibitor of cellular adenosine deaminase [71].

Miller and coworkers reported a diastereoselective synthesis of spirono-raristeromycin 62-(±) using an acylnitroso Diels–Alder reaction [87,88]. (SeeFigure 1.6.) The affinity-labeling probes were prepared for the elucidationof the molecular mechanism of SAHHs. Kitade and coworkers reportednovel noraristeromycin analogs possessing epoxy functional groups at the3′,4′-positions (63–65 in Figure 1.6) [89] as potential affinity-labeling probes,for the elucidation of the catalytic site of SAHH and their affinities with bothHsSAHH and PfSAHH, as shown in Schemes 1.14 and 1.15. The values ofKi and Kinact, which are useful for evaluating the affinity and reactivity of anaffinity-labeling reagent, are summarized in Table 1.4. The Ki and Kinact valuesof epoxy compound 64 against HsSAHH were weaker than those of previouslyreported FDPHA. In addition, compound 64 did not show any Kinact againstPfSAHH. It is noteworthy that 3′,4′-epoxy-2-fluoronoraristeromycin 65 hadmoderate Kinact against HsSAHH and PfSAHH.

Figure 1.7 Favorable metabolic profile of carbocyclic 3-deaza nucleoside.


Scheme 1.14 Synthesis of 2′,3′-β-epoxynoraristeromycins [89]. Reagents and conditions:(a) TBDMSCl, imidazole, DMF, rt 5 h, 33%; (b) methanesulfonyl chloride, DMAP,Et3N, CH2Cl2, 0◦C, 0.5 h, 86%; (c) Bu4 NF, THF, rt, 0.5 h, 86%; (d) t-BuOK, DMF,rt 0.5 h, 42%.

Scheme 1.15 Synthesis of 2′,3′-α-epoxynoraristeromycins [89]. Reagents and conditions:(a) HC(OEt)3, p-toluenesulfonic acid monohydrate, acetone, 1 h, rt 88–97%; (b) methane-sulfonyl chloride, DMAP, Et3N, CH2Cl2, 0◦C, 0.5 h, 79–97%; (c) TFA/H2O (1:1), rt, 2 h,95–99%; (d) t-BuOK, DMF, rt 0.5 h, 42–79%.

TABLE 1.4 Ki values and Kinact values of compounds (64–66) against HsSAHHand PfSAHH [89,90]

HsSAHH PfSAHH

Compound Ki (μM) Kinact (min) Ki (μM) Kinact (min)

FDPHA 8.8 0.09 — —64 12.4 0.552 ND ND65 20.3 0.133 14.3 0.09966 5 ± 0.9 — — —


The blending of key structural features from the purine and pyrimidinenucleobase scaffolds gives rise to a new class of hybrid nucleosides. Thepurine–pyrimidine hybrid nucleosides can be viewed as either N-3 ribosylatedpurines or 5,6-disubstituted pyrimidines, thus recognition by both purine- andpyrimidine-metabolizing enzymes is possible. Given the increasing reports ofthe development of resistance in many enzymatic systems, a drug that couldbe recognized by more than one enzyme, could prove highly advantageous inovercoming resistance mechanisms related to binding site mutations. In thisregard, Seley-Radtke and coworkers designed and synthesized carbocyclic uracilderivatives with either a fused imidazole or thiazole ring compound 66 and 67(Figure 1.6) [90]. The targets were screened for their ability to inhibit SAHaseand DNA methyltransferase (DNA MeTase). Weak to no activity was observedagainst DNA MeTase (data not shown) for either compound; however, 66exhibited good activities against SAHase, whereas 67 showed no appreciableactivity. The Ki value for compound 66 was found to be 5.0±0.9 μM againstSAHase in the hydrolysis direction based on a Km value of 7.9 μM for theSAH substrate.

Modification in the vicinity of the 2′-hydroxy of the ribose in naturalribonucleosides can produce effective RNA chain terminator. For example,2′-C -methylcytidine [80] and 2′-C -methyladenosine [79] were discovered aspotent anti-HCV agents, and 2′-C -methylcytidine had studied in phase II clinicaltrials. The 4′-homologated stavudine and thiostavudine analogues are moleculesof considerable interest as antiviral and antitumor agents. Modeling studiesdemonstrated the presence of a narrow, relatively hydrophobic 4′-pocket that canaccommodate these substitutions, contributing to the observed enhancement inpotency. On the basis of these findings, that the branched nucleoside analoguesexhibited excellent anti-HCV activities, Hong and coworkers have synthesize4′(α)-ethyl-2′(β)-methyl carbodine analogues (68 and 69) [91]. The synthesizednucleoside analogues 68 and 69 were assayed for their ability to inhibit HCVRNA replication in a subgenomic replicon Huh7 cell line (LucNeo#2). However,the synthesized nucleosides did not show either any significant antiviral activityor toxicity up to 50 μM.

Chu and coworkers synthesized enantiomerically pure cyclopentyl cytosine[(-)-carbodine (72); Scheme 1.16] [92] from d-ribose and evaluated for its anti-influenza activity in vitro in comparison to the (+)-carbodine (71), (±)-carbodine(70), and ribavirin. The (-)-Carbodine (72) exhibited potent antiviral activityagainst various strains of influenza A and B viruses as shown in Tables 1.5and 1.6.

The same group also synthesized carbocyclic-6-benzylthioinosine analoguesas part of their continuing efforts to develop potent and selective antitoxoplasmicagents (Scheme 1.17) [93]. Various substituents on the aromatic ring of the 6-benzylthio group resulted in increased binding affinity to the enzyme as comparedto the unsubstituted compounds. The nature of the substituent at the para positionseems to have a substantial impact on binding to the enzyme. A cyano substi-tution at the para position decreased binding, whereas the methyl substitution


Scheme 1.16 Synthesis of (-)-carbodine [92]. Reagents and conditions: (a) MeSO2Cl,TEA, DCM, rt, quantitative; (b) NaN3, DMF, 140◦C, 4 h, 89%; (c) 10% Pd/C,EtOH, rt, 30 psi, 2 h, quantitative; (d) β-methoxyacryloyl isocyanate, DMF,—20◦Cto rt, 10 h, 72%; (e) 30% NH4OH, EtOH/dioxane (1:1), 100◦C, 18 h, 85%; (f)2,4,6-triisopropylbenzenesulfonyl chloride, DMAP, Et3N, 30% NH4OH, rt, 17 h, 78%;(g) TFA/H2O (2:1), 60◦C, 3 h, 82%.

was the best substrate in this series. Modeling studies showed carbocyclic 6-benzylthioinosine and 6-benzylthioinosine have similar sugar ring puckering withC2′-endo and anti -base conformation, which led to their binding into the activesite of T. gondii adenosine kinase. Experimental investigations and theoreticalcalculations further support that an oxygen atom of the sugar is not critical forthe ligand-binding. In agreement with its binding affinity, compounds 73 and74 demonstrated potent antitoxoplasma activity IC50 11.9 and 14.5 μM in cellculture, respectively, without any apparent host toxicity (Table 1.7).

1.2.2.2. Neplanocin A Analogs Neplanocin A analogs are specificallydesigned to improve the selectivity and reduce the toxicity of this classof nucleosides. Chemical modifications mainly focused on the 5′- and/or6′-positions and base moiety (Figure 1.8).

As mentioned earlier (Table 1.1), 3-deazaneplanocin A (4) exhibited potentinhibitory effect toward SAH hydrolase (Ki 0.05 nM, vida supera , Table 1.1)without toxicity after 24-h exposure up to 100 μM [86]. In newborn mice, itshowed marked protective effect against a lethal infection with VSV at a dose of0.5 mg/kg/day. This compound is one of the most potent SAH inhibitors knownso far and has received particular attention from researchers. Chu and coworkersrescreened the antiviral activity of 3-deazaneplanocin A (4) and noticed interest-ing antiviral activity against HCV and HBV (Table 1.8).

It has been recognized that the introduction of a halogen atom at the2-position of adenine nucleosides allowed resistance to the adenosine deaminase;for instance, arabinosyl-2-fluoroadenine and 2-chloro-2′-deoxyadenosine are

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Scheme 1.17 Synthesis of 6-mercaptopurine derivatives [93]. Reagents and Conditions:(a) NaBH4, MeOH, 0◦C, 1 h, 96%; (b) (i) MsCl, Et3N, CH2Cl2, 0◦C, 1 h, 92%; (ii)NaN3, DMF, 150◦C, 6 h, 85%; (iii) H2, 10% Pd/C, MeOH, 30 psi, 2 h; (iv) 5-amino-4,6-dichloropyrimidine, Et3N, n-PrOH, reflux, 24 h, 68% (two steps); (c) CH(OEt)3, p-TsOH,rt, 14 h, 65%; (d) thiourea, EtOH, reflux, 3 h; (e) TFA/H2O (2:1, v/v), 50◦C, 3 h, 52%(two steps); (f) appropriate benzylhalide, NH4OH/H2O, rt, 11h.

TABLE 1.7 The effect of carbocyclic 6-(p-methylbenzylthio)inosine (73),carbocyclic 6-(p-chlorolbenzylthio)inosine (74), and therapeutic compounds onhost-toxicitya and percent survivalb of wild-type (RH) and adenosine kinase deficient(TgAK−3) strains of Toxoplasma gondii grown in human fibroblasts in culture [93]

Concentration (μM)

Compound Infection 0 5 10 25 50 IC50 (μM)

73 (p-CH3) wild type (RH) 100 43 2.5 0 0 11.9 ± 0.4TgAK−3 100 99 100 100 100none 100 100 100 100 100

74 (p-Cl) wild type (RH) 100 73 12 0 0 14.5 ± 1.3TgAK−3 100 100 100 100 100none 100 100 100 100 100

pyrimethaminec wild type (RH) 100 99 55 25 23 16.1 ± 2.5none 100 101 100 108 108

sulfadiazinec wild type (RH) 100 93 58 53 46 27.3 ± 3.3none 100 98 100 100 102

aHost toxicity of uninfected cells was measured by MTT method in at least two independent exper-iments each of three replica as previously described [15–21].bPercent survival of parasites was measured by incorporation of [5, 6-13H]uracil in at least twoindependent experiments of three replica each as previously described.cTherapeutic compounds.

potent antitumor agents resistant to adenosine deaminase. The 2-Halo deriva-tives of adenosine have also been known to inhibit AdoHcy hydrolase. On thebasis of these results, Matsuda and coworkers designed and synthesized 2-fluoro-NPAs (135) as adenosine deaminase–resistant equivalents of NPA (Scheme 1.18)[94,95]. The 2-Fluoroneplanocin A (135) showed an antiviral potency and a


Figure 1.8 Biologically active neplanocin A analogs.

Scheme 1.18 Synthesis of 2-fluoroneplanocine [94]. Reagents: (a) K2CO3, 18-crown-6,2-fluoroadenine, DMF; (b) BCl3, CH2Cl2; (c) HCl, MeOH.


TABLE 1.8 Antiviral activity of 3-deazaneplanocin A (4)

Entry Virus Assay Cell line EC50 (μM) CC50 (μM) SI

1 Measles NR CV-1 0.4 12 30Visual CV-1 0.9 10 11

2 Vaccinia CPE HFF 2.9 >300 >1033 HCMV CPE HFF 0.36 183 5084 HCV HRR Huh7 ET 1.44 0.89 0.625 HBV VIR Huh7 ET 0.59 178 326 Tacaribe NR Vero 76 <0.1 0.1 >1

Visual <0.1 22 >220

NR, neutral red; CPE, cytopathic effect; HRR, HCV RNA replicon; SI, selective index (EC50/CC50).

spectrum that was comparable to that of neplanocin A (3) (Table 1.9). It wasparticularly active against vaccinia virus, vesicular stomatitis virus, parainfluenzavirus, reovirus, arenaviruses, and human cytomegalovirus (i.e., those viruses thatfall within the scope of the S -adenosyl-L-homocysteine hydrolase inhibitors).

In addition to these analogs, a series of 6′-position modified neplanocinA analogs were prepared by the same group [96–98]. Compound (6′R)-6′-methylneplanocin A (136, RMNPA) demonstrated excellent antiviral potencyand selectivity superior to that of the neplanocin A (Table 1.9). In mice study,136 (EC50 1.0 mg/kg/day) [99] was found to be superior to chloroquine (EC50

1.8 mg/kg/day). Interestingly, its diastereomer, (6′S)-6′-methylneplanocin A(137, SMNPA), was completely biologically inactive. The synthesis of RMNPA136 was accomplished starting from neplanocin A, as depicted in Scheme 1.19.Protection of 2′- and 3′-positions as well as the amino group on the base moietyleft a free 6′-OH, which was oxidized, alkylated, and then separated by HPLCto provide optically pure 136 and 137. A recent report described an improvedasymmetric synthesis of 136 via a chelation-controlled stereoselective additionreaction [99].

TABLE 1.9 Antiviral activity and cytotoxicity of compounds neplanocin A 3,2-F-neplanocin (135) and 6′-(R)-(RMNPA) in vero cells

Antiviral activity, IC50 Cytotoxicity,(μg/mL)a CC50 (μg/mL)b

Compounds VSV Measles Mumps Vero cells

Neplanocin 3 0.25 0.10 0.11 1552-F-neplanocin 135 0.25 0.03 1.2 >2006′-(R)-(RMNPA) 136 0.25 0.09 0.19 >200

aInhibitory concentration, required to reduce virus-induced cytopathicity (VSV) or virus plaqueformation (measles and mumps) by 50%.bThe 50% cytotoxic concentration, required to reduce the number of viable cells by 50%.


Scheme 1.19 Synthesis of RMNPA [98]. Reagents and conditions: (a) (i) TMSCl, Py, (ii)BzCl, (iii) NH4OH; (b) HClO4, acetone; (c) BaMnO4; (d) Me3Al; (e) (i) 90% HCOOH;(ii) NH4OH, Dioxane; (f) HPLC (C18) separation.

The 5′-nor derivative 138 or 139 [46], prepared via enone 8 through a conver-gent approach (Scheme 1.20), has been demonstrated to be approximately 10-foldbetter than the parent compound 3 in terms of antiviral activity (CC50/IC50)

[100].A comparative study analyzed the antiviral activity and toxicity of neplanocin

A (3), 5′-norneplanocin A (138) and 5′-nor-3-deazaneplanocin A (139) against awide range of DNA and RNA viruses [100], in which 138 and 139 showed greaterselectivity than neplanocin A against vesicular stomatitis virus and rotavirus(Table 1.10).

Schneller and coworkers, in their pursuit of neplanocin A analogues withnontoxic, antiviral potential, found the 6′-isoneplanocin A analogues 140–141(Figure 1.8) were synthesized [101].

Another interesting compound is 6′-homoneplanocin A (142), which displayedparticular activity against human cytomegavirus (EC50 0.15–0.5 μg/mL), vac-cinia virus (EC50 0.1 μg/mL), and vescular stomatitis virus (EC50 1.0 μg/mL)

Scheme 1.20 Synthesis of 5′-norneplanocin analogs [46]. Reagents and conditions: (a)NaBH4, CeCl3; (b) (i) TsCl, Et3N; (ii) base, NaH; (c) HCl.


TABLE 1.10 Antiviral activity against VV and cytotoxity of 138, 139, and NPA

Compounds IC50 (μM) ID50 (μM) Antiviral selectivity (ID50/IC50)

Compd 138 0.28 15 61Compd 139 0.95 56 59Neplanocin A (3) 0.08 0.5 6

[102]. Starting from enone 16, the key intermediate 187 was obtained via anaddition/reduction/rearrangement sequence. Treating mesylated alcohol 187 withadenine or 3-deazaadenine salt, followed by deprotection, afforded desired 6′-homoneplanocin analogs (Scheme 1.21).

Jeong and coworkers synthesized fluoroneplanocin A 144 (Scheme 1.22),which was believed to inhibit SAH hydrolase based on the type II mechanism[26]. The fluorosugar was prepared starting from eneone 23, which was convertedto the iodo derivative 190 by treating with I2/CCl4/pyridine. Compound 190 wasthen reduced, protected, fluorinated, and deprotected to provide desired key inter-mediate 191. The preparation of target nucleoside 144 was accomplished afterobtaining the compound 191 in hand, via SN2 type coupling reaction followed bydeprotection steps. The 5′-Fluoroneplanocin A (144) has been found to exhibit

Scheme 1.21 Synthesis of 6′-homoneplanocin A [102]. Reagents and conditions: (a) (i)(TMS)2 NH, BuLi, EtOAc; (ii) LiBH4; (iii) TBSCl, Im; (iv) Ac2O, DMAP, Et3N; (v)PdCl2(MeCN)2, p-benzoquinone; (vi) K2CO3; (b) (i) MsCl, DMAP; (ii) base, NaH, 15-crown-5; (c) HCl.

23 190 191 144

Scheme 1.22 Synthesis of fluoroneplanocin A [26]. Reagents and conditions: (a) I2, CCl4,Py; (b) (i) NaBH4, CeCl3; (ii) TBDPSCl, Im; (iii) n-BuLi, N -fluorobenzenesulfonimide;(v) TBAF; (c) (i) MsCl, Py; (ii) adenine, K2CO3, 18-crown-6; (iii) BBr3; (iv) Ac2O, Py;(v) NH3/MeOH.


better and irreversible SAHH inhibition and more potent antitumor activity thanneplanocin A. According to their results, after conversion of 3′-hydroxy groupto a keto group by NAD+ the fluorine atom acts as a leaving group during aMichael addition-elimination reaction induced by a nucleophile present in SAHH,as shown in Figure 1.9. Generally, an iodide is a better leaving group than a flu-oride. Therefore, it was of great interest to synthesize 5′-iodoneplanocin A (145in Figure 1.8) [103,104]. Moon and coworkers synthesized 5′-iodoneplanocin A(145), an isostere of 5′-fluoroneplanocin A, and its analogs, 146 and 147 [104],having different purine nucleobases and to evaluate their antiviral activity andcytotoxicity. But none of them showed antiviral activity.

Considering that neplanocin A and its fluoro-substituted analogues showedgood biological activities, the cyclopentene template of these nucleosides canbe assumed to be a good template for phosphorylations by kinases. That’s whyMoon and coworkers designed and synthesized 2′-β-C -methylneplanocin A (148)[105]. Disappointingly, compound 148 did not show significant activity againstHCV.

The antiviral and antitumor activity of NPA (3) seems to be interesting dueto an efficient inhibition of S -adenosylhomocysteine (AdoHcy) hydrolase. How-ever, the main drawback for the therapeutic utilization of NPA as an antiviralagent comes from its significant cytotoxicity. In contrast to NPA, ara-neplanocinA (149; ara-NPA) has shown less cytotoxicity while still retaining reasonableantiviral activity [106]. However, a thorough literature search indicated that lit-tle attention has been paid to the ara-neplanocin family of compounds. Apart

Figure 1.9 A plausible inhibitory mechanism of SAHH by fluoro-neplanocin A viaMichael addition-elimination process (SAHH, S -adenosylhomocysteine hydrolase; Ade,Adenine) [104].


from the adenine derivative 149 (ara-NPA), only the cytosine derivative 150(ara-NPC) has been reported so far, which was evaluated only for its antitumoractivity. Whereas the ara-NPA was obtained from a divergent approach startingfrom NPA in four steps, the ara-NPC was obtained from a 2,2′-anhydro-uridineintermediate, which was the by-product of 2′-deoxygenation reaction. Consider-ing the poor versatility of the reported approaches, Chu and coworkers decidedto develop a convergent strategy for the synthesis of ara-neplanocin A analogueshave been developed [106]. Microwave-assisted Mitsunobu reaction proved tobe an essential tool both for the 2′-β-hydroxy inversion and for the couplingreaction with the heterocyclic bases, as shown in Scheme 1.23. The exploitationof the present approach allowed generating a family of ara-neplanocins of whichbiological potential is still unexplored.

In comprehensive structure activity relationship studies of d- and l-neplanocinanalogs (vide supra , Scheme 1.6), Chu and coworkers showed that the cytosine(155) and 5F-cytosine analog (156) exhibited potent anti-orthopoxvirus as wellas anti-West Nile virus activities as shown in Table 1.11 [56,107].

On the basis of inhibitory activity of truncated cyclopentenyl cytosine againstadenosylhomocysteine hydrolase (SAH), its fluorocyclopentenyl pyrimidinederivatives (157 and 158) were efficiently synthesized from d-ribose viaelectrophilic fluorination as a key step by Jeong and coworkers (Scheme 1.24)[108]. The final nucleosides were evaluated for SAH inhibitory activity, amongwhich the uracil derivative 157 showed significant inhibitory activity (IC50 8.53

192 193 194 195

196197154

Scheme 1.23 Synthesis of ara-neplanocin A (3) analogues under subzero microwave-assisted conditions [106]. Reagents and conditions: (a) BzCl, pyridine, rt, 2 h; (b) Dowex,MeOH, MW, open vessel, 65◦C, 30 min (two cycles); (c) t-Bu2Si(OTf)2, DMF, 0◦C,30 min; (d) p-NO2PhCO2H, Ph3P, DIAD, benzene, MW, 180◦C, sealed tube, 5 min; (e)THF/ MeOH/H2O (3:2:1), LiOH, rt, 30 min; (f) Ph3P, DIAD, THF, MW, −40◦C, 100 W,5 min; (g) Et3N · 3HF, rt, 30 min; (h) Sat. NH3 in MeOH, rt, 1 h; (i) Sat. NH3 in MeOH,90◦C, 18 h; (j) 2N HCl/MeOH, 60◦C 15 h; (k) (i) formic acid, 90◦C, MW, 5 min; (ii) Sat.NH3 in MeOH, rt, overnight.


TABLE 1.11 Antiviral activity and toxicity of nepalnocin A (3), 155 and 156[56,107]

Activity (μg/mL) Toxicity (μg/mL)

Compds Virus MK2 Vero MK2 Vero

Nepalnocin A (3) Smallpox (7124) 0.10 0.03 23 50Smallpox (BSH) 0.10 0.14 36 10Cowpox 100 >100 100 20Monkeypox 0.26 0.21 42 57Vaccinia virus 2.62 >100 31 >100West Nile virus N/A >51 (μM) N/A 3.5 (μM)

Cytosine analog 155 Smallpox (7124) 0.08 <0.05 44 100Smallpox (BSH) 0.03 >100 3 30Cowpox 0.06 0.05 10 1Monkeypox 0.1 <0.05 21 4Vaccinia virus 0.12 0.04 36 40West Nile virus N/A 0.2 (μM) N/A 1.95 (μM)

5-F Cytosine analog 156 Smallpox (7124) 1.73 2.63 >100 39Smallpox (BSH) 0.51 1.17 >100 100Cowpox 0.43 1.35 70 >100Monkeypox 0.61 2.05 >100 100Vaccinia virus 0.53 1.46 100 >100West Nile virus N/A 15 (μM) N/A 27.4 (μM)

Scheme 1.24 Synthesis of truncated fluorocyclopentenyl pyrimidines (157 and 158)[108]. Reagents and conditions: (a) I2, pyridine, CCl4, rt, 1.5 h; (b) NaBH4, CeCl3-H2O,MeOH, 0◦C, 40 min; (c) TBDPSCl, imidazole, DMF, rt, overnight; (d) NFSI, n-BuLi,THF, −78◦C, 10 min; (e) TBAF, THF, rt, 1 h. (f) N3-benzoyl-uracil, PPh3, DEAD,THF, rt, 2.5 h; (g) NH3 in MeOH, rt, overnight; (h) 50% aq. TFA, rt, 3 h; (i) 4-chlorophenyldichlorophosphate, 1,2,4-triazole, pyridine, rt, overnight; (ii) 1,4-dioxane:28% NH4OH = 1:2, rt, 3 h.


μM). They were also evaluated for cytotoxic effects in several human cancercell lines such as fibro sarcoma, stomach cancer, leukemia, and colon cancer,but they did not show any cytotoxic effects up to 100 μM, indicating that4′-hydroxymethyl groups are essential for the anticancer activity.

A number of apio nucleosides, in which 4′-hydroxymethyl group are shifted toC3′ position have been synthesized to search for novel antiviral agents. Apio-ddA,mimicking the parent compound ddA, exhibited comparable anti-HIV activityto ddA. On the basis of these findings, apio-neplanocin A (159) and its ana-logues (160–162) [109] were asymmetrically synthesized, but unlike compounds3 and 135, these compounds did not show inhibitory activity against AdoHcyhydrolase. Fluoroneplanocin A (135, F-NPA), which was synthesized by Moon’sgroup, exhibited more potent inhibition of AdoHcy hydrolase than NPA (3), aswell as a significant antiviral activity, but also exhibited high cytotoxicity to thecells. Cytotoxicity of NPA (3) and F-NPA (135) seems to be derived from theinhibition of cellular polymerases by their 5′-triphosphate metabolites. On theother hand, it has been known that the inhibitory ability of AdoHcy hydrolase isderived from nucleosides by themselves, such as NPA (3) and F-NPA (135), nottheir triphosphates. Therefore, nucleosides, which show potent inhibitory abil-ity against AdoHcy hydrolase but which cannot be phosphorylated by kinases,have been considered a promising target for the development of new antiviralagents. On the basis of these findings, apio fluoroneplanocin A (apio F-NPA, 163)[110] and its uracil analog 164 was synthesized as a potential AdoHcy hydrolaseinhibitor.

On the basis of chemical and biological properties of C-nucleosides as well ascarbocyclic nucleosides, it was of interest to synthesize hybrid nucleosides, carbo-cyclic C-nucleosides. Although some carbocyclic nucleosides and C-nucleosidesare naturally occurring, so far no natural carbocyclic C-nucleosides have beenidentified. Therefore, it was of great interest to synthesize optically active carbo-cyclic C-nucleosides, possessing a cyclopentenyl moiety as analogs of neplanocin.Chu and coworkers reported the enantiomeric synthesis of purine and pyrimidinecyclopentenyl C-nucleosides 165–168 (Scheme 1.25) [111] from the key interme-diate, 2,3-(isopropylidenedioxy)-4-(trityloxymethyl)-4-cyclopenten-1-O-mesylate(204), which was prepared from d-ribose in eight steps and evaluated as poten-tial antiviral agents against HIV, SARSCoV, Punta Toro, West Nile, and cowpoxviruses. However, only 9-deazaneplanocin A (165) exhibited moderate anti-HIVactivity (EC502.0 ± 1.1) [111].

Recently, Chu’s group also reported that the triazole analog 169 (Scheme 1.26)was a potent antiviral agent against vaccinia virus with an EC50 of 0.4 μM,whereas the positive control cidofovir had an EC50 of 6 μM [112].

Recently, the same group also described the synthesis and antiviral activityof 7-deazaneplanocin A (7-DNPA) against orthopoxviruses (vaccinia and cow-pox virus) with EC50 values of 1.2 and 3.4 μM, respectively [113]. In addition,the further screening of the compound 170 revealed significant anti-HBV andanti-HCV activity with low cytotoxicity. In view of these interesting biological


Scheme 1.25 Enantioselective synthesis of purime cyclopentenyl C-nucleoside (165 and166) [111]. Reagents and conditions: (a) NCCH2CO2Et, NaH, THF, rt then to 55◦C,40 h; (b) (i) DIBAL-H, Et2O,−78◦C, 30 min; (ii) H2NCH2CN.H2SO4, NaOAc.3H2O,MeOH, rt, 24 h; (c) (i) ClCO2Et, DBU, CH2Cl2 0◦C then to reflux, 6 h; (ii) K2CO3,MeOH, rt, 1 h; (d) HC(=NH)NH2.AcOH, EtOH, reflux, 8 h; (e) (i) 12% HCl/MeOH,rt, 2 h; (ii) NaHCO3/MeOH, rt, 2 h; (f) (i) DIBAL-H, Et2O,−78◦C, 30 min; (ii)H2NCH2CO2Et.HCl, NaOAc.3H2O, MeOH, rt, 24 h; (g) (i) BzN = C = S, CH2Cl2,0◦C, 1 h; (ii) MeI, DBN, CH2Cl2, rt, 2 h; (iii) NH3/MeOH, 95◦C, 16 h.

Scheme 1.26 Synthesis of triazol analog [112]. Reagents and conditions: (a) (i) MsCl,Et3N, (ii) NaN3; (b) (i) methyl propiolate, CuI, Et3N; (ii) NH3/MeOH; (c) HCl.

results, it was of interest to explore the structure-activity relationships of 7-substituted-7-DNPA analogues as potential antiviral agents. These 7-substitutionswere introduced by using 7-substituted-7-deaza heterocyclic base precursors (F,Cl, Br, and I) or via substitution reactions after the synthesis of the carbo-cyclic nucleosides (Scheme 1.27) [114]. Among the synthesized compounds,170–176 exhibited significant anti-HCV activity (EC50 ranged from 1.8 to 20.1μM, Table 1.12) [114]. 7-DNPA 170 displayed anti-HCV activity with an EC50


Scheme 1.27 Synthesis of 7-deazaneplanocin analogues [114]. Reagent and conditions:(a) PPh3, DIAD, THF, room temp; (b) NH3, MeOH, 80◦C; (c) HCl, MeOH, THF,50◦C; (d) (i) NaH, p-methoxybenzyl chloride, DMF; (ii) HCl/MeOH/THF; (iii) Ac2O,pyridine; (iv) DDQ, CH2Cl2, H2O; (e) Bu3Sn(CHCH2), Pd(PPh3)4, Et3N, CuI, DMF; (f)TMSCtCH, Pd(PPh3)4, Et3 N, CuI; (g) Bu3SnCN, (PPh3)4Pd, ClCH2CH2Cl, reflux; (h)H2O2, NH4OH.


value of 2.5 μM without any cytotoxicity at a concentration up to 100 μM(Table 1.12). In the additional studies of the anti-HCV activity of 7-DNPA,170 is at least comparable to, if not better than, the positive controls, 2′-C -Me-cytosine (2′-C -Me-C) and 2′-F-C -Me-cytosine (2′-F-C -Me-C; Table 1.13)[114]. Compounds 172–174 showed moderate to potent anti-HBV activity (EC50

0.3–3.3 μM). Among synthesized nucleosides, the 7-ethylnyl substituted com-pound 174 exhibited interesting anti-HBV activity against wild type as well asseveral lamivudine and adefovir-associated HBV mutants, including rtL180M,rtM204I, rtM204V and rtN236T as shown in Table 1.14 [114].

1.2.3. Neplanocin F

Neplanocin F, a minor constituent of the family of neplanocin antibiotics, wassynthesized as a racemate from racemic cyclopentenone 23, which in turnwas available from d-ribonolactone by Marquez and co-workers as shown inScheme 1,28 [115]. The carbocyclic ring of neplanocin F corresponds to the

TABLE 1.12 Anti-HCV activity of 7-DNPA analogues based on an HCV RNAreplicon assay and cytotoxicity [114]

Anti-HCV activity, Cytotoxicity,Compounds EC50 (μM) CC50 (μM) SI

170 2.5 >100 >40171a 9.5% 112.7%172 2.1 15.0 7.1173a 49.2% 99.7%174 20.1 48.9 2.4175a 0.5% 103.3%176 1.8 11.8 6.62′-C -Me-adenine 0.15 >10.0 >66.7

aCompounds assessed at a single-concentration of 20 (μM) with percentage donating inhibition levelcompared to control cells.

TABLE 1.13 Additional studies of anti-HCV activity and cytoxicity of 7-DNPA 170in the hcv rna replicon huh7 assay in comparison to the known agents(2′-F-C -Me-C and 2′-C -Me-C) [114]

Anti-HCV activity, Cytotoxicity,Compounds EC50 (μM)a EC90 (μM)a CC50 (μM)b

170 0.9 8.2 >502′-F-C -Me-cytosine 1.7 5.3 >502′-C -Me-cytosine 3.5 11 >50

aEffective concentrations required for reducing HCV level by 50% and 90% in 5 days.bCytotoxicity concentration required for reducing the rRNA levels by 50% in 5 days.


TABLE 1.14 In vitro antiviral activity of compound 174 against HBV mutantsbased on the intracellular HBV DNA replication assay [114]

EC50 (μM)a

Strain Anti-HBV activity 174 3TC Adefovir

WT 2.5 0.2 1.3rtL180M 2.0 10.0 1.6rtLM/rtMV 2.8 >100 1.2rtM204I 3.0 >100 1.8rtM204V 2.1 >100 1.5rtN236T 5.6 0.3 7.7

artLM/rtMV) rtL180M/rtM204V double mutant.

Scheme 1.28 Synthesis of (±)-neplanocin F (223) [115]. Reagents and conditions: (a)(i) 40% TFA; (ii) Ac2O/Et3N/DMAP; (b) MeSO2C1/Et3N; (c) (i) LiN3/DMSO/110C;(ii) NaOMe/MeOH; (iii) NaH/DMF, BnBr; (d) [H2]/Lindlar catalyst/MeOH, rt;(e) 5-Amino-4,6-dichloropyrimidine/Et3N/n-BuOH/145◦C; (f) (i) (EtO)3CH/HCl/rt; (ii)BCl3/CH2Cl2/−78◦C; (g) NH3/MeOH/110◦C.

allylic rearranged isomer of neplanocin A. Regiospecific reduction of the racemiccyclopentenone 23 and protection of the resulting α-alcohol as a benzyl ether231 produced, after removal of the isopropylidene moiety, a compound (232)having allylic and homoallylic secondary alcohol functionalities. Differences inthe reactivity of these two secondary alcohols were successfully manipulatedto prepare the homoallylic substituted azide 234, which was then reduced andconverted to the desired adenine ring by conventional methods. In contrast to itsbioactive isomer, neoplanocin A, neplanocin F was devoid of cytotoxicity andin vitro antiviral activity (Figure 1.10).

Rodrıguez and coworkers described an improved procedure for the synthesisof (+)-neplanocin F (224) as shown in Scheme 1.29 [116]. The cyclopentenol,which was achieved from d-ribono-1,4-lactone 7, reacted with benzyl bromidein DMF at 0◦C to afford the corresponding benzyl ether 238 in good yield,


Figure 1.10 Neplanocin F and its analogues.

Scheme 1.29 Synthesis of (+)-neplanocin F [116]. Reagents and conditions: (a) ref[117]; (b) (i) 60% AcOH, 50◦C; (ii) trimethyl orthoformate, CH2Cl2, CAN, rt, 2h;(iii) DIBAL,−78◦C, 1h; (c) 6-chloropurine, DEAD, PPh3, THF, rt, 16h; (d) (i)CF3COOH, CH2Cl2, rt, 16h; (ii) MeOH/NH3, 70◦C, 5h; (iii) BCl3, CH2Cl2,−78◦C; (iv)MeOH,−78◦C.

which after treatment with acetic acid at 60◦C followed by MOM protection atthe allylic position exclusively, gave rise to compound 239. The 6-chloropurinewas then coupled with 239 via the Mitsunobu reaction to produce solely N-9alkylated product 240. The compound 240 was treated first with trifluoroaceticacid, NH3/MeOH and then followed by BCl3 in methylene chloride gave rise totarget molecule (+)-neplanocin F (224).

To evaluate the biological properties of such a carbanucleoside againstemerging pathogens, its enantioselective synthesis was reconsidered. In addition,(-)-neplanocin F (225) could be considered an attractive template giving anaccess, through appropriate chemical modifications, to new series of nucleosidederivatives displaying potential biological properties. Rodrıguez and coworkerssynthesized (-)-neplanocin F (225) as shown in Scheme 1.30, in which thesynthesis of (-)-neplanocin F (225) was stereospecificaly achieved from theknown cyclopentenone 14, which was obtained from commercially available2,3-O-isopropylidene-d-1,4-ribonolactone 7 according to literature protocols[118]. Briefly, treatment of 14 with [(benzyloxy)methyl](tributyl)stannane in thepresence of n-BuLi in THF at−78◦C yielded stereoselectively the 1,2-additionproduct, which on benzoylation provided compound 241. Palladium-catalyzedrearrangement of 241 gave the corresponding isomeric allylic benzoate 242


Scheme 1.30 Synthesis of (-)-neplanocin F (225) [118]. Reagents and conditions: (a)(i) BnOCH2SnBu3, n-BuLi/THF, −78◦C; (ii) BzCl/pyridine, rt; (b) (i) PdCl2[CH3CN]2,p-benzoquinone/THF, 85◦C; (ii) NaOH 1%, MeOH, rt; (iii) BnBr/NaH/DMF, rt; (c) (i)60% AcOH, 50◦C; (ii) HC(OMe)3Ce(NH4)2(NO3)6, CH2Cl2, rt; (iii) DIBAL,−78◦C; (iv)Tf2O/DMAP/CH2Cl2, 0◦C; (d) adenine, K2CO3, 18-crown-6 ether, DMF, 60◦C; (e) (i)18% TFA in CH2Cl2, rt; (ii) BCl3, CH2Cl2,−78◦C.

with good yield. Introduction of the heterocyclic base was achieved via thepreparation of the triflate 243, which on reaction with adenine gave solelythe N-9 alkylated product. Removal of the MOM group by treatment withTFA/CH2Cl2 as well as the two benzyl ethers by treatment with BCl3/CH2Cl2at −78◦C provided the target molecule (-)-neplanocin F (225).

Chu and coworkers describe the synthesis and antiviral activity of (±)-5′-deoxyneplanocin F analogues 226–230 as shown in Scheme 1.31 [119]. Amongthe compounds 226–230, 5′-deoxyneplanocin F adenine (226) exhibited moderateanti-HIV activity (EC50 13.8 μM) in human lymphocytes without any markedcytotoxicity up to 100 μM.

1.2.4. Carbovir, Abacavir and Their Analogs

1.2.4.1. Carbovir and Abacavir Vince and coworkers first reported (±)-Carbovir with potent anti-HIV activity and low cytotoxicity [120]. Although the(-)-d form is approximately 75-fold more potent than its enantiomer, triphosphatesof (-)-d and (+)-l-carbovir are equally active against HIV reverse transcriptase[120,121]. Therefore, the reduced activity seen with (+)-l-carbovir in vitro could,in part, be attributed to low levels of conversion to its phosphates. Cytosolic 5′-nucleotidase converts (-)-d-carbovir to its triphosphate, which can incorporateinto viral DNA and disturb viral replication but have no interaction with hostcell DNA polymerase α, β, and γ. Unfortunately, the low aqueous solubility andpoor oral bioavailability, as well as inefficient central nervous system penetration,prevented it from further developing as anti-HIV agents [122–124]. To improve


Scheme 1.31 Synthesis of (±)-neplanocin F analogues (226–230). Reagents andconditions: Synthesis of adenine derivatives. Reagents and conditions: (a) (i)(Boc)2O, DMAP, Et3N, CH2Cl2, rt, 3 h; (ii) m-CPBA, CH2Cl2, refluxed, 2 h; (b) MeONa,MeOH, rt, overnight; (c) (i) CF3COOH, CH2Cl2, 0◦C, 2.5 h; (ii) Et3N, EtOH, 4,6-dichloro-5-nitropyrimidine, 0◦C, 2 h; (d) (i) SnCl2.2H2O, EtOH; 80◦C, 10 min; (ii)CH(OCH3)3, CH3SO3H, rt, 1 h; (e) (i) DIBAL-H, THF, CH2Cl2,—78◦C, 4 h; (ii)NH3/CH3OH, 110◦C, 24 h; (f) p-Nitrobenzoic acid, DIAD, Ph3P, THF, 0◦C, 6 h; (g) (i)DIBAL-H, BF3.Et2O, CH2Cl2,—78◦C, 2.5 h; (ii) Ac2O, pyridine, CH2Cl2, 0◦C, 3 h; (h)(i) CF3COOH, CH2Cl2, 0◦C, 2.5 h; (ii) N -(4,6-dichloro-5-nitropyrimidin-2-yl)acetamide,Et3N, EtOH, 3 h; (i) (i) SnCl2.2H2O, EtOH, 60◦C, 30 min; (ii) CH(OCH3)3, CH3SO3H,rt, 3 h, in two steps; (iii) HSCH2CH2OH, CH3ONa, CH3OH, reflux, 7 h; (j) (i)CF3COOH, CH2Cl2, 0◦C, 2.5 h; (ii) Et3N, methacryloyl isocyanate, rt, overnight; (k)(i) CF3COOH, CH2Cl2, 0◦C, 2.5 h; (ii) Et3N, 3-methoxy-2-methylacryloyl isocyanate,toluene, 0◦C, 3 h; (l) NH4OH, CH3OH, 85◦C, 24 h.


Figure 1.11 Carbovir and abacavir.

its preclinical profiles, a number of prodrug of carbovir were prepared, includinga 6-cyclopropylamino substituted analog, which was later known as the clinicallyuseful drug abacavir (Figure 1.11) [123].

Abacavir exhibits significant anti-HIV activity with low cytotoxicity [123],and more important, it has excellent pharmacokinetic as well as toxicologi-cal profiles. The unique activation process of abacavir to its triphosphate isdescribed in Figure 1.12: (1) adenosine phosphortransferases are responsible forthe monophosphorylation; (2) deamination to carbovir monophosphate is per-formed by cytosolic deaminase; and (3) cytosolic enzymes are responsible forthe conversion of carbovir monophosphate to triphosphate [125].

In December 1998, abcarvir was approved by the FDA for the treatment ofHIV infection under the trade name of Ziagen™. It has been used in combinationswith AZT and 3TC (Trizivir™) and later with 3TC (Epizcom™).

Vince’s procedure [120,126] (Scheme 1.32) utilized the racemic compound245 as the starting material, which underwent a sequence of hydroly-sis/esterification/reduction/deprotection to generate the key intermediate 258.

Figure 1.12 Activation pathway of abacavir [125].

Scheme 1.32 Synthesis of racemic carbovir [120,126]. Reagents and conditions: (a) (i)H+; (ii) MeOH, H+; (iii) esterification; (b) (i) LiBH4; (ii) H+; (c) base construction.


The compound 258 was converted to the target nucleoside (±)- as well as otheranalogs by reported methodology [127].

Later, it was found that bicycle compound (±) 245 can be resolved by usingPsedomomonas solanacearum [128,129]. Pig liver esterase (PLE) distinguishesthe two enantiomers of (±) 257 [130], and adenosine deaminase recognizes either(-) or (+) 261 at different temperature. By using these enzymatic methods, opti-cally pure (-) 255 could be prepared (Scheme 1.33) [126].

Enantioselective synthesis of (-)-carbovir was reported by Jones and coworkersfrom Glaxo [131]. Starting from the same chiral epoxide 262, two different routeswere developed, in which the first one (route a) generated a double bond at thenucleoside level, whereas the second one (route b) produced a vinyl epoxide268 at the very beginning of the sequence. However, both routes needed anextra step to remove the 6′-hydroxyl group by Barton-McCombie radical reaction(Scheme 1.34).

Crimmins’s approach (Scheme 1.35) [132] to prepare (-)-carbovir relies onthe Trost’s palladium-catalyzed nucleophilic coupling reaction [44]. The racemichomoallylic chloride 273 was converted to optically active endocyclic vinyl com-pound 277 in four steps by using a chemical resolution method. The compound277 was then coupled with base moiety under Trost’s conditions (allylpalladiumdichloride dimer/Ph3P) to give nucleoside 278, which was further hydrolyzed toafford the target compound 255.

Scheme 1.33 Enzymatic resolution reactions in the synthesis of carbovir [126,128–130].Reagents and conditions: (a) Psedomomonas solanacearum; (b) PLE; (c) adenosine deam-inase.


Scheme 1.34 Synthesis of carbovir from epoxide 262 [131]. Reagents and conditions: (a)PMBCl, NaH; (b) Ph3P, DIAD, base; (c) (i) PhOCSCl, DMAP, (ii) BuSnH, AIBN; (d)DDQ; (e) (i) MsCl, DMAP, (ii) NaOCH2CH2OMe; (f) base derivation and deprotection;(g) (i) MsCl, DMAP, (ii) TBAF; (h) BF3 • Et2O, Ac2O; (i) NaNO2, AcOH.

Scheme 1.35 Synthesis of carbovir from chloride compound 273 [132]. Reagents andconditions: (a) Mg, CO2, recrystallization as (-)-(α-phenylethyl)amine salt; (b) LAH; (c)BuLi, CO2, I2; (d) DBU; (e) 2-amino-6-chloropurine, allylpalladium chloride dimmer,PPh3; (f) NaOH.


Scheme 1.36 Chiral auxiliary assisted asymmetric synthesis of carbovir [132].Reagents and conditions: (a) n-BuLi, pentenoic pivalic mixed anhydride; (b)Bu2BOTf, Et3N, CH2 = CHCHO; (c) Grubbs’ catalyst; (d) LiBH4; (e) Ac2O, Et3N,DMAP; (f) 2-amino-6-chloropurine, Pd(PPh3)4.

A chiral auxiliary assisted asymmetric synthesis of carbovir and abacavir wasaccomplished by the same group (Scheme 1.36) [132]. Pentenoic pivalic mixedanhydride was coupled with 279 to obtain 280, which was subjected to the synaldol condensation to provide the diene 281. A metathesis/reduction/esterificationsequence afforded 284, which underwent Pd(0) catalyzed a coupling reactionand a base derivation to smoothly generate abacavir or carbovir. More recently,Crimmins and coworkers optimized the selectivity of the coupling reaction by asolid phase synthesis [133].

Trost and coworkers reported improvements in the asymmetric desymmer-trization reaction in 1996 (Scheme 1.37) [134]. A unique ligand 288, a tertiaryamine base (pempidine), and a modified guanine equivalent 289, were employedin the coupling step. By this method, both enantioselectivity and regioselectivitywere significantly improved.

Scheme 1.37 Improved asymmetric synthesis of carbovir [134]. Reagents and conditions:(a) (C3H5PdCl)2, pempidine, 288, 289, DMSO/THF; (b) [Pd2(dba)3]-CHCl3, Ph3P; (c)(i) tetramethylgunidine; (ii) tetrabutylammonium oxone, Na2CO3; (iii) Ca(BH4)2; (iv)NH4OH.


Riera and coworkers developed a new enantioselective approach to (-)-carbovirand (-)-abacavir from the Pauson–Khand (PK) adduct 292 (Scheme 1.37) [135].The chiral cyclopentenone 292 is readily accessible in enantiomerically pureform via PK reaction of trimethylsilylacetylene 291 and norbornadiene, using N -benzyl-N -diphenylphosphinotert-butyl-sulfinamide as a chiral P,S ligand, show-ing its usefulness as a cyclopentenone synthon [136]. Starting from PK adduct292, the hydroxymethyl group was efficiently added via photochemical conjugateaddition. Protection as a triisopropylsilyl ether followed by the retro-Diels-Alderreaction afforded the protected hydroxymethyl cylopentenone 295. Diastereose-lective reduction to the corresponding allyl alcohol and palladium catalyzed sub-stitution afforded an advanced intermediate 297, which was converted into enan-tiomerically pure (-)-carbovir and (-)-abacavir as shown in Scheme 1.38 [135].

Chu and coworkers developed another sequence to prepare l-carbovir analogsas shown in Scheme 1.39 [137]. Starting from l-enone 16, which could be readily

Scheme 1.38 Synthesis of (-)-carbovir and (-)-abacavir from enantiomerically purePauson-Khand (PK) adduct 292 [135]. Reagent and conditions: (a) (i) KCN, NH4Cl,DMF, H2O, rt, 3h; (ii) 2,2-Dimethyl-1,3-propanediol, p-TsOH cat., toluene, reflux, 12h;(b) (i) DIBAL-H, THF, 0◦C, 1h; (ii) NaBH4, MeOH, 0◦C, 2h; (iii) HCl (1M), acetone,rt, 3h; (iv) TIPS-Cl, imidazole, DMF, rt, 3h; (c) AlMeCl2, CH2Cl2, 55◦C, 2h; (d) (i)DIBAL-H, THF,−78◦C, 1h; (ii) ethyl chloroformate, pyridine, CH2Cl2, rt, 40 min; (e) 6-chloro-9H-purin-2-amine, NaH, Pd(PPh3)4, DMF, 45◦C, 3h; (f) cyclopropyl amine, EtOH,reflux, 4h; (g) TBAF, THF, rt, 1h; (h) NaOH, H2O, 100◦C, 5h.


Scheme 1.39 Synthesis of l-carbovir analogs [137]. Reagents and conditions: (a) (i)tert-butyl methyl ether, sec-BuLi, t BuOK, CuBr · Me2S; (ii) NaBH4, CeCl3 · 7H2O; (iii)BzCl; (iv) HCl; (b) (i) CH(OMe)3/Py/p-TSA; (ii) Ac2O, 120◦C–130◦C; (iii) NaOH; (c)Mitsunobu couplings and base derivation.

synthesized from d-ribose in several steps (vida supra , Scheme 1.2), the diol300 was obtained via a four-step sequence in high yield. Treating the diol 300under pyrolytic elimination condition successfully provided the allylic alcohol301. Condensation of the allylic alcohol 301 with proper base moieties, suchas 6-chloropurine, under the Mitsunobu conditions provided desired nucleosides,which was subjected to base derivation and deprotection steps to furnish thetarget l-carvovir analogs (302). This scheme is very straightforward and suitablefor scale up.

1.2.4.2. Carbovir and cbacavir analogs In light of the fact that noraris-teromycin and norneplanocin increased selectivity, Huang and coworkers [138]prepared racemic 5′-norcarbovir 303 and 5′-norabacavir 304 with the anticipationthat these two desmethylene derivatives might have anti-HIV activity similar tocarbovir and abacavir. However, it was found that only norabacavir 304 showedmoderate anti-HIV-1 activity with an EC50 5.0 μg/mL, but it was toxic to hostcells. The synthesis of both compounds started from an epoxide 337, whichunderwent the Trost type coupling reaction to give nucleoside 338. Further basederivation afforded desired 303 and 304 (Scheme 1.40).

Katagiri and Kaneko reported (-)-BCA 305, an unnatural l-carbocylic nucle-oside as a potent anti-HIV-1 agent with an EC50 of 0.71 μM in MT-4 cells[139–141]. However, no updated information of this compound is available(Figure 1.13).

The enzymatic resolution by Rhizopus delemar lipase (RDL) was appliedin the synthesis of optically pure intermediate 339, which was then oxidizedand underwent Curtius rearrangement to give carbamate 341. A deprotectionstep, followed by a well-known base construction procedure, afforded the target

Scheme 1.40 Synthesis of norcarbovir and norabacavir [138]. Reagents and conditions:(a) Pd(OAc)2, TPP, base; (b) NaOH; (c) EtOH, cyclopropylamine.


Figure 1.13 Carbovir and abacavir analogs.

nucleoside 305 (Scheme 1.41). Nucleosides having a 4′-carbon-substituenthave attracted much attention due to the reported potent anti-HIV activityof 4′-cyanothymidine and 4′-ethynyl-2′-deoxycytidine. Also, the 4′-ethynylanalogue (4′-Ed4T) of anti-HIV agent stavudine (d4T) was found to show ahigher activity against HIV than the parent compound, stavudine. Based on

Scheme 1.41 Synthesis of (-)-BCA [141]. Reagents and conditions: (a) Rhizopus delemarlipase; (b) (i) MOMCl; (ii) K2CO3/MeOH; (iii) PCC and then NaClO2; (iv) DPPA; (c)(i) KOH; (ii) Base construction; (iii) NH3.


these facts, Kumamoto and his coworkers synthesized 4′-branched (±)-BCAderivatives (4′-hydroxymethyl 306, 4′-vinyl 307, and 4′-ethynyl 308) [142]that can be regarded as a hybrid of 305 and 4′-carbon-substituted nucleosides(4′-cyanothymidine, 4′-ethynyl-2′-deoxycytidine, and 4′-ethynyl analogue (4′-Ed4T)). Compounds 306–308 were tested for their potential to inhibit replicationof HIV-1 and HCV in cell culture, but no significant inhibition was observed.

Toyota and coworkers reported the synthesis of 3′-fluorine substituted carboviranalog 309 in 1998; however, no biological data were provided [143]. Since then,Chu and coworkers have accomplished the asymmetric synthesis of both 2′- and3′-fluorine substituted analogs including d- and l-nucleosides, and antiviral datawere reported (Table 1.15) [144,145]. Among 2′-F substituted nucleosides, l-adenine derivative 310 was the most potent anti-HIV agent with an EC50 of 0.77μM without toxicity at a concentration up to 100 μM. In the other series, D-3′-Fguanosine analog 311 exhibited marked anti-HIV activity (EC50 0.41 μM) withmarginal toxicity. Interestingly, this compound (311) showed significant cross-resistance to the HIV M184V mutant (FI 38.8; Table 1.15), which was believedto be the result of template/primer realignment as indicated in the molecularmodeling studies [145].

To synthesize 2′-F compounds, 2′-F-allylic alcohol 347 was prepared from acommon enone intermediate 14 in 13 steps (Scheme 1.42). Compound 14 wasthen condensed with bases under Mitsunobu conditions and subjected to basederivations to afford target nucleosides. However, the 3′-F analog of 347 wasdifficult to prepare due to its instability. Therefore, in this series the double bondwas generated in the final stage of the whole sequence by microwave-assistedreactions. The l-series compounds were prepared in the same manner.

Modifications of 4′- and 6′-position of carbocyclic ring have also generatedcompounds 314 [146,147] and312–313 [148] (Scheme 1.43). However, neitherof them showed significant biological activity.

Two-directional ring-closing metathesis (RCM) was applied successfully forthe synthesis of 4′-vinylated carbocyclic nucleoside analogues from the trivinylintermediate 362, which was readily made using a sequential Claisen rearrange-ment and RCM starting from Weinreb amide 360, as depicted in Scheme 1.44

TABLE 1.15 Antiviral activity of compounds 310 and 311 against HIV-wild type(xxBRU) and M184V mutant [144,145]

xxBRU M184V

Compds EC50 (μM) EC90 (μM) EC50 (μM) EC90 (μM) FIa

310 0.77 8.34 75.3 >100 983TCb 0.027 0.25 >100 >100 >100

311 0.098 0.58 3.8 14.9 38.8Carbovirc 0.087 0.27 0.20 1.1 2.3

aFI is the fold increase (EC50HIV-1M184V/EC50HIV-1xxBRU.)bPositive control for compound 310.cPositive control for compound 311.


Scheme 1.42 Synthesis of 2′-F and 3′-F d4 carbocyclic nucleosides [144,145]. Reagentsand conditions: (a) (i) tert-butyl methyl ether, sec-BuLi, tBuOK, CuBr · Me2S; (b) (i)NaBH4, CeCl3 · 7H2O; (ii) BnBr; (iii) HCl; (c) AIBBr, K2CO3; (d) LAH; (e) (i) TrCl, Py;(ii) PDC; (iii) DAST; (f) (i) tBuOK, THF; (ii) HCl; (iii) TBSCl, Im; (iv) Na/NH3 (liq.);(g) Mitsunobu conditions and base derivations; (h) TrCl, DMAP; (i) Super-hydride; (j)PDC; (k) neat DAST; (l) (i) TMSI; (ii) TBDPSCl, Im; (m) (i) Mitsunobu conditions andbase derivations; (ii) deprotection; (n) microwave assisted elimination, t BuOK, DMF.

Scheme 1.43 General schemes for the synthesis of compounds 312–314 [146,148].


Scheme 1.44 An efficient synthesis of 4′-vinylated carbocyclic nucleosides [149].Reagents: (a) Grubbs’ catalyst (II), CH2Cl2 α-isomer:X=H, Y=OH, β-isomer:X=OH, Y=H; (b) ClCO2Et, pyridine, DMAP; (c) nucleosidic bases, Pd2(dba)3 ·CHCl3, P(O-i-Pr)3, NaH, THF/DMSO; (d) (i) TBAF, THF; (ii) 2-mercaptoethanol,NaOMe, MeOH; (iii) CH3COOH; (ii) and (iii) applicable only for compound 317.

[149]. An antiviral evaluation of the compounds 315–317 against various virusessuch as HIV-1 (MT-4 cells), HSV-1 and HSV-2 (CCL 18 cells), and HCMV(AD-169) revealed that the guanine analogue 317 exhibited moderate anti-HIVactivity in the MT-4 cell line (EC50 = 10.2 μM; Table 1.16) [149]. It is believedthat the arrangement in the carbocyclic guanine nucleoside analogue 317 may beconformationally similar to that in natural nucleosides containing ribose. Hence,this arrangement will enhance the level of phosphorylation by kinase to producethe active monophosphate form.

Based on these findings of branched nucleosides, a class of nucleosides com-prising 4′α-quaternary carbocyclic nucleosides with an additional methyl groupat the 6′-position was synthesized (Scheme 1.45) [150]. The quaternary car-bon at the 4′-position of carbocyclic nucleoside was installed successfully via aClaisen rearrangement. The stereocontrolled construction of a methyl group inthe 6′α-position was directed through the Felkin-Anh rule. A Bis-vinyl compound368 was cyclized successfully using Grubbs’ catalyst II to provide a carbocycle

Scheme 1.45 Stereocontroled synthesis of quaternary carbon containing novel abacaviranalogue 318 [150]. Reagents and conditions: (a) second-generation Grubbs’ catalyst,CH2Cl2, reflux, overnight, major 369; (b) (i) ClCO2Et, DMAP, pyridine, rt, overnight;(ii) 2-amino-6-chloropurine, Pd2(dba)3 · CHCl3, P(O-i-Pr)3, NaH, THF/DMSO, reflux,overnight; (iii) TBAF, THF, rt; (c) cyclopropylamine, EtOH, reflux.


TABLE 1.16 Antiviral activity of compounds 315–318, 321–324, and 326–329[149,150,152,154]

HIV-1 HSV-1 HSV-2 HCMV CytotoxicityCompounds EC50 (μM) EC50 (μM) EC50 (μM) EC50 (μM) CC50 (μM)

315 95 >100 >100 >100 95316 77.9 >100 90.5 23.7 >100317 10.2 65.8 >100 41.5 99318 10.67 >100 >100 >100 >100321 90 >100 >100 >100 90322 45.7 98 >100 19.3 98323 99 >100 >100 99 >100324 11.91 88 >100 36.4 99326 13.1 67.3 >100 ND >100327 72.1 >100 >100 ND >100328 66.2 99 99 ND 99329 34.8 >100 >100 ND >100AZT 0.01 ND ND ND 4.50GCV ND 2.2 2.2 0.8 >10ACV ND 0.2 ND ND >100

Note: AZT, azidothymidine; GCV, ganciclovir; ACV, acyclovir. ND, not determined. EC50 (μM).concentration required to inhibit 50% of the virus induced cytopathicity. CC50 (μM), concentrationrequired to reduce the cell viability by 50%.

nucleus for the target compound. The antiviral evaluations against various virusessuch as HIV-1, HSV-1, HSV-2, and HCMV were performed. The synthesizedcompound 318 showed moderate anti-HIV activity (EC50 10.67 μM, MT-4 celllines) without any cytotoxicity up to 100 μM (Table 1.16) [150].

Chu and coworkers reported the hitherto unknown synthesis of 3-deazacarbovir319 [151] and its adenosine analogue 320 as shown in Schemes 1.46 and 1.47[151]. The major highlight in the synthesis of adenosine analogs was to use6-N,N -diboc protected 3-deazapurines for regioselective Mitsunobu coupling aswell as unexplored palladium catalyzed coupling with these substrates. Synthesis

371 372 373 319

Scheme 1.46 Synthesis of 3-deazacarbovir 319 [151]. Reagents and conditions: (a) (i)Diphenyl carbamoyl chloride, diisopropyl amine, pyridine, 1 h, 85%; (ii) K2CO3, MeOH,45 min; (b) 374, Pd(PPh3)4, THF/DMSO (1:1), rt, 45 min–1 h; (c) NH3, MeOH, 80◦C,16 h.


375 320374

Scheme 1.47 Synthesis of 3-deazaadenosine analogue 320 [151]. Reagents and condi-tions: (a) N,N -di-Boc-3-deazaadenine, Pd(PPh3)4, THF/DMSO (1:1), rt, 1 h; (b) (i) DCM,TFA, rt, 5–7 h; (ii) K2CO3/MeOH, 1 h, rt.

of 3-deazacarbovir 319 has been accomplished by the regioselective palladiumcatalyzed coupling of 6-N,N -diphenylcarbamoyl protected 3-deazaguanine base372 with dicarbonate 374. All the target nucleosides were screened for anti-HIV-1 activity, and none of them have significant activity as well as toxicity up to100 μM.

Hong and coworkers developed a synthetic route for carbocyclic versionsof stavudine analogues (321–324; Scheme 1.48) [152]. The construction of anethynylated quaternary carbon at the 4′-position of carbocyclic nucleosides wasaccomplished using Claisen rearrangement and ring-closing metathesis (RCM) ofdienyne 377 as key transformations. The synthesized compounds 321–324 wereevaluated against HIV-1 (MT-4 cells), HSV-1 (CCL-81 cells), HSV-2 (CCL-81 cells), and HCMV (AD-169, Davis cells). Among them, only the guanineanalogue 324 is moderately active against HIV-1 in the MT-4 cell line (EC50

11.91 μM), and the thymine analogue 322 showed weak antiviral activity againstHCMV (Table 1.16).

Scheme 1.48 Synthetic route of carbocyclic versions of stavudine analogues (321–324)[152]. Reagents: (a) Grubbs’ catalyst (II), CH2Cl2; (b) ClCO2Et, pyridine, DMAP;(c) (i) pyrimidine nucleosidic bases, Pd2(dba)3 · CHCl3, P(O-i-Pr)3, NaH, THF/DMSO;(ii) TBAF, THF; (d) 2-amino-6-chloropurine, Pd2(dba)3 · CHCl3, P(O-i-Pr)3, NaH,THF/DMSO; (e) (i) TBAF, THF; (ii) 2-mecaptoethanol, NaOMe, MeOH; (iii) CH3COOH.


Synthesis of (±)-4′-ethynyl-5′,5′-difluoro-2′,3′-dehydro-3′-deoxy-carbocyclic-thymidine (325) [153] was carried out by Kumamoto and coworkers, as shownin Scheme 1.49. The difluoromethylylidene group of 325 was constructed by theelectrophilic fluorination to the cyclopentenone 382 by using Selectfluor to theenolate derived from 381. The resulting difluoroenone 383 was stereoselectivelyreduced to the allyl alcohol followed by manipulation of the methoxycarbonylgroup of 383 allowed to prepare 384. Introduction of thymine base was inves-tigated based on the Mitsunobu reaction by employing cyclopentenyl allyl alco-hols variously substituted at the 4-position. It was found the 4-methoxycarbonylderivative 383 gave the highest selectivity in terms of both regio- and stereo-chemistry. A brief conformational analysis of 325 was also carried out based onits X-ray crystallographic data.

Hong and coworkers [154] synthesized 4′-modified cyclopentenyl pyrimidineC-nucleosides via C–C bond formation using SN2 alkylation via the key interme-diate mesylates 388 and 389 (Scheme 1.50), which were prepared from acyclicketone derivatives. When antiviral evaluation of synthesized compound was per-formed against various viruses such as HIV-1, HSV-1 and HSV-2, isocytidineanalogue 326 found moderate active against HIV-1 in CEM cell line with anEC50 13.1 μM [154] (Table 1.16).

Qing and coworkers stereoselectively synthesized 3′,3′-Difluoro-2′-hydroxymethyl-4′,5′-unsaturated carbocyclic nucleosides 330–332 [156] fromester 400, which can be conveniently prepared from 2,3-isopropylidene-d-glyceraldehyde 398 (Scheme 1.51). The whole synthesis highlighted thestereoselective Reformatskii-Claisen rearrangement, ring-closing metathesis(RCM), and palladium-catalyzed allylic alkylation, in which the regioselectivitywas reversed from that of nonfluorinated substrates.

Scheme 1.49 Synthesis of (±)-4′-ethynyl-5′,5′-difluoro-2′,3′-dehydro-3′-deoxy-carbo-cyclic-thymidine (325) [153]. Reagents and conditions: (a) PDC, CH2Cl2(78%); (b) (i) TMSCl, Li/HMDS, THF,−78◦C; (ii) Selectfluor, MeCN; (c) (i)NaBH4, CeCl3.7H2O, MeOH, THF,−78◦C; (ii) DIBAL-H, CH2Cl2,−78◦C; (iii)P(O)(OMe)2C(N2)COMe, K2CO3, MeOH; (d) (i) N3-benzoylthymine, DIAD, Ph3P,THF; (ii) NH3/MeOH.


Scheme 1.50 Synthesis of 4′-modified cyclopentenyl pyrimidine C-nucleosides via C–Cbond formation using SN2 alkylation via the key intermediate mesylates 388 and 389[154,155]. Reagents and conditions: (a) MsCl, TEA, CH2Cl2; (b) (i) NaH, CH2(CO2Et)2,THF; (ii) LiCl, DMSO; (c) (i) LDA/THF, HCOEt; (ii) H2NC(=NH)NH2.carbonate,NaOEt/EtOH, for comp 394 and 396; (d) (i) LDA, HCOEt; (ii) MeI, DMF; (e)NH2CONH2, t-BuOK, THF, for comp 397 and 395; (f) TBAF, THF.

Boojamara and his coworkers have discovered a diphosphate of a novelcyclopentyl based nucleoside phosphonate with potent inhibition of HIV reversetranscriptase (RT) (336, IC50 = 0.13 μM) [157]. In cell culture the parentphosphonate diacid 335 demonstrated antiviral activity EC50 = 16 μM, aprodrug of which is currently under clinical investigation and within fivefold oftenofovir (PMPA). A favorable resistance profile toward K65R was achievedbut was offset by a decreased susceptibility by the prevalent M184V mutant.Further development of 335 toward optimal phosphonamidate prodrugs willlikely allow efficient delivery the phosphonate to the lymphatic system andprovide a novel nucleotide RT inhibitor for the treatment of HIV.

1.2.5. Entecarvir and Analogs

1.2.5.1. Entecavir Because of the slow kinetics of viral clearance and thespontaneous genetic variability of hepatitis B virus (HBV), antiviral therapy ofchronic hepatitis B remains a clinical challenge. Despite the recent develop-ment of lamivudine, adefovir dipivoxil, and pegylated interferon alpha for thetreatment of chronic HBV infection, there are still critical need for new antivi-ral compounds. Entecavir (416) (2′-deoxy carbocyclic guanosine analog with anexocyclic double bond on the 6′-position; Figure 1.14) has been approved inUnited States for the therapy of chronic hepatitis B [158]. Extensive studies havebeen performed to characterize its antiviral activity in enzymatic and tissue cul-ture models, as well as in animal models of HBV infection. In clinical trails,entecavir administration was associated with a significantly more potent viral


Scheme 1.51 Synthesis of 3′,3′-Difluoro-2′-hydroxymethylunsaturated carbocyclic nucle-osides (330–332; R = Me) [156]. Reagents and conditions: (a) (i) (EtO)2POCH2CO2Et,NaH, THF; (ii) AcOH/H2O; (iii) Ag2O, BnBr, CH2Cl2; (b) (i) DIBAL-H, CH2Cl2,−78◦C;(ii) ClF2COOH, CH3Cl, 90◦C; (c) (i) Zn, TMSCl, MeCN, 105◦C; (ii) EtOH, H2SO4,50◦C; (d) HN(Me)OMe.HCl, n-BuLi, THF,−78◦C; (e) (i) allylmagnesium chloride,THF,−78◦C; (ii) Et3N, THF; (iii) NaBH4, CeCl3.7H2O, MeOH, 0◦C; (f) Grubbs CatalystII, toluene, 100◦C; (g) (i) MeOCOCl, pyridine, CH2Cl2, 0◦C; (ii) 3-benzoyaluracil or 3-benzoyalthymine, Pd(PPh3)4, PPh3, THF, 60◦C; (h) (i) NH3, MeOH; (ii) BCl3, CH2Cl2,−40◦C; (i) (i) NaIO4, MeOH, H2O; (ii) NaBH4, 0◦C.

416 Entecavir

N

NHN

NH2

O

N6′

1′

2′3′

4′

5′

OH

HO

Figure 1.14 Structure of entecavir (416).


suppression compared to lamivudine and a significant advantage in terms of bio-chemical and histological improvement in comparison to lamivudine. Entecavirwas tolerated as well as lamivudine in the phase III trials. No case of resistancewas detected after two years of therapy in nucleoside naıve patients. Treatment ofpatients with lamivudine failure requires a higher dosage of entecavir (1.0 mg vs.0.5 mg for drug naıve patients) and induces a significant decline in viral loads.The availability of entecavir as a new treatment option is providing clinciansmore choice to keep both viral replication and liver disease under control. Thisprovides new hope for improved treatment concepts for chronic HBV infection.

Entecavir undergoes rapid intracellular phosphorylation to the active triphos-phate form [159], which inhibits HBV replication by acting as a nonobligatechain terminator in priming, at the RNA-dependent DNA synthesis as well asDNA-dependent DNA synthesis stages [160]. In vitro studies demonstrated thatentecavir was the most potent inhibitor of HBV replication in comparison to otheranti-HBV agents (EC50 3.75 nM in HepG 2.2.15 cell assay) [161–164]. Resis-tance studies indicated that 3TC/FTC-double mutant (rtM204V/I and rtL180M)reduced the viral susceptibility to entecavir by 20 to 30-fold (Table 1.17), whereasadefovir-resistant mutant (rtN236T) retained full susceptibility to this compound[165,166].

In the clinical trial, a small proportion (6%) of patients developed entecavir-associated mutation after a long-term administration. However, most of them didnot experience confirmed virological rebounds [167,168]. The main resistancemutations of entecavir are rtT184G, rtS202I, and rtM250V on a background oflamivudine-resistant mutations [169,170].

Phase II trials showed that entecavir administration at dose of 0.5 or1.0 mg/day for 4 weeks produced significant reduction of serum HBV DNA inboth nucleoside-naıve and lamivudine-experienced patients, and the viral loadrebound was slower than lamivudine treatment after cessation of the therapy[171–174]. In three randomized, multicenter phase III trials, nucleoside-naıveor -experienced patients (HBeAg positive or negative) were included. After2 years of administration, 81% of entecavir recipients (0.5 mg/day) had aviral load below 300 copies/mL versus only 39% in the lamivudine recipients

TABLE 1.17 Antiviral activity of entecavir (416) and 3TC against HBV (wild typeand mutations) [165]

EC50 (μM)

HBV Entecavir (416) 3TC

Wild type 0.0004 0.56Mutants rtL180M 0.0005 >10

rtM204I 0.06 >80rtM204V 0.003 33

rtL180M/rtM204I 0.25 >10rtL180M/rtM204V 0.28 >80


(100 mg/day), whereas ALT normalization ratio and clearance of HBsAg ratiowere 79% versus 68% and 5% versus 3%, respectively. In addition, entecaviradministration at 1.0 mg/day produced significant viral load reduction inlamivudine refractory patients in comparison to the control group [172]. Basedon these impressive results from the clinical trials, the U.S. FDA has approved0.5 and 1.0 mg doses of entecavir as an oral, once-daily drug (Baraclude®) forthe treatment of chronic hepatitis B infection.

Recently Huang and his coworkers studied genotypic evoluation of HBV quasispecies in nucleoside-/nucleotide-naıve patient who developed resistance to ente-cavir [175]. The lamivudine resistant quasi species (rtM204V ± rtL180M), absentat baseline, were emerged as early as 48 weeks after entecavir administration.Entecavir-resistant quasi species (rtM204V ± rtL180M plus S202G) were foundafter week 112 and gradually became the predominant mutations afterward. Thelamivudine- and entecavir-resistant mutations emerged closely in combinationwith the rtV207L, rtA222T, rtP237T, or rtI163V substitutions. Their results indi-cated that the lamivudine-resistant mutations were developed first and may serveas a prequisite for subsequent entecavir-resistant mutations in this nucleoside-/nucleotide-naıve patient.

The first synthesis of entecavir was accomplished by Bisacchi et al . [161],in which chiral epoxide 418 was prepared from sodium cyclopentadienide 417by an asymmetric hydroboration/epoxidation/protection sequence (Scheme 1.52).Treatment of epoxide 418 with a purine salt provided nucleoside 419 in 60%yield with desired regioselectivity. After the protection of the amino group of419, Dess-Martin oxidation and Nysted methylenation afforded the exocyclicdouble-bond compound 422, which was converted to entecavir (416) after basederivation and deblocking steps.

Ziegler and Sarpong studied the radical cyclization of the important inter-mediate 428 toward the synthesis of entecavir (Scheme 1.53) [176]. Compound

Scheme 1.52 Synthesis of entecavir (416) [161]. Reagents and conditions: (a) (i)BnOCH2Cl; (ii) diisopinylcampheylborane; (iii) NaOH, H2O2; (iv) VO(acac)2, t-BuOOH;(v) BnBr, NaH; (b) NaH, Base; (c) MMTrCl; (d) Dess-Martin reagent; (e) Nysted reagent,TiCl4; (f) base derivation and deprotection.


Scheme 1.53 Synthesis of carbocyclic core 428 as an intermediate for preparing entecavir[176]. Reagents and conditions: (a) (MeO)2POCN2COMe, K2CO3; (b) (i) TBSOTf, 2,6-lutidine; (ii) mCPBA; (c) Cp2TiCl; (d) PivCl, DMAP; (e) HOAc.

423 was prepared from d-diacetone by a known procedure [177]. Ohira’s pro-tocol [178] was applied to convert unsaturated 423 into terminal acetylene 424,which was protected with a TBS group and treated with mCPBA to provideepoxide 425. Compound 425 underwent intramolecular radical cyclization in thepresence of Cp2TiCl. Desired carbocyclic intermediate 428 was obtained afterstandard protection group manipulations.

An alternative route to prepare entecavir was developed by Chu and cowork-ers starting from enone 14, which was transformed to 342 using 1,4-additionmethod (Scheme 1.54) [50]. The exocyclic double bond was constructed understandard Mannich reaction/Hoffman elimination protocol, which led to α, β-unsaturated ketone 429. Compound 429 was subjected to reduction, protection,and deprotection steps afforded triol 430. The protection of the 3′- and 5′-hydroxyl

Scheme 1.54 New schemes toward the synthesis of entecavir by Chu and coworkers.Reagents and conditions: (a) tert-butyl methyl ether, sec-BuLi, tBuOK, CuBr · Me2S; (b)(i) LDA, Eschenmoser’s salt; (ii) MeI, NaHCO3; (c) (i) NaBH4, CeCl3 · 7H2O; (ii) BnBr,NaH; (iii) HCl; (d) TIPDSCl2/Imidazole; (e) (i). NaH, CS2, MeI; (ii) Bu3SnH, AIBN;(iii) Na/Liq NH3; (f) DIAD, TPP, base; (g) base derivation and deprotection.


groups was followed by Barton-McCombie deoxygenation and Birch reductionto yield key intermediate 432. Standard Mitsunobu coupling, base derivation, andprotecting-group manipulations furnished entecavir (416).

Despite the significant successes in the area of anti-HBV agents, resistanceand cross-resistance against available therapeutics are the major hurdles in drugdiscovery. Chu and his group studied the molecular basis of drug resistanceconferred by the B and C domain mutations of HBV-polymerase on the bind-ing affinity of entecavir [179]. In this regard, homology modeled structure ofHBV-polymerase was used for minimization, conformational search and inducedfit docking followed by binding energy calculation on wild type, as well as onmutant, HBV-polymerases (L180M, M204V, M204I, L180M+M204V, L180M-M204I) shown in Table 1.18. Their studies suggest a significant correlationbetween the fold resistances and the binding affinity of entecavir [179]. Thebinding mode studies reveal that the domain C residue M204 is closely asso-ciated with sugar/pseudosugar ring positioning in the active site, and furthermutation of M204 to V204 or I204 reduces the final binding affinity, whichleads to the drug resistance. The domain B residue L180 is not directly close(∼6A) to the entecavir, but indirectly associated with other active-site hydropho-bic residues such as A87, F88, P177, and M204. These five hydrophobic residuescan directly affect the incoming nucleoside analogs in terms of its associationand interaction that can alter the final binding affinity. There was no carbocyclicring shifting observed in the case of entecavir. The exocyclic double bond ofentecavir occupied in the backside hydrophobic pocket (made by residues A87,F88, P177, L180, and M204), which enhances the overall binding affinity, asshown in Table 1.18.

Further molecular modeling studies have been carried out with 3TC-resistantHBV to investigate the active site interactions to understand the resistant profileof ETV. The 3TC-associated L180 mutant disfavors the shifting of oxathiolanering, whereas there is no significant movement of the carbocyclic ring of ETV incomparison to dGTP. Thus, it is obvious that the L180M mutation will not affectits binding to the active site. In addition, the mutant M204V does not have muchof an effect on the binding mode of ETV as well. The mutation of M204 to V204can result in only partial filling of the small hydrophobic pocket, which may bethe reason for the reduction of potency of ETV in M204V HBV RT. There is apossible partial steric clash between the exocyclic alkene and the I204 residue,which supports the reduction of potency of ETV in comparison to M204V HBV.Despite of the partial steric clash, there is no forward movement observed forthe exocyclic sugar ring of ETV as it was clearly observed in the case of 3TC.

1.2.5.2. Entecavir analogs During the course of the development of ente-caivr, its regio-isomers, 434 and 435, were synthesized and screened againstHBV. Unfortunately, both compounds proved to be inactive against HBV [180](Figure 1.15).

The antiviral activity of (±)-436, a carbocyclic adenine analog with exocyclicdouble bond, was first described in 1988 [82]. It was found that this compound


TABLE 1.18 Multiligand bimolecular association with energetic (eMBrAcE)calculation of entecavir triphosphate (ETV-TP) in comparison to dGTP afterinduced fit docking and minimization in HBV-polymerase [179]

Energy Difference results (�E: kJ/mol)

HBV-polymerase Electrostatic Vdwa Total �E b

Wild type (WT)dGTP –5161.7 139.5 –366.8 –37.9ETV-TP –5465.8 196.6 –404.7

L180MdGTP –5010.2 173.7 –422.7 2.6ETV-TP –5009.4 163.1 –420.1

M204VdGTP –4977.5 179.7 –417.4 –10.3ETV-TP –4972.7 163.3 –427.7

M204IdGTP –5233.9 169.0 –429.9 –8.6ETV-TP –5277.4 180.3 –438.5

L180M-M204VdGTP –5293.3 179.5 –449.1 –3.0ETV-TP –5321.3 170.7 –452.1

L180M-M204IdGTP –5267.1 172.2 –438.5 1.0ETV-TP –5297.6 185.0 –437.5

avan der Waals interaction.b�E = energy difference (-ive: favorable) in comparison to the dGTP.

Figure 1.15 Entecavir analogs.

was active against vaccinia virus. Recently, Chu and coworkers have accom-plished the asymmetrical synthesis of the whole series of d-form compoundsfor a complete SAR study. However, no interesting biological activity of tar-get nucleosides were observed except marked cytotoxicity effect of (-)-436. Thesynthesis of 436 in Chu’s protocol utilized the allylic alcohol 438, which wasobtained by reducing α, β-unsaturated ketone 429 depicted in Scheme 1.55. TheMitsunobu coupling of alcohol 438 with proper base equivalents, followed bybase derivations and deprotection steps, yielded the desired nucleosides.


Scheme 1.55 Synthesis of (-) 436. Reagents and conditions: (a) NaBH4, CeCl3 · 7H2O;(b) DIAD, Ph3P, Base; (c) base derivation and deprotection.

During the course of our drug discovery programs, introduction of fluorineatom onto the sugar moiety generated a number of novel nucleosides with inter-esting biological interesting nucleosides. Therefore, it is of great interest toexplore the substitution of fluorine atom on the carbocyclic nucleosides witha 6′-exocyclic alkene (6′-methylene) (437) by Chu and coworkers, as shown inScheme 1.56 [181]. Fluorinated nucleoside 437 was synthesized according to thenewly developed procedure. Interestingly, adenine derivative 437 was not onlyactive against HBV-WT but also retained full potency against lamivudine- andadefovir-resistant mutants as shown in Table 1.19. Such potent anti-HBV activity,as well as excellent resistance profile, makes this type of nucleosides attractivecandidates as potential anti-HBV agents. Further structure-activity studies of thisclass of 2′-F carbocyclic nucleosides are in progress.

1.2.6. Carbocyclic Arabino- and Xylo-Nucleosides

Cyclaradine 444, a carbocyclic analog of ara-A, was discovered by Vince andcoworkers [182]. It was resistant against adenosine deaminase and exhibitedantiviral activity against HSV-1 (EC502.8–9.0 μM) and vaccinia virus (EC50

Scheme 1.56 Synthesis of 2′-F-6′-methylene carbocyclic nucleoside (437) [181].Reagents and conditions: (a) DAST, CH2Cl2, rt; (b) (i) TBAF/HOAc, THF, rt; (ii) BzCl,Py, rt; (c) BCl3, CH2Cl2,−78◦C; (d) DIAD, Ph3P, 6-chloropurine, THF, rt.


TABLE 1.19 In vitro anti-HBV activity of 437 against lamivudine and adefovirdrug-resistant mutants on the intracellular HBV DNA replication assay [181]

Anti-HBV activity 437 (μM) Lamivudine (μM) Adefovir (μM)

Strain EC50 EC90 EC50 EC90 EC50 EC90

WT 1.5 4.5 0.2 0.6 1.3 7.1rtM204V 1.8 4.7 >100 >100 1.6 7.0rtM204I 1.0 5.0 >100 >100 1.9 8.0rtL180M 2.1 5.1 1.5 22 5.5 7.7rtLM/rtMVa 2.2 5.5 >100 >100 2.1 8.5rtN236T 1.7 4.6 0.2 0.9 7.8 36

artLM/rtMV = rtL180M/rtM204V double mutant.

9.0 μM) [182]. Carbocyclic xylo-nucleosides 445 was reported to exhibit potentantitumor activity with EC50 0.38 μM. Its guanine analog 446 was active againstHSV-1 (EC50 1.8–3.0 μM) (Figure 1.16).

The synthesis of both classes of compounds started from the same epoxide447, which was hydrolyzed to yield two products: arabino-type 448 and xylo-type450 (Scheme 1.57). Two intermediates were deprotected and further convertedto the desired arabino- and xylo-nucleosides by known chemistry.

Figure 1.16 Biological active carbocyclic arabino- and xylo-nucleosides.

Scheme 1.57 Synthesis of carbocyclic arabino- and xylo-nucleosides [182]. Reagents andconditions: (a) (i) H2SO4, (ii) Ac2O; (b) (i) HCl, (ii) OH−; (c) bases construction.


1.2.6.1. Carbocyclic 2′-Deoxy-Nucleosides and Related Nucleosides1.2.6.1.1. Carbocyclic 2′-deoxy nucleosides (E)-5-(2-Bromovinyl)-2′-deoxyuridine (452, BVDU) is highly active against anti–herpes zoster [183].However, the fast degradation to (E)-5-(2-Bromovinyl)-2′-deoxyuracil (BVU)catalyzed by pyrimidine nucleoside phosphorylases limits the therapeutic usageof BVDU [184]. The carbocyclic counterpart, C -BVDU (453), however, is nolonger a substrate for phosphorylases while it maintains the antiviral potency.Its analog, (E)-5-(2-iodovinyl)-2′-deoxyuridine (454, C -IVDU), exhibits similarselectivity as well as antiviral activity [185] (Figure 1.17).

The synthesis of C -BVDU and C -IVDU started from aminotriol 5, whichwas converted to anhydrouridine 475 in three steps (Scheme 1.58). Treatment of475 with acetyl bromide followed by dehalogenation, and deprotection providedcarbocyclic 2′-deoxyuridine 476. Introduction of 5-vinylbromide was accom-plished via an iodination/coupling reaction/hydrolyzation/bromination sequence.C -IVDU was prepared in a similar manner [185].

Figure 1.17 Biologically active carbocyclic 2′-deoxy nucleosides.


Scheme 1.58 Synthesis of C -BVDU and C -IVDU [185]. Reagents and conditions: (a)(i) silver cyanate, β-methoxyacryloyl chloride; (ii) NH4OH; (iii) diphenyl carbonate;(b) (i) acetyl bromide; (ii) Bu3SnH, AIBN; (c) (i) I2, nitric acid; (ii) methyl acrylae,Pd(OAc)2, Ph3P, Et3N; (iii) KOH; (iv) NBS for 453 or I2, iodic acid, K2CO3 for 454.

Another approach to the synthesis of C -BVDU was described by Wyatt et al .as outlined in Scheme 1.59, which is comparably concise with the original one[186].

Another interesting compound in this series is carbocyclic 2′-deoxyguanosine(455, C -dG), which demonstrated potent antiviral activity against herpes simplexvirus (HSV-1 and 2) [187], human cytomegalovirus (HCMV), and HBV [188]. C -dG apparently is activated by virus-encoded kinase to exhibit anti-herpes activity,although it is a poor substrate for cellular phosphorylating enzymes [189].

Racemic C -dG was synthesized by a linear method (Scheme 1.60) [187]. It isnoteworthy that the enantiomeric synthesis of C -dG was accomplished by Liangand Moser via an enzymatic approach [190]. Protection and hydroformylationof a racemic vinyl diol 482 led to an aldehyde 483, which was reduced toan alcohol 484. A standard protecting-group manipulation provided compound485. Enzymatic resolution of 485 with Pseudomonas fluorescens lipase (PFL) inthe presence of vinyl acetate followed by the deprotection of the acetate groupfurnished optically pure compound 487. Cyclic sulfate 488 was then preparedand condensed with base moieties to provide the target carbocyclic nucleosides.

Borthwick et al . described another enzymatic resolution method soon afterLiang and Moser’s report [191]. Chu and coworkers developed a solid phase syn-thesis of L-C -dG as well as its analogs (Scheme 1.61) [192]. Standard protecting-group manipulation and radical deoxygenation led to compound 494, which was

Scheme 1.59 Alternative route of the synthesis of C -BVDU [186]. Reagents and condi-tions: (a) (i) Et3N, (ii) HCl; (b) Ac2O, DMAP; (c) NBS or Br2; (d) NaOH.


Scheme 1.60 Synthesis of carbocyclic 2′-deoxynucleoside [187]. Reagents and condi-tions: (a) (i) (t-Bu)2SiOTf2, 2,3-lutidine; (ii) [RhCl(PPh3)3], H2/CO, 80 bar; (b) NaBH4;(c) (i) TrCl, DMAP, Et3N; (ii) TBAF; (d) PFL, vinyl acetate; (e) ethylenediamine; (f) (i)SOCl2, Et3N; (ii) RuCl3, NaIO4; (g) (i) NaH, base; (ii) deprotection.

Scheme 1.61 Solid phase synthesis of l-carbocyclic 2′-deoxynucleosides [192]. Reagentsand conditions: (a) TIPDSCl2, Py; (b) (i) NaH, CS2, MeI; (ii) Bu3SnH, AIBN; (c) (i)Pd(OH)2, H2; (ii) DHP, PPTS; (d) (i) TBAF; (ii) TBDMSCl, Im; (e) (i) BzCl, Py; (ii)TBAF; (f) 498, DMAP, DIPEA; (g) PPTS, 1-butanol/1,2-dichloroethane; (h) DIAD, Ph3P,Bases; (i) K2CO3.

coupled with p-nitrophenyl carbonate resin 498 to yield compound 495. Fullyprotected 495 was subjected to the acidic hydrolysis to remove the THP group toprovide alcohol 496 ready for Mitsunobu coupling reaction. In the coupling reac-tion, it was found that both regioselectivity and yield were generally improvedunder the solid phase condition in comparison with solution phase synthesis[192].


The most interesting analogs of carbocyclic 2′-deoxyribonucleosides are aseries of 2′-fluoro substituted compounds. Among these analogs, the adeninederivative 461 was approximately 10-fold more active than cyclaridine (444)against herpes viruses (HSV-1 and 2) [193]. Guanine derivative 460 initially dis-played significant antiviral activity against HSV-1 and 2 with EC50′s of 0.006and 0.05 μg/mL, respectively, and did not show any toxicity up to 300 μg/mL[194]. Unfortunately, compound 460 was found to be toxic in later studies. Soonafter this discovery, the same group in Glaxo described the synthesis and antivi-ral activity of fluorinated pyrimidine analogs [195]. However, only C -FMAU(457) and C -FIAU (458) exhibited moderate anti-HSV-1 activities, which wereboth significantly lower than that of the parent compound FMAU (456). Also,6′-Fluoro substituted guanosine analogs were prepared. The β-F analog 463(EC500.16 and 0.77 μg/mL for HSV-1 and 2, respectively) was approximately50- to 100-fold more potent than α-F isomer 462.

The synthesis of these fluorinated carbocyclic nucleosides all followed a linearmethodology via 499, 500, or 501 as key intermediates that can be prepared fromcompound 5 by standard methods (Figure 1.18) [196].

The replacement of the 2′-hydrogen of the natural ribonucleosides with amethyl group yielded compounds with excellent chain-terminating properties.Among them, 2′-C -methyladenosine and 2′-C -methylcytidine demonstratedpotent anti-HCV agents in clinical trials [79,80]. More recently, 2′-C -fluoro-2′-C -methylcytidine was discovered as a hepatitis C virus RNA-dependant RNApolymerase (HCV RdRp) inhibitor and showed better inhibitory activity in theHCV replicon assay than 2′-C -methylcytidine, with low cellular toxicity. Onthe basis of potent anti-HCV activity of 2′-modified nucleosides, Hong andcoworkers designed and synthesized 2′(α)-C-fluoro-2′(β)-C -methyl carbodinederivatives from 2-methyl cyclopentenone 502 [197]. Geminal substitution atthe 2′-position might impose favorable steric as well as electronic effect onthe interaction with HCV polymerase. The key fluorinated intermediate 504was prepared from the epoxide intermediate 503 via selective ring-openingfluorination of epoxide using hydrofluoric acid. Coupling of 504 with nucleosidicbases under the Mitsunobu reactions followed by deprotection afforded thetarget carbocyclic nucleoside analogues 464 and 465 (Scheme 1.62). Thesecompounds were evaluated as inhibitors of the hepatitis C virus (HCV) in Huh-7cell line in vitro. The cytosine analogue 464 weakly inhibited the replication ofthe replicon, NK-R2AN, in huh-7 cells by 50% at 18.2 μM [197].

500 501499

Figure 1.18 Key intermediates for the preparation of fluorinated nucleosides.


Scheme 1.62 Preparation of 2′(α)-C -fluoro-2′(β)-C -methyl carbodine derivatives(464–465) [197]. Reagents: (a) (i) BnBr, NaH, DMF; (ii) 47% HF, (NH4)2SiF6, CsF;(b) N6-bis-Boc-adenine, PPh3, DIAD, 0◦C; (c) (i) TFA, DCE/MeOH, rt; (ii) Pd(OH)2,cyclohexene, MeOH, reflux; (d) N4-Bz-cytosine, PPh3, DIAD; (e) (i) NaOMe/MeOH; (ii)Pd(OH)2, cyclohexene, MeOH, reflux.

Hughes and coworkers [198] analyzed novel nucleoside reverse transcriptase(RT) inhibitors (NRTIs) for their ability to inhibit DNA synthesis of excision-proficient HIV-1 RT mutants. A major pathway for HIV-1 resistance to NRTIsinvolves reverse transcriptase mutations that enhance ATP-dependent pyrophos-phorolysis, which excises NRTIs from the end of viral DNA. d-Carba-thymidine(466) [198] is a carbocyclic nucleoside that has a 3′-hydroxyl on the carbocyclicring. The 3′-hydroxyl group allows RT to incorporate additional dNTPs, whichshould protect d-carba TMP from excision. d-Carba thymidine (466) can beconverted to the triphosphate form by host cell kinases with moderate efficiency.d-Carba thymidine-TP is efficiently incorporated by HIV-1 RT; however, the nextdNTP is added slowly to a d-carba TMP at the primer terminus. d-carba thymi-dine (466) effectively inhibits viral vectors that replicate using NRTI-resistantHIV-1 RTs, and there is no obvious toxicity in cultured cells as shown inTable 1.20.

Meier and his coworkers synthesized several carbocyclic l-nucleosides (e.g.,l-carbadU 467, l-carba-dT 468, l-carba-FdU 469, l-carba-BVDU 470, and

TABLE 1.20 Antiviral efficacy of AZT and d-carba T vs wild-type and select NRTIresistant HIV-1 viral vectors [198]

d-carba T, 466 (μM) AZT (nM)

HOS HOS 313 HOS

Wild type 3.2 ± 1.7 0.83 ± 0.21 1.4 ± 0.1M184V 1.8 ± 1.0 0.19 ± 0.03 3.6 ± 0.3AZT-R 1.3 ± 0.4 0.87 ± 0.13 20.1 ± 1.8SSGR + T215Y 4.6 ± 1.2 1.0 ± 0.1 39.6 ± 7.7


l-carba-dC 471) [199] by coupling a cyclopentane-system with heterocyclesaccording to a modified Mitsunobu-protocol (Scheme 1.63). This reaction gavetwo regioisomers, the N1-alkylated product and an unwanted O2 product. Asimple SN2-reaction has been investigated as an alternative for such couplings.The key step is the stereoselective synthesis of the cyclopentane system509. Starting from cyclopentadiene an alkylation with BOMCl followed by astereoselective hydroboration introduces the 3′-hydroxy group of the needednucleoside sugar moiety [199]. This step allows the subsequent synthesis of l-or d-nucleosides using (+)-diisopinocampheylborane or (-)-(ipc)2-borane.

Carbocyclic nucleoside analogues are catabolically stable because these areresistant to the phosphorolytic cleavage by pyrimidine nucleoside phosphorylase.The carbocyclic analogue (C-BCNA, 473) [200] of a highly potent and selectiveanti-VZV bicyclic nucleoside analogue (BCNA, 472) 6-pentylphenylfuro[2,3-d]pyrimidine-2′-deoxyribose was synthesized using carbocyclic 2′-deoxyuridineas starting material by McGuigan and his coworkers [200]. C-BCNA (473) wasfound to be chemically more stable than the furano lead (472); however, it wasshown to be significantly less antivirally active than its parent nucleoside ana-logue, as shown in Table 1.21. It was noted to have a 10-fold lower inhibitoryactivity against the VZV-encoded thymidine kinase. This reduction of activitymay be attributed to the different conformation of the sugar and base, as pre-dicted by computational studies and supported by NMR studies. However, otherfactors besides affinity for VZV-TK may account for the greatly reduced antiviralpotency.

Imidazolone (dIz) nucleoside is an abundant, highly mutagenic, and ratherunstable DNA lesion that can cause dG →dC transversion mutations. dIz is gen-erated in DNA by a variety of oxidative processes such as type I photooxidation.Carell and his coworkers synthesized the carbocyclic imidazolone nucleoside(cdIz, 474) [201], which is a stabilized version of the oxidatively generated

Scheme 1.63 Synthesis of 2′-deoxy-l-carbonucleosides (453–457) [199]. Reagents andconditions: (a) (1) (+)- (ipc)2BH or (2) (-)- (ipc)2BH, −60◦C to rt, 15 h 45%; (b) (i)NaH, BnBr, TBAI, THF, rt, 12 h, 91%; (ii) 9-BBN, THF, rt, 12 h, 3N NaOH, 30% H2O2,0◦C, 79%; (iii) PPh3, DIAD, benzoic acid 0◦C to rt, 96%; (c) (i) PPh3, DIAD, N3-benzoylphyrimidines, CH3CN, −40◦C to rt, 15 h, 1% NaOH in CH3OH, rt, 4 h; (ii) Pd/C,H2, EtOH.


TABLE 1.21 Antiviral and cytostatic activity of 452, 472–473 compounds [200]

EC50a (μM) VZV MCC50

b (μM) CC50c (μM) IC50

d (μM)

TK+ OKA TK+ YS TK−07 TK− YS HEL HEL VZV-TK

472 0.0003 0.0001 >5 >5 >20 >50 4.9473 0.49 0.28 >50 — >50 >50 44452 0.005 0.005 >200 >200 >200 >200 1.3

aThe 50% effective concentration, or compound concentration, required to inhibit VZV-inducedcytopathicity by 50%.bMinimal inhibitory concentration, or compound concentration, required to cause a microscopicallyvisible alteration of cell morphology.cThe 50% cytostatic concentration, or compound concentration, required to inhibit HEL cell prolif-eration by 50%.dThe 50% inhibitory concentration, or compound concentration, required to inhibit VZV-TK-catalyzed dThd (1 μM) phosphorylation by 50%.

DNA lesion imidazolone (dIz). The carbocyclic modification protects this lesionanalogue from anomerization. Replacement of the 2′-deoxyribose moiety by thecyclopentane unit stabilizes this lesion analogue against cleavage by base exci-sion repair (BER) enzymes because of the lack of a glycosidic bond, but thismodification does not prevent degradation of the heterocycle, for example, togive the oxazolone (dZ) hydrolysis product.

1.2.7. Conformationally locked carbocyclic nucleosides

Neplanocin C (511), a naturally occurring carbocyclic nucleoside isolated fromAmpullariela regularis in the early 1980s, provided a prototype of conformation-ally locked nucleoside with a cyclopropane ring fused on the 4′, 5′ position of acyclopentane ring [202] (Figure 1.19).

The [3,1,0]-bicyclic system adopted a predominant Northern conformationas indicated in the X-ray analysis [203]. The synthesis of the neplanocin Cstarted from a known cyclopentenol 24 (Scheme 1.64) [117]: The cyclopentenol24 was condensed with 6-chloropurine and deblocked the acetonide group toprovide nucleoside 542, which was further subjected to the epoxidation, aminationfollowed by debenzylation to furnish the target neplanocin C.

Inspired by the novel structure of neplanocin C, a number of carbocyclicanalogs have been prepared. Among these compounds, the most systematicallyand extensively studied are those 1′,5′-methano (522–526) as well as the 4′,5′-methano carbocyclic nucleosides (517–521) as shown in Figure 1.19.

d-form adenine derivative 512 was found to adopt a typical Northern confor-mation and exhibited moderate anti-HIV activity while its enantiomer was devoidof the antiviral activity. The synthesis of the target nucleosides was accomplishedvia a cyclopropane fused cyclopentanal 545, which was prepared by treating com-pound 544 with chloroiodomethane in the presence of samarium (+2) at −78◦C(Scheme 1.65). Condensation of various base moieties with 545 followed by basederivation provided the desired nucleosides 512–516 [204,205].


Figure 1.19 Conformationally locked carbocyclic nucleosides.

Scheme 1.64 Synthesis of neplanocin C [117]. Reagents and conditions: (a) (i) DIAD,Ph3P, 6-chloropurine; (ii) AcOH; (b) m-CPBA; (c) (i) NH3/MeOH; (ii) H2, Pd/C.

Soon after the report of conformationally locked carbocyclic dideoxynucleo-sides as described earlier, Altmann et al . also accomplished the synthesis of both4′,5′- and 1′,5′-methano-2′-deoxy carbocyclic thymidine (519 and 524, Scheme1.66a; and 1.66b) [206]. The synthesis of 519 was accomplished starting fromallylic alcohol 24, which was subjected to the Simmons-Smith cyclopropanationto give ring fused compound 547 with the desired stereochemistry due to the


Scheme 1.65 Synthesis of compounds 512–516 [204,205]. Reagents and conditions: (a)CH2ICl, Sm, HgCl2; (b) DIAD, Ph3P, Bases; (c) Base derivation and deprotection.

Scheme 1.66 Synthesis of compounds 519 and 524 [206]. Reagents and conditions: (a)Zn/Cu, CH2I2; (b) (i) TsCl, Et3N, DMAP; (ii) NaN3; (iii) H2, Lindar’s catalyst; (c) baseconstruction; (d) (i) HCl; (ii) H2, Pd/C; (e) (i) TIPDSCl2, Im; (ii) BOM-Cl, DBU; (f)(i) CH3C6H4OC(S)Cl, DMAP, Et3N; (ii) Bu3SnH, AIBN; (g) (i) TBAF; (ii) H2, Pd/C;(iii) NaOMe; (h) (i) TMSBr, ZnBr2; (ii) N -methyl acetamide; (i) t BuOK, t-BuOH; (j) (i)KOH; (ii) DPPA, Et3N; (iii) H2, Pd/C; (k) base construction and deprotection.

directing effect of the allylic hydroxyl group [207]. After the alcohol 547 wasconverted into the amine 548, the heterocyclic moiety was constructed understandard conditions to give nucleoside 550, which was protected and subjectedto the Barton–McCombie deoxygenation to afford target compound 519 (Scheme1.66a). On the other hand, the synthesis of 1′,4′-methano-2′-deoxy carbocyclicthymidine utilized the bicyclic lactone 553 as a key intermediate, which wastreated with TMSBr followed by N -TBDMS to provide compound 554 (Scheme1.66b). Formation of the three-membered ring went smoothly under basic con-dition. Subsequent deprotection, Curtius rearrangement, deprotection and base


construction furnished target nucleoside 524. The X-ray analysis indicated that4′,5′-methano-2′-deoxy carbocyclic thymidine 519 preferred boatlike Northernconformation, whereas the isomer 524 existed predominately as boatlike Southernconformation.

In the series of Northern 2′-deoxy nucleosides, the adenine analog 516 wasprepared by Siddiqui and Marquez et al . (Scheme 1.65) and showed good antivi-ral activity against HCMV and EBV [208]. To explore the full potential of thisclass of molecules, a series of nucleoside bases (adenine, uracil, cytosine, andguanine) built on the Northern bicycle[3,1,0]hexane pseudo-sugar ring were syn-thesized using a convergent approach [209]. Nucleoside 519 displayed excellentantiherpes activity with EC50 of 0.03 and 0.09 μg/mL against HSV-1 and HSV-2, respectively. It was nontoxic to host cells at concentration up to 100 μg/mL.Interestingly, the isomer of compound 524, Southern bicycle[3,1,0]hexane thymi-dine, was devoid of antiviral activity. Conformational analysis revealed that notonly was the ring pucker of these two conformationally rigid nucleosides quitedifferent (519: Northern, 524: Southern), but also the base rotation angle (χ) ofcompound 524 was more stiff in comparison to 519. All these disparities togethermight explain, to some extent, the difference in the antiviral activity of thesetwo compounds. Based on these findings, Marquez and coworkers performed asystematic SAR study of a number of conformationally locked carbocyclic nucle-osides (527–529) [210–214]. Their data showed that herpes thymidine kinasehad a strong preference to the Southern conformation and antibase disposition inthe monophosphorylation step but insensitive to the presence or absence of 3′-OH. However, in the diphosphorylation step, the 3′-OH was extremely importantto this enzyme, and Southern conformation was still preferred. Cellular DNApolymerase and HIV reverse transcriptase favored exclusively the triphosphateof the Northern conformers [210,212,214].

Marquez and his coworkers found that chemically synthesized 5′-triphosphatesof bicyclo-[3.1.0] hexane models of 2′-deoxynucleosides that were restricted inthe North conformation (517–520) [209] were readily incorporated by HIV-1 RTand other polymerases, none of the nucleosides were effective anti-HIV agentsdue to inefficient cellular phosphorylation. On the other hand, the conforma-tional constraint in South-bicyclo [3.1.0] hexane nucleosides, which influences thenucleobase to adopt the biologically unsuitable syn conformation that is favoredby intramolecular hydrogen bonding in a hydrophobic environment, such as theenzyme’s binding site, resulted in compounds 522–525 that were not phospho-rylated by cellular kinases, despite their excellent substrate recognition by themore tolerant, viral HSV-1 kinase.

The incorporation of the main structural feature of D4T, which is itsplanarity, into the structure of the very potent anti-HSV compound, North-methanocarbathymidine (519) resulted in the very effective anti-HIV thymidineanalogue (532) with a novel bicycle[3.1.0]hexene pseudosugar that included thecritical double bond. The 4- to 10-fold decrease in potency of 532 relative toD4T was tentatively correlated with the nearly 10-fold reduction in the level ofplanarity (νmax = 6.818) of the embedded five-membered ring segment of the


bicyclo[3.1.0]hexene template relative to the nearly flat pseudosugar ring of D4T(νmax = 0.618) [215]. As each of the cellular kinases that perform the criticalfirst phosphorylation step have different preferences for the various nucleobases,we decided to complete our study by incorporating the rest of the nucleobases(A, C, and G) to the same bicyclo[3.1.0]hexene template (530–533) (Scheme1.67) derived from the conformationally locked North-bicyclo [3.1.0]hexanenucleosides 517–520 [215]. It is important to remember that in terms ofsugar conformation, once the amplitude of the puckering measured by νmax

reaches a small value, such as ∼6◦ in 530–533, the designation of Northand South loses its significance. Based on that argument, they decided toinvestigate the synthesis and anti-HIV activities of the complementary set ofbicyclo[3.1.0]hexane nucleosides (534–537) (Scheme 1.68) derived from theantipodal, conformationally locked, South-bicyclo[3.1.0]hexane nucleosides522–525 bearing the four natural nucleobases. The transformation of abicyclo[3.1.0]hexane nucleoside into a bicyclo [3.1.0]hexene nucleoside flattensthe five-membered ring of the bicyclic system and rescues anti-HIV activity forNorth-D4T (532), North-D4A (530), and South-D4C (537). The relationshipbetween planarity and the anti/syn disposition of the nucleobase that is favoredby a particular pseudosugar platform are proposed as key parameters incontrolling biological activity.

2′-C -methyladenosine and 2′-C -methylguanosine showed potent anti-HCVactivity in a cell-based HCV replicon assay, in which 2′-methyl group preventsthe incorporation of incoming nucleosides triphosphates [80]. These nucleosideswere reported to adopt a Northern C3′-endo conformation (pseudorotation angle,

Scheme 1.67 Synthesis of north-bicyclo-[3.1.0]hexene nucleoside 530–533 [215].Reagents and conditions: (a) DIAD, PPh3, base THF, 0◦C → rt; (b) Et3N · 3HF, MeCN, �.

Scheme 1.68 Synthesis of south-bicyclo-[3.1.0]hexene nucleoside 534–537 [215].Reagents and conditions: (a) TBDPSCl, imidazole, DMF or TBDPSCl, DMAP, pyri-dine; (b) MsCl, pyridine; (c) (i) 1,8-diazabicyclo [5.4.0]undec-7-ene (DBU), PhMe orDMF, Base; (ii) TBAF, THF.


P = 15.6◦). Carbocyclic nucleosides in which the cyclopropane ring is fusedbetween C4′ and C6′ also fix the conformation of the carbasugars to a North-ern C3′-endo conformation (P = 0±18◦). Thus, this conformational informationprompted Moon and his coworkers to design and synthesize stereoselectivelycyclopropyl-fused-carbanucleosides 538–541 (Scheme 1.69) [216].

The cyclopentenone 567 was stereoselectively reduced to α-allylic alcohol fol-lowed by modified Simmons–Smith cyclopropanation gave bicyclo[3.1.0]hexanederivative 568 as a single stereoisomer. Selective protection of the least hinderedalcohol in diol with a TBDPS group followed by Swern oxidation of the remain-ing alcohol afforded the ketone 569. The cyclic sulfate was obtained by stere-oselective Grignard reaction, removal of both silyl protecting groups followedby SOCl2 treatment in presence of triethyl amine, and oxidation of the resultingcyclic sulfite with sodium periodate in presence of RuCl3-3H2O. Compounds538–541 were synthesized by utilizing regioselective cleavage of the isopropyli-dene group and cyclic sulfate (571) chemistry as key steps [216]. Antiviral assayof 538–541 against HCV was performed, but these compounds did not show anysignificant anti-HCV activity in a cell-based HCV replicon assay.

Scheme 1.69 Synthesis of 2-C -methylcarbanucleoside 538–541 [216]. Reagents and con-ditions: (a) (i) NaBH4, CeCl3.7H2O, MeOH, 0◦C, 30 min.; (ii) Et2Zn, CH2l2, CH2Cl2, rt,5 h; (b) (i) Me3AI, CH2Cl2, rt, 4 d; (ii) TBDPSCl, imidazole, CH2Cl2, rt, 20 min.; (iii)(COCl)2, DMSO, TEA, CH2Cl2, rt, 1 d; (c) (i) MeMgl, Et2O, rt, 2.5 h; (ii) TBAF, THF,rt, 1 d; (iii) TBDMSCI, imidazole, CH2Cl2, 0◦C, 2 h; (d) (i) SOCl2, TEA, CH2Cl2, 0◦C,10 min.; (ii) RuCl3-3H2O, NalO4, CCl4 : CH3CN:H2O = 1 : 1 : 1.5, rt, 10 min.; (e) (i)adenine or 2-amino-6-chloropurine, NaH, DMF; (ii) 20% aq H2SO4; (f) 70% CF3COOH,rt, 50 min.; (g) NaNO2, AcOH, rt, 3 h; (h) 3N aq HCl, rt, 2 d; (i) cyclopropylamine,ethanol, rt, 1 d.


1.2.7.1. Conformationally locked norborane and Isoxazoline-basedcarbocyclic nucleoside A modification, which increases resistance againstenzymatic degradation, is substitution of the furanose ring of the sugar moietyby a carbocyclic ring. Many of such modified analogues carbocyclic nucleosidesexhibit interesting antiviral activity. Recently, a series of carbocyclic analoguescontaining bicycloalkanes, bicycloheteroalkanes or tricycloheteroalkanes withactivity against Coxsackie viruses was prepared by Sala and coworkers asshown in Figure 1.20 [217,218]. They were reported synthesis of novel racemicconformationally locked nucleosides with bicyclo[2.2.1]heptene (norbornene)or heptane (norbornane) ring system substituted with nucleobase at position 7with syn-configuration as well as with anticonfiguration. Two methodologieswere employed for introduction of the chloropurine moiety to the scaffold:(i) the Mitsunobu reaction of the chloropurine nucleobase with appropriatealcohols and (ii) a built-up strategy from appropriate amines. These compoundscould be considered as the 6-chloropurines substituted at position 9 withdifferent substituted bicyclic scaffolds (bicyclo[2.2.1]heptane/ene-norbornaneor norbornene). These bicyclo systems (norbornene or norbornane), like theoxabicyclo[2.2.1]heptane, represent conformationally locked carbapentofuranosering systems. All compounds were evaluated for antiviral activity againstCoxsackie virus B3, as shown in Table 1.22. Most analogues showed activity inthe low micromolar range with minimal cytotoxicity to the cell line used in thisstudy. Studies on the mode of action of the most promising compounds are inprogress.

Figure 1.20 Conformationally locked norborane and Isoxazoline-based carbocyclic nucle-oside.


TABLE 1.22 Antiviral evaluation against CVB3 of the 9-substitutedpurines in Verocells, all data are mean values ± standard deviation for at least three independentexperiments [217,218]

Compounds IC50 (μM) EC90 (μM) CC50 (μM)

574 1.13 ± 0.33 NDa >50575 1.86 ± 1.13 ND >50576 1.00 ± 0.04 ND >50577 0.88 ± 0.11 ND >50578 1.00 ± 0.01 ND >50579 0.98 ± 0.17 ND >50580 0.91 ± 0.07 ND >50581 1.10 ± 0.03 ND 47.85 ± 3.05582 0.81 ± 0.20 1.82 ± 0.91 >50583 1.05 ± 0.01 ND >50584 0.95 ± 0.02 1.96 ± 0.98 >50585 1.00 ± 0.02 ND >50586 1.16 ± 0.24 34.40 ± 27.97 >50587 0.83 ± 0.05 1.70 ± 0.85 >50588 46.9 ND >326589 0.90 ± 0.04 1.78 ± 0.89 >50590 1.03 ± 0.20 3.46 ± 2.91 >50591 0.66 ± 0.35 >50 >50

aND: not determined.

Quadrelli and coworkers synthesized the isoxazoline-based carbocyclicnucleosides 592 and 593 by the linear construction of the desired purineand pyrimidine bases on the regioisomeric aminols 600 (Scheme 1.70) [219]obtained through elaboration of the hetero-Diels–Alder (HDA) cycloadducts598 of cyclopentadiene with nitrosocarbonyl intermediates (RCONO). Thecomplex course of 1,3-dipolar cycloaddition between benzonitrile oxide and thecyclohexadiene adduct 598 of (nitrosocarbonyl) benzene afford regioisomericmixtures of exo and endo cycloadducts. The exo cycloadducts of 599 aresuitable starting materials for isoxazoline-cyclohexane nucleoside synthesis.Detachment of the benzoyl group and reductive cleavage of the N–O bondprovided the stereodefined aminols 586, which were used for the linearconstruction of purine nucleosides by well-established synthetic protocols.Substitution with 5-amino-4,6-dichloropyrimidine and subsequent condensationwith orthoformates afford the chloropurines 592 and 593 [219]. These werefurther derivatized by replacement of the chlorine with amines and alkoxides togive suitable samples for antiviral tests in very good yields.

The same group was synthesized isoxazoline-carbocyclic nucleosides hav-ing a hydroxymethylene side chain, and a variety of analogues was attainedstarting from the stereodefined heterocyclic aminol 605, which are readily avail-able through exo selective 1,3-dipolar cycloadditions of benzonitrile oxide to2-azanorborn-5-enes 601 and elaboration of the cycloadducts 602 (Scheme 1.70).

THREE-MEMBERED CARBOCYCLIC NUCLEOSIDES 73

Scheme 1.70 Preparation of aminols for the synthesis of isoxazoline-carbocyclic nucleo-sides (592–596) [219,220].

The stereodefined heterocyclic aminols 605 afford the carbocyclic skeleton forthe linear construction of the purine rings. Functionalization of the chloropurines594–596 [220] with a variety of amines extended the synthetic potential of thisstrategy, allowing for a fine-tuning of their biological and antiviral activity as wellas comparison with the corresponding nor-nucleosides. Biological evaluation ofthe newly obtained compounds 594–596 is in progress.

1.3. THREE-MEMBERED CARBOCYCLIC NUCLEOSIDES

In general, three-membered carbocyclic nucleosides can be divided in two classes.The first ones have the base moiety directly attached to the ring, whereas the otherones have a spacer between the base and the ring (Figure 1.21).

Chu and coworkers accomplished the first asymmetric synthesis of d- andl-cyclopropyl nucleosides, which belong to the first category (Scheme 1.71)[221]. Protected d-mannitol was converted to the vinyl alcohol 618 by stan-dard oxidation/wittig reaction/reduction sequence. The requisite cyclopropyl ringwas installed by Simmons-Smith cyclopropanation following oxidation, Curtiusrearrangement and deprotection protocol to yield cyclopropyl amine 620. Thetarget d-nucleosides 606 were obtained by a linear methodology. l-Cylcopropylnucleosides 607 were synthesized in similar fashion using l-gulonic γ-lactoneas chiral starting material. Unfortunately, no significant biological activity wasexhibited by synthesized nucleosides.

On the other hand, in the second category, several biologically interestingnucleosides were discovered. Ashton et al . reported that the conformationallyconstrained acyclovir analog 608 showed similar anti-HSV-1 and 2 activities tothe parent nucleoside [222,223]. Tsuji and coworkers explored extensive SAR ofcarbocyclic nucleosides bearing a methylene spacer between the base and car-bocyclic ring [223,224]. The guanine derivative 609 was active against HSV-1


Figure 1.21 General structures of three-membered ring carbocyclic nucleosides and somerepresentative molecules.

Scheme 1.71 Synthesis of cyclopropyl nucleosides 606 and 607 [221]. Reagents andconditions: (a) (i) Pb(OAc)4; (ii) Ph3P = CHCOOMe; (iii) DIBAL-H; (b) ZnEt2, CH2I2;(c) (i) RuO2/NaIO4; (ii) ClCO2Et, Et3N; (iii) NaN3; (iv) BnOH, heat; (v) H2, Pd/C; (d)base construction, deprotection.

THREE-MEMBERED CARBOCYCLIC NUCLEOSIDES 75

and HSV-2 with EC50s of 0.0093–0.035 and 0.12–0.24 μg/mL, respectively, incomparison to 0.27–1.0 and 0.25–1.3 μg/mL for acyclovir and 0.54–2.0 and1.2–2.7 μg/mL for penciclovir. Furthermore, this nucleoside was 8- to 20-foldmore potent than acyclovir and penciclovir against VZV, and the selectivity indexof nucleoside 609 was also high. Studies demonstrated that 609 can be phospho-rylated by HSV-1 thymidine kinase (TK) very efficiently. As an extension of theresearch, a series of 5-substituted uracil derivatives were prepared, and some ofthe target nucleosides exhibited potent anti-VZV activity (Scheme 1.72) [225].Particularly, 5-bromovinyl nucleoside 610 was about 40-fold more potent thanacyclovir and had good oral bioavailability in rats (68.5%). The enantiomericsyntheses of compounds 609 and 610 were accomplished using chiral cyclo-propane lactone 621 as starting material. The key intermediate was condensedwith base moiety via classic SN2 reaction followed by deprotection/derivation toafford target nucleosides [225].

Zemlicka and coworkers described another type of interesting carbocyclicnucleosides, in which the spacer between the base and the ring is an unsatu-rated double bond [226,227]. Compounds, such as 611 and 612 Figure 1.21),displayed broad-spectrum antiviral activity.

The pair of enantiomers (611 and 612) was synthesized through enzymatic aswell as chemical resolutions (Schemes 1.73a and 1.73b). It was interesting to findthat nucleosides 611 and 612 exhibited equipotent anti-HCMV activity (EC502.9and 2.4 μM, respectively). However, compound 611 was somehow more potentthan 612 against HSV-1 and 2 with EC50s of 8.8 vs. 38 μM and 35 vs.>50 μM,respectively. On the contrary, compound 611 was less effective (EBV) or devoidof activity (HIV-1) in comparison to 612. Further modifications of the spacergenerated spiropentane nucleoside 613 (Scheme 1.73c). Although no antiviralactivity was found at the nucleoside level, phosphoralaninate nucleotide of 613showed significant antiviral activity against HCMV, HSV-1 and 2, VZV, andEBV HIV-1, as well as HBV, which indicated the inefficient phosphorylation ofspiropentane of nucleosides in vitro [228].

The same group also synthesized chiral E and Z-stereoisomers of (1,2-dihydroxyethyl) methylenecyclopropane analogues of 2′-deoxyadenosine and2′-deoxyguanosine and evaluate for their antiviral activity (Scheme 1.74)[229]. (R)-Methylenecyclopropylcarbinol (636) was converted in few stepsto reagents 642, which were used for alkylation-elimination of adenine and

Scheme 1.72 Synthesis of cyclopropyl nucleosides 609 and 610 [225]. Reagents andconditions: (a) (i) NaBH4; (ii) Ph2CN2, DDQ; (iii) LiBH4; (iv) CBr4, Ph3P, Et3N; (b)BVU, K2CO3; (c) HCl.


Scheme 1.73 Synthesis of cyclopropyl nucleosides 611–613 [226,227]. Reagents andconditions: (a) N2CHCO2Et, Rh(OAc)4; (b) K2CO3; (c) K2CO3, heat; (d) adenosinedeaminase, pH 7.5; (e) (i) Ac2O, Py; (ii) [ Me2N = CHCl]+ Cl−; (f) NH3/MeOH; (g)i -BuOCOCl, Et3N; (ii) (R)-2-phenylglycinol, separation; (h) (i) H2SO4; (ii) HCl, EtOH;(iii) Br2; (iv) DIBAL-H; (v) Ac2O, Py; (i) (i) K2CO3, adenine; (ii) NH3/MeOH; (j) (i)LAH; (ii) Ac2O, Py; (k) N2CHCO2Et, Rh(OAc)4; (l) (i) NaOH, separation; (ii) Ac2O,Py; (iii) (PhO)2P(O)N3, Et3N, tBuOH; (iv) K2CO3, aq. MeOH; (v) separation; (vi) HCl,MeOH; (m) base construction and deprotection.

2-amino-6-chloropurine to get ultimately analogues from 614–617. TheZ-isomer 615 was an inhibitor of plaque reduction assay against Towne andAD169 strain of human cytomegalovirus (HCMV) with EC50 of 6.8 and 7.5μM [229]. It was also active in murine cytomegalovirus (MCMV) assay (EC50of 11.5 μM). It was less active against HCMV with mutated gene UL97. Itinhibited Epstein-Barr virus (EBV) with EC50 of 8 μM. It is not cytotoxic, andits efficacy is somewhat lower than that of ganciclovir. None of the E-isomersof the present analogues were effective against HCMV.

1.4. FOUR-MEMBERED CARBOCYCLIC NUCLEOSIDES

A natural nucleoside, oxetanocin A 644 (OXT-A, Figure 1.22) is a four-member-ring nucleoside produced by Bacillus megaterium [230,231]. The broad-spectrum

FOUR-MEMBERED CARBOCYCLIC NUCLEOSIDES 77

Scheme 1.74 Synthesis of E and Z-(1,2-dihydroxyethyl)methylenecyclopropane Ana-logues of 2′-deoxyadenosine and 2′-deoxyguanosine by Zemlicka and coworkers[229]. Reagents and conditions: (a) (i) (COCl)2, DMSO, CH2Cl2,—60◦C; (ii) NaCN,NH4Cl, H2O, CH2Cl2; (b) Conc. HCl, CH2Cl2; (c) BnBr, K2CO3, Bu4NI, DMF; (d)DIBALH,THF, 0◦C; (e) (i) Ac2O, pyridine; (ii) pyridine.HBr3, CH2Cl2; (f) (i) adenine,K2CO3, DMF, 100–105◦C; (ii) K2CO3, MeOH; (g) 2-amino-6-chloropurine, K2CO3,DMF, 100–105◦C; (h) (i) NH3, MeOH; (ii) 80% HCOOH, 80◦C.

antiviral activity of the compound has prompted considerable attention to thisclass of nucleosides [232].

Preparation of carbocyclic analogs of the natural counterparts was first reportedby Honjo [233]. A [2+2] formation provided cyclobutane intermediate 655,which underwent a series of manipulations to afford cyclobutylamines 656. Theracemic C -OXT-A was constructed through a linear approach (Scheme 1.75a.).In the same year, the synthesis of optically pure C -OXT-G was accomplished byNarasaka and coworkers by using an asymmetric [2+2] addition as a key step(Scheme 1.75b) [234].

Among synthesized nucleosides, the guanine (646, C -OXT-G) and adenine(645, C -OXT-A) derivatives were active against HIV in ATH18 cells (EC50 1–2μM) [235]. In addition, the d-enantiomer of C -OXT-G (647, lobucavir, LBV)could be phosphorylated to its triphosphate by viral TK as well as protein kinase[236] and exhibited broad-spectrum antiviral activity against HBV and herpesviruses [237]. Lobucavir was advanced to clinical trials as an anti-HBV agentby the Bristol-Myers Squibb Phamaceuticals. However, the clinical studies weresuspended due to oncogenicity in rodents.

Further modifications based on the structure of C -OXTs generated series ofinteresting four-membered carbocyclic nucleosides. Monofluoro nucleoside (-)


Figure 1.22 Four-membered carbocyclic nucleosides.

648 showed significant antiviral activity against HSV-1 and 2 (EC50 0.7–1.8μM), VZV (EC50 1.8–3.5 μM), and HCMV (EC50 3.5–35 μM); however, itwas toxic to cells [238,239]. Removal of the 4′-methylene group of C -OXTs(649) resulted in a considerable decrease of anti-HSV and anti-VZV activityin comparison to the parent compounds [240]. Interestingly, the triphosphate ofnucleoside 650, which did not have a 2′-hydroxylmethyl group, was reported tobe active against wild-type HIV-RT as well as M184V mutant [241].

Novel spiro-carbocyclic nucleosides 651 and 652 have been prepared by Chuand coworkers via enzymatic resolution (Scheme 1.76). Both d- and l- nucleo-sides exhibited some anti-HIV activity with EC50 values of 22.4 and 48.6 μM,respectively, whereas l-enantiomer was less toxic than its d counterpart [242].

1.5. SIX-MEMBERED CARBOCYCLIC NUCLEOSIDES

Herdewijin and coworkers have prepared a number of cyclohexenyl and cyclohex-anyl analogs, such as nucleosides 668–672 [243–246]. However, no biological

SIX-MEMBERED CARBOCYCLIC NUCLEOSIDES 79

(a)

(b)

Scheme 1.75 Synthesis of racemic and optically pure C -OXT-A and C -OXT-G[233,234]. Reagents and conditions: (a) CH3CN, heat; (b) (i) LAH; (ii) BzCl, Py; (iii)p-TsOH, acetone; (iv) NH2OH; (v) H2, PtO2; (c) base construction and deprotection.

Scheme 1.76 Synthesis of optically pure spiro-carbocyclic nucleosides [242]. Reagentsand conditions: (a) P. cepacia lipase, AcOCH=CH2; (b) (i) Amberlite IR-120; (ii) TrCl,Py; (iii) TBDPSCl, Im; (iv) BF3 • OEt2; (v) Me3P(OPh3)I; (vi) DBU; (vii) TBAF; (c)Et2Zn, CH2I2; (d) Mitsunobu coupling and base derivations and deptrotection; (e) Ac2O,Py, then followed the procedure for compound 663.

activity was noticed with the exceptions of guanine derivatives of C3-hydroxylcyclohexenyl 671 and672, which were shown to be potent and highly selec-tive antiviral agents against herpes virus (HSV-1 and 2 and VZV) with EC50

vaules comparable to acyclovir and ganciclovir as shown in Table 1.23 [246].The NMR conformational studies suggested that the nucleosides antiviral activitywas correlated with their predominant conformation (Figure 1.23).


TABLE 1.23 Antiviral activity of d- and l-cyclohexenyl-G (671 and 672) incomparison with approved antiviral drugs [246]

671 672 Acyclovir GanciclovirVirus (IC50 μg/mL) (IC50 μg/mL) (IC50 μg/mL) (IC50 μg/mL)

HSV-1 (KOS)a 0.002b 0.003b 0.01b 0.001b

HSV-1 (F)a 0.002b 0.003b 0.003b 0.001b

HSV-1 (McIntyre)a 0.004b 0.004b 0.005b 0.001b

HSV-1 (TK− KOSACV)a

0.38b 1.28b 9.6b 0.48b

HSV-1 (TK−/TK+VMW1837)a

0.01b 0.01b 0.07b 0.01b

HSV-2 (G)a 0.05b 0.07b 0.02b 0.002b

HSV-2 (196)a 0.07b 0.1b 0.02b 0.001b

HSV-2 (Lyons)a 0.07b 0.07b 0.02b 0.001b

VZV (YS)c 0.49d 1.2d 1.1d NDe

VZV (OKA)c 0.64d 1.9d 0.8d NDVZV (TK− 07/1)c 2.1d 5.8d 13d NDVZV (TK− YS/R)c 2.8d 6.8d 28d NDCMV (AD 169)c 0.6d 1.5d ND 0.6d

CMV (Davis)c 0.8d 1.7d ND 0.8d

aActivity determined in E6SM cell cultures.bMinimum inhibitory concentration (μg/mL) required to reduce virus-induced cytopathogenicity by50%. Virus input was 100 50% cell culture infective doses (CCID50).cActivity determined in HEL cells.dMinimum inhibitory concentration (μg/mL) required to reduce virus plaque formation by 50%.Virus input was 20 plaque-forming units (PFU).eND: not determined. The values are means of two independent determinations.

Figure 1.23 Six-membered carbocyclic nucleosides.

SIX-MEMBERED CARBOCYCLIC NUCLEOSIDES 81

Recently, it has been described that both enantiomers of cyclohexenylguanine671 and 672 display potent and selective anti–herpes virus activity (HSV-1, HSV-2, VZV, CMV) [246]. It is well known that opposite enantiomers can display dif-ferent pharmacological and toxicological properties. Hence, Alibs and coworkersreport an enantiodivergent approach to d- and l-4-hydroxycyclohexenyl nucleo-sides 673–676 [247], starting from the common intermediate 679, which bearsa hydrobenzoin moiety as the chiral auxiliary (Schemes 1.77 and 1.78). They

Scheme 1.77 Synthesis of Cyclohexenyl adenine nucleoside 673 and 674 [247]. Reagentsand conditions: (a) (R,R)-hydrobenzoin, p-TsOH, benzene, reflux; (b) (i) Br2, ether, from10◦C to 0◦C; (ii) DBU, dioxane, 100◦C; (iii) (S)-2-Me-CBSCB, CH2Cl2, −78◦C to rt;(c) 6-chloropurine, DBAD, PPH3, THF, −10◦C to rt; (d) (i) TFA-H2O (14:1), 0◦C; (ii)(R)-2-Me-CBS CB, CH2Cl2, −78◦C to rt; (e) (i) p-NO2C6H4COOH, DBAD, PPH3, THF,−78◦C to rt; (ii) NH3, MeOH, 90◦C.

Scheme 1.78 Synthesis of Cyclohexenyl uracil nucleoside 675 and 676 [247]. Reagentsand conditions: (a) N3-benzoyluracil, DBAD, PPh3, THF,—10◦C to rt; (b) (i) TFA-H2O(14:1), 0◦C; (ii) (S)-2-Me-CBS CB, CH2Cl2,−78◦C to rt; (c) (i) p-NO2C6H4COOH,DBAD, PPh3, THF,−78◦C to rt; (ii) Me2 NH, EtOH; (d) (i) ClCO2Et, Py, DMAP, CH2Cl2,rt; (ii) N3-benzoyluracil, (η3 − C3H5PdCl)2, dppe, DMF, 80◦C; (e) (i) TFA-H2O (14:1),0◦C; (ii) (R)-2-Me-CBS CB, CH2Cl2,−78◦C to rt.


have designed a divergent synthesis that involves two main transformations: (1)introduction of the nucleobase with either inversion (Mitsunobu methodology) orretention (Pd-catalyzed coupling reaction) of configuration, followed by removalof the chiral auxiliary, and (2) stereoselective reduction of the carbonyl groupto deliver the target cyclohexene nucleosides with the cis relative configuration.As a preliminary test, the new synthesized nucleoside analogues were evaluatedon MT4 cells for anti-HIV-1 activity against wild-type NL4-3 strain as well ascytotoxicity using AZT and AMD3100 as the positive control. Unfortunately,none of the compounds was found anti-HIV active.

1.6. CONCLUSION

Carbocyclic nucleosides have been a subject of great interest in the syntheticmedicinal chemistry for the past decades. Particularly, the discovery of aba-cavir and entecavir as clinically effective antiviral agents prompted the studiesof various carbocyclic nucleosides. Although the synthesis of carbocyclic nucle-osides has advanced dramatically, more efficient and practical methods are stillin demand for the preparation of biologically active compounds as well as chi-ral key intermediates. In the future, novel biologically interesting carbocyclicnucleosides will likely be continuously discovered to improve the existing list ofchemotherapeutic agents.

1.7. ACKNOWLEDGMENT

This article was partially supported by the grant (AI 25899) from National Insti-tute of Allergy and Infectious Diseases, NIH.

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