Nucleosides with Transposed Base or 4′-Hydroxymethyl Moieties

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Nucleosides with Transposed Base or 4′-Hydroxymethyl Moieties and Their Corresponding Oligonucleotides Kiran Toti,†,§,∥ Marleen Renders,‡,∥ Elisabetta Groaz,‡,∥ Piet Herdewijn,‡ and Serge Van Calenbergh*,† †

Laboratory for Medicinal Chemistry, Faculty of Pharmaceutical Sciences, Ghent University, Ottergemsesteenweg 460, 9000 Ghent, Belgium ‡ Laboratory for Medicinal Chemistry, Rega Institute for Medical Research, Katholieke Universiteit Leuven, Minderbroedersstraat 10, 3000 Leuven, Belgium ABSTRACT: This review focuses on 4′-hydroxymethyl- or nucleobase-transposed nucleosides, nucleotides, and nucleoside phosphonates, their stereoisomers, and their close analogues. The biological activities of all known 4′-hydroxymethyl- or nucleobasetransposed nucleosides, nucleotides, and nucleoside phosphonates as potential antiviral or anticancer agents are compiled. The routes that have been taken for the chemical synthesis of such nucleoside derivatives are described, with special attention to the innovative strategies. The enzymatic synthesis, base-pairing properties, structure, and stability of oligonucleotides containing nucleobase- or 4′-hydroxymethyl-transposed nucleotides are discussed. The use of oligonucleotides containing nucleobase- or 4′hydroxymethyl-transposed nucleotides as small oligonucleotide (e.g., human immunodeficiency virus integrase) inhibitors, in applications such as antisense therapy, silencing RNA (siRNA), or aptamer selections, is detailed.

CONTENTS 1. Introduction 2. Biological Activity of Nucleobase- or 4′-Hydroxymethyl-Transposed Nucleosides and Nucleotides 2.1. Scope of This Section 2.2. Nucleosides with a Transposed 4′-Hydroxymethyl Group 2.3. Apionucleoside Phosphonates 2.4. Isonucleosides 2.5. Isonucleoside Phosphonates 2.6. Homonucleosides and Homonucleoside Phosphonates 2.7. C-Nucleosides and Nucleosides with Extremely Modified Non-Natural Bases 3. Chemical Synthesis of Nucleobase- or 4′-Hydroxymethyl-Transposed Nucleosides and Nucleotides 3.1. Scope of This Section 3.2. Apio- and 2′- and 3′-Deoxyapionucleosides 3.3. 2′,3′-Dideoxyapio-, 2′,3′-Dideoxy-4′-thioapio-, and 2′,3′-Didehydro-2′,3′-dideoxyapionucleosides 3.4. Rigidified and 2′-Substituted Apio- and 4′Thioapionucleosides 3.5. Substituted 2′,3′-Dideoxyapio- and 2′,3′Dideoxy-4′-thioapionucleosides 3.6. Isonucleosides and Deoxyisonucleosides 3.7. 3′- and 4′-Substituted Isonucleosides and 4′-Thioisonucleosides 3.8. Extra Transposed Nucleosides © XXXX American Chemical Society

3.9. Hydroxymethyl-Deleted and Base-Transposed Nucleoside Phosphonates 4. Oligonucleotides Containing Nucleobase- or 4′Hydroxymethyl-Transposed Nucleotides 4.1. Scope of This Section 4.2. Apionucleic Acids (apioNAs) 4.2.1. Enzymatic Polymerization of Apionucleotides 4.3. Apionucleoside Phosphonate Oligomers 4.3.1. Enzymatic Polymerization of 3′-O-(Phosphonomethyl)-L-threofuranosyl Nucleotides 4.4. Isonucleic Acids (INAs) 4.4.1. Base-Pairing Properties 4.4.2. Structure 4.4.3. Enzymatic Stability 4.4.4. Biological Activity 4.4.5. Enzymatic Polymerization 4.5. 3′−2′-α- L -Threofuranosyl Nucleic Acids (TNAs) 4.5.1. Base-Pairing Properties 4.5.2. Structure 4.5.3. Nonenzymatic Polymerization 4.5.4. Enzymatic Polymerization 4.5.5. Biological Activity 4.5.6. Considerations on the Origin of Life and the “Pre-RNA World” 5. Summary and Concluding Remarks

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Received: September 21, 2015

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Review

NMP is converted to the nucleoside 5′-diphosphate (NDP) (or nucleoside phosphonate to NP-MP) by the action of nucleoside monophosphate kinases (NMPKs). Subsequent phosphorylation by NDPKs yields the NTP (or NP-DP). The triphosphate analogue interacts with viral DNA/RNA polymerase either as a competitive inhibitor or as a substrate. Since antiviral nucleosides often lack a 3′-hydroxyl group, their incorporation eventually leads to DNA/RNA chain termination. Due to the close structural similarity of the triphosphorylated antiviral nucleosides and the natural NTPs, the former may also interact with host cell DNA/RNA polymerases, resulting in toxicity. This is the main cause of mitochondrial toxicity. The toxicity may be diminished to a large extent in the case in which the virus produces its own kinases, making it possible to design viral kinase/polymerase-specific nucleosides. Non-natural Lnucleoside analogues have also proven successful in lowering mitochondrial toxicity due to differential uptake by mitochondrial transporter proteins.6 In recent years, nucleoside analogues have also been found useful to inhibit crucial enzymes such as S-adenosylhomocysteine (SAH) hydrolase, viral/cellular RNA helicase, and inosine monophosphate dehydrogenase. Also other applications of nucleoside analogues, for instance, to modulate purinergic Gprotein-coupled receptors, prove promising with several compounds under clinical investigation.13 Nucleoside analogues are also being developed as inhibitors of enzymes that play a crucial role in bacteria responsible for emerging infectious diseases.14 In naturally occurring nucleosides and nucleotides, the purine or pyrimidine base is attached to C-1′ of ribose or 2deoxyribose, while the hydroxymethyl group is attached to C4′. This review will cover the biological activity and synthetic accessibility of nucleosides in which either the 4′-hydroxymethyl group (or the 4′-(phosphonooxy)methyl group or an isoster of the latter) or the base moiety has been transposed to the neighboring (or another) carbon (Figure 3). The former class will essentially focus on so-called apionucleosides, which are derived from the plant-specific 3-branched-chain monosaccharide apiose. To evade the aminal structure, which is present in natural nucleosides and prone to hydrolytic and enzymatic cleavage, analogues were conceived in which the base moiety is transposed. Such analogues are commonly referred to as isonucleosides, a term first introduced by Montgomery et al. in 1976.15 Although both types of modifications have been extensively explored, reviews on this topic are absent, with the exception of a review by Nair and co-workers which is restricted to isomeric dideoxynucleosides of D- and L-related stereochemistry and already stems from 1995.16 In this paper, we review the effects of such modifications (and additional manipulations of the sugar and base moieties) on the biological activity and relevant synthetic pathways toward the target compounds. In section 4 we discuss the bioorganic chemistry of oligonucleotides containing iso- and apionucleotides.

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1. INTRODUCTION Nucleosides consist of a heterocycle (often called a nucleobase) attached to a sugar moiety through a glycosidic bond (Figure 1). When a nucleoside contains a phosphate residue at any oxygen of the sugar, it is termed a nucleotide. Endogenous nucleosides generally contain one of the five natural nucleobases bound to a D-ribofuranose or D-2′-deoxyribofuranose sugar to form RNA and DNA building blocks, respectively.1 These molecules are biosynthesized via two pathways. In the de novo pathway,2 nucleosides are built up from basic small molecules. Purine nucleosides are constructed from activated carbohydrates, amino acids, N10-formyl-THF (as a formate source), and carbonic acid. Pyrimidines are made from carbamoyl phosphate, amino acids, activated carbohydrate, and N10-formyl-THF (as a 5-methyl donor). The major difference between the purine and pyrimidine pathways is that the former are built on the sugar moiety, while the pyrimidine core structure is built first and later attached to sugar, to form different pyrimidine nucleosides. In the salvage pathway,3 nucleosides are recovered from degradation products. Comparatively, the latter is less energy demanding. Nucleos(t)ides are the basis of genetic material, play an important role in signaling pathways, and are also involved in energy transfer and storage. The chemical modification of nucleosides has proven to be and remains a valuable strategy toward antiviral and anticancer drugs.4 Modified nucleosides were first used in cancer chemotherapy. Cytarabine (also known as arabinofuranosyl cytidine or ara-C) was approved by the FDA in 1969. Today several other nucleosides (fludarabine, cladribine, clofarabine, gemcitabin, 5-F-dU, 2-deoxycoformycin, capecitabine, nelarabine, and decitabine) are clinically used for the treatment of a variety of hematological malignancies. The use of nucleosides as antivirals became widespread only after the discovery of the human immunodeficiency virus (HIV), which fueled antiviral drug development.5 At present, there are more than 25 approved nucleoside/nucleotide analogues in use as antivirals (for a table refer to ref 4). Generally, these nucleos(t)ides closely resemble natural nucleosides, but may contain slightly modified bases and sugars, non-natural L-sugars, bis-heterosugar moieties, and acyclic or carbocyclic sugar surrogates.6−10 In a strict sense, most nucleoside drugs are prodrugs. They are metabolized by cellular or viral kinases to their corresponding nucleoside 5′-triphosphates (NTPs), which are the actual inhibitors of DNA or RNA polymerases (Figure 2).8 A nucleoside first undergoes phosphorylation by nucleoside kinases to its 5′-monophosphate (NMP). Very often, this is the rate-determining step in the activation process, 2′,3′-dideoxy3′-azidothymidine (AZT) being a well-known exception.11 Nucleoside phosphonates (NPs) or prodrugs have been developed to bypass this first phosphorylation step.12 The B

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Figure 1. Structure of nucleos(t)ides.

Figure 2. General mode of action of nucleosides/phosphonates.

2. BIOLOGICAL ACTIVITY OF NUCLEOBASE- OR 4′-HYDROXYMETHYL-TRANSPOSED NUCLEOSIDES AND NUCLEOTIDES

nomenclature of the cyclic forms of apiose unconventionally requires the use of two chirality designations (D, L) to distinguish between the two possible C-3-epimers. Alternatively, the prefixes “threo” and “erythro” may be used. DApio-D-furanose (3b) then corresponds with 3-C-(hydroxymethyl)-D-erythrofuranose. In the literature, the natural D-apioD-furanose is normally simply referred to as “D-apiose”. In this paper, the term “D/L-furano” is used to unambiguously specify the configuration at C-3. The sugar attachment position for purines (e.g., A and G) is N-9 and that for pyrimidines (e.g., C, T, and U) is N-1 unless noted otherwise. Apiose derivatives, including nucleosides such as 1′-adenin-9yl β-D-apio-D-furanoside (4a; Figure 6), are found in plants.17 The first syntheses of apionucleosides were reported by Riest et al.18 and Carey et al.19 Detailed studies on the chemistry, physical properties, and evaluation of apionucleosides were carried out by Tronchet and colleagues. It was demonstrated that apionucleosides mainly adopt the E3′ (S-type) conformation and that the β-D-apio-D-furanoadenine nucleoside 4a is

2.1. Scope of This Section

In this section, the biological activities of all known 4′hydroxymethyl-transposed (Figure 4, structure 1) and nucleobase-transposed (representative structure 2) nucleosides, nucleotides, and nucleoside phosphonates are compiled, including those of stereoisomers and close analogues. 2.2. Nucleosides with a Transposed 4′-Hydroxymethyl Group

Apiose (3a) is one of the best known C-branched natural sugars. D-Apio-D-furanose can be considered as a regioisomer of D-ribofuranose in which the hydroxymethyl group is moved from the 4-position to the 3-position. Noteworthy, apiose has one asymmetric center in the open form but acquires an extra asymmetric carbon at position 3 in the cyclic furanose form (in addition to the anomeric center) (Figure 5). Hence, the C

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Most 2′,3′-dideoxydidehydroapionucleosides 16a−f show moderate to potent anti-HCMV activity without cytotoxicity, the 5-fluorouracil derivative 16c being the most potent (EC50 = 30.2 μM in AD-169 cells and 12.3 μM in Davis cells). However, the corresponding thioapio analogues lack significant antiHCMV activity.38 Thioapio-2′,3′-dideoxynucleosides 17a−g and their anomers 18a−g (both tested as enantiomeric mixtures) were assayed against HIV-1, but found unpromising due to cytotoxicity. In particular, the 6-Cl-purine analogues 17e and 18e show cytotoxicity in MT4-cells (CC50 < 8.5 and 5 μM, respectively). This led the authors to assess the potential of 6chloropurine analogue 18e in colon-2 cancer cells (IC50 = 14 μM).39 Jeong et al. synthesized a series of N-type apionucleosides (i.e., 20a−d and 21a−d; Figure 7), somewhat related to the potent antiherpetic agent (N)-methanocarba-T (19). 20b demonstrates weak toxicity-dependent anti-HIV-1 and antiHIV-2 activity (EC50 = 190.6 μM).40 The 2′,3′-cyclopropylfused α-nucleosides 22a−d are devoid of antitumor activity, but no antiviral data have been reported.41 Compounds 23a−c, which may be considered as ring-expanded oxetanocin analogues, and unusual azasugar nucleosides 24a−c are inactive against HIV-1, HSV-1, and HSV-2.42,43 Likewise, the 2′-azido and fluoro analogues 25a,b were found inactive against a panel of viruses.27,28 This was also the case for the racemate of 26 and its anomers.44 The 4′-thio analogues 27a,b are inactive against HIV-1, HSV-1, and HSV-2.42,43 No biological data have been reported for compounds 28a,b and their enantiomers.45 Compound 29a (Figure 8) shows interesting activity against HIV (EC50 = 2.55 μM). Remarkably, its anomer 30a shows even much improved activity, but also significant toxicity (EC50 = 0.073 μM and IC50 = 1.0 μM in PBM cells). None of these compounds show any significant anti-HBV activity up to a concentration of 100 mM.46 Biological data for their 4′-thio congeners are not presented.47 Compound 32b is weakly active against HIV-1 (EC50 = 72.6 μM) and HCMV (EC50 = 187.6 μM), whereas the T and U anomers (31a,b) showed inferior anti-HIV-1 activity (EC50 = 238.6 and 331.2 μM, respectively).48 Replacing the 3′-vinyl moiety in 32b by a phenyl group renders the molecule inactive.49 Substituting this vinyl by a hydroxymethyl group resulted in (±)-9-(4,4-bis(hydroxymethyl)tetrahydrofuran-2-yl)adenine (33a), which demonstrates weak anti-HIV-1 activity (EC50 = 75.8 μM), while its thymine congener is even weaker (199 μM).50,51 The racemic mixture of 34a was found active against HBV (EC50 = 3.5 μM). The (R,R)-isomer was synthesized stereoselectively and found to be inactive; hence, it was inferred that the activity is due to the (S,S)-enantiomer or L-apio enantiomer. No other analogues of this type show activity against HBV, HIV-1, HSV-1, HSV-2, HCMV, and poliovirus or cytotoxicity.52−54 The 3′-methoxy- and 3′-fluoro-2′,3′-dideoxyapionucleosides 37−40 are among the most promising in the apionucleoside class. These compounds are highly active against HBV. In vitro activity data are reported for selected compounds only (T, C, 5F-C, and 5-I-U). Among these, cytosine analogue 39e is the most potent (EC50 = 0.011 μM) with a selectivity index of over 10000-fold (control compound 2′,3′-dideoxycytidine, EC50 = 5.57 μM).55−58 Notably, enantiomers of 39e and 40e (and their adenine analogues) do not show significant activity against HIV, HSV-1, HSV-2, and poliovirus, but oddly enough their ability to inhibit HBV is not reported.59

Figure 3. Nucleoside modifications discussed in this review.

Figure 4. General structure of 4′-hydroxymethyl-transposed (1) and nucleobase-transposed (2) nucleosides and nucleoside phosphonates.

Figure 5. D-Apiose and its C-3-epimers.

resistant to adenosine deaminase, while its α-D-apio-L-furano counterpart 5a is a substrate of this enzyme.20−22 Later studies showed that the pyrimidine counterparts 4b−d are inactive against a panel of viruses (e.g., HIV-1, HIV-2, HSV-1, HSV-2, VV, and VSV).23 Watson et al. evaluated the epimers 5a−e against T-lymphocyte proliferation and HSV.24 Compound 5a significantly reduces HSV-2 plaque formation in BHK cells (42% reduction at 4 μM), while 4a does not. All investigated αD-apio-L-furanonucleosides (5a−e) inhibit rat T-lymphocyte and human MGL8 lymphoblast proliferation. 2′-Deoxyapionucleosides with natural pyrimidine bases (6a− c) were reported to be inactive against a panel of viruses and slightly toxic to Vero cells. Also the enantiomeric 2′-deoxy compounds 7a−c and their anomers 8a−c were also found inactive.23,25 The D- and L-3′-deoxyapionucleosides 9a−e and 10a−c failed to inhibit DNA or RNA viruses (e.g., HIV-1, HIV-2, HSV-1, HSV-2, VV, and VSV) and, unlike their 2′-deoxy regiomers 6a−c, show no cytotoxicity.26 Later Jung et al. found 9b active against HSV-1(KOS) (MIC50 = 39.6 μM) and 9e active against HSV-1 in two different cell lines, i.e., KOS and 2(G) (MIC50 = 7.1 and 35.9 μM, respectively).27,28 The cytidine congener 11 was shown to be inactive against HIV.29 Initially, the adenine analogue 13a (L-furano-L-dideoxyapioadenosine) was reported to show weak anti-HIV activity in MT4 cells (data not provided),30 but subsequently, it was found that series 12, 13, and 14 are neither cytotoxic nor active.31−34 Recently, we have discovered that phosphoramidate prodrugs of 12a inhibit HIV replication in vitro. In particular, the benzyl alanilate ProTide analogue of adenine (12a) showed noticeable anti-HIV-1,2 activity (EC50 = 0.5 and 1−1.5 μM, respectively) with a moderate selectivity index, and the corresponding triphosphate acted as a viral DNA chain terminator. These data suggest that the inactivity of 2′,3′-dideoxy-D-apionucleoside 12a, and possibly also related compounds, is due to an inefficient first intracellular phosphorylation step. Surprisingly though, ProTides of dideoxyapiothymine analogues did not inhibit HIV at 250 μM.35 The homologous 2′,3′-dideoxyapioadenosine (15) and its enantiomer were found to be inactive, resistant to adenosine deaminase, and hydrolytically more stable than 2′,3′-dideoxyadenosine (ddA).36,37 D

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Figure 6. Overview of the apio-, 2′- or 3′-deoxyapio-, 2′,3′-dideoxyapio-, 2′,3′-didehydro-2′,3′-dideoxyapio-, and 2′,3′-dideoxy-4′-thioapionucleosides discussed in the text.

of 43a is 2-fold weaker compared to 42a against all viruses. Among the pyrimidine analogues, only compound series 42 show anti-HCMV activity, 42c, 43c, and 43b show anti-HSV-1 activity, and 42b and 42d show anti-HSV-2 activity.60 Compound 44a inhibits the replication of HCV (IC50 = 19 μM).61

In comparison to 33a, 41a completely loses its activity, while the cytidine derivative 41d gains activity against HIV-1 (EC50 = 75.0 μM) and HCMV (EC50 = 149.2 μM).50,51 If we take into account the data from compounds 29 to 40, it is evident that the 3′-substitution is a crucial feature for activity, possibly by imposing a favorable conformation or molecular orientation, e.g., for efficient enzymatic conversion to the corresponding triphosphates and/or incorporation of the latter by reverse transcriptase (RT) or DNA polymerase. Compound 42a is moderately potent against HIV-1 (EC50 = 36.2 μM) and also shows moderate HSV-1 (EC50 = 116.7 μM) and HCMV (EC50 = 148.9 μM) activity. The antiviral activity

2.3. Apionucleoside Phosphonates

Herdewijn et al. were the first to report the synthesis and encouraging anti-HIV properties of L-2′-deoxythreose nucleoside phosphonates 46a−d (Figure 9),62 which may be considered bioisosteres of 2′,3′-dideoxy-D-apionucleoside E

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Figure 7. Rigidified and modified apionucleosides and 4′-thio analogues.

monophosphates. Specifically, the adenine and thymine analogues showed promising anti-HIV-1 and anti-HIV-2 activity (EC50 = 2.36 and 6.59 μM, respectively) without significant toxicity (>320 μM). The experiments were carried out using adefovir (9-(2-(phosphonomethoxy)ethyl)adenine, PMEA; HIV-1 EC50 = 5.5 μM, HIV-2 EC50 = 14.0 μM) and tenofovir (R)-9-(2-(phosphonomethoxy)propyl)adenine, PMPA; HIV-1 EC50 = 3.4 μM; HIV-2 EC50 = 3.5 μM)) as positive controls. The diphosphate derivative of 46a (9-(3-O(phosphonomethyl)-2-deoxythreosyl)adenine, PMDTA) is incorporated by human DNA polymerase α only at an enzyme concentration 100 times higher than required for the dATP substrate. On the other hand, HIV RT accepts this diphosphophosphonate readily with only 2.5 times slower kinetics compared to dATP. This makes 46a a promising candidate for further development. Its diastereomer 47 and its enantiomer 48 are inactive against HIV.63 Interestingly, substituting the 3′-oxygen with a methylene decreases the efficacy (49a; EC50 = 12.6 μM) while increasing the toxicity (30.4 μM in CEM cells).64 The 3′-thioether 5065 and homologues 51a,b66 did not exhibit anti-HIV, anti-HCV, and anti-RSV activities or cytotoxic properties. Although the 2′-hydroxy analogues 52a−d,67 53a,b,65 and 54a,b66 failed to inhibit viral replication, analogues 52a−d were readily accepted by the Therminator polymerase to form oligophosphonates. Many 3′-substituted erythro analogues were reported, such as the unsaturated phosphonates 55a,b and 56, the cyclic apiosyl phosphonates 57a,b, and the 3′phosphonopropyl-modified β-D-erythrosylnucleosides 58a,b. None of these exhibit anti-HIV, anti-HCV, and anti-RSV activities or have cytotoxic properties.68 The 2′-azido derivative of 46a (59) was also found inactive against HIV.63

Several 2′-deoxy-3′-substituted apio phosphonates have been reported. Racemic 60a is moderately active against HIV-1 (22.2 μM) but suffers from cytotoxicity in PMB (42.4 μM) and CEM (30.4 μM) cells.69 The recently reported 3′-trifluoromethyl phosphonates 61a,b and 62a,b are also moderately active against HIV-1 with IC50 values ranging from 36 to 53 μM.70 Similar to compound 49a, 64b combines significant anti-HIV-1 activity (EC50 = 10.2 μM) with cytotoxicity (IC50 = 45.5 μM in PMB and 32.0 μM in CEM cells). Other phosphonates (60b, 63a,b, 64a, and 65a,b) exhibit weak anti-HIV-1 activity (EC50 = 45−80 μM) without cytotoxicity (>100 μM). A modeling study points toward a shift of the phosphonate moiety of 64b compared to that in PMDTA, while 60a shows an additional slight shift of the base.69,71 Compounds 66−68 are all inactive against HIV. The diphosphate derivative of the locked phosphonate 67 is ineffectively incorporated by HIV RT. Only at very high substrate and enzyme concentrations (400 μM and 1.44 U/ μL), 18% of the template could be elongated.63 2.4. Isonucleosides

In an effort to discover nucleosides that can withstand chemical and enzymatic glycosidic cleavage, Montgomery and Thomas synthesized the first isonucleosides 69a and 69b (Figure 10).72 They were found inactive against murine leukemia L1210 cells. Later studies showed that compound 69a demonstrates reasonable activity against HSV-1 and HSV-2 (EC50 = 78.8 and 93.9 μM, respectively) and HCMV (EC50 = 76.4 μM), in addition to L1210 cell toxicity (IC50 = 71.6 μM). The guanine analogue 69d is 4 times more potent than 69a against HSV-1 and HSV-2 (EC50 = 15.3 and 22.8 μM, respectively) without noticeable cytotoxicity, while the other bases are inactive.73 The inhibition of HSV-1 by the enantiomeric 70a and 70c is weaker (IC50 = 124 and 68.5 μM, respectively), while 70c also F

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Figure 8. Substituted 2′,3′-dideoxyapionucleosides.

exhibits weak anti-HSV-2 activity (IC50 = 137 μM). These compounds showed interesting antiproliferative effects on human promyelocytic leukemia (HL-60) cells (ED50 = 5.08, 1.6, and 8.06 μM for 70a, 70c, and 70d, respectively). Additionally, the corresponding monophosphate esters display antileukemia activity. With ED50 values of 4.5 and 9.3 μM, compounds 71b and 71d clearly surpass the activities of 71a and 71c (ED50 ≥ 170 μM). It is inferred from the 1H−1H coupling constant that these compounds adopt an S-type conformation (in DMSO).74 The triphosphate derivatives of 70a−d are readily accepted by many polymerases (thermostable) and act as chain terminators.75 The isonucleoside 5′homologues 72a−e and their enantiomers 73a−e76,77 failed to show promising anti-HIV activity. Ten years after the first synthesis of isonucleoside 69a, its 3′deoxy enantiomer, known as (R,R)-isoddA (74), was reported to possess promising anti-HIV activity (ED50 = 5−20 μM in

different cell lines).78 The guanine counterpart is less active, while other bases do not show any anti-HIV activity. Compound 74 was shown to be highly resistant to adenosine deaminase and to be activated intracellularly to its triphosphate more efficiently than ddA.79 Turning 74 into phosphoramidate and SATE (S-acyl-2-thioethyl) prodrugs increases the anti-HIV activity almost 400-fold.80 In fact, the ProTide of 74 is as effective as AZT (zidovudine) (EC50 = 0.04 μM, MT-4 cells) against HIV-2. However, this ProTide also possesses considerable toxicity with a selectivity index of only 65.81 Additionally, compound 74 displays weak binding affinity for A2A and A3 receptors, while it was found to be inactive at the A1 receptor.82 Interestingly, the triphosphate of the 8-azaadenine congener 75 inhibits recombinant RT almost 8 times more effectively (IC50 = 4.3 μM) than the triphosphate derivative of 74 (IC50 = 35 μM), while 74 is much more potent in acutely infected cells. G

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Figure 9. Threose and erythrose nucleoside phosphonates.

This anomaly could be due to a different uptake of the two compounds in infected cells.81 The dideoxyisoguanine 76 is marginally active against HSV-1 and HSV-2 (MIC50 = 8−16 μM) and virtually inactive against vaccinia virus and thymidine kinase (TK)-deficient mutants of HSV-1, a trend that is similar to that of acyclovir.83 Nair and co-workers reported that (S,S)-isoddA (78) displays 20 times higher anti-HIV-1 (IC50 = 0.67 μM) activity than its enantiomer 74.84,85 The inhibition of HIV-1 RT by the triphosphate of 78 (Ki = 16 nM) is comparable to that caused by the triphosphate derivative of AZT (Ki = 4 nM). (S,S)IsoddATP and ddATP exhibit complementary inhibitory activities of human DNA polymerases. ddATP is a strong inhibitor of DNA polymerase β and γ (Ki = 1.1 and 0.018 μM, respectively), whereas isoddATP effectively inhibits DNA polymerase α (Ki = 0.63 μM). (S,S)-IsoddA is insensitive to deaminases and even acts as a weak inhibitor of this enzyme. However, it was found to inhibit the growth of human bone marrow progenitor cells. Isonucleoside 78 also exhibits HBV activity in vitro (IC50 = 3.3 μM, CC50 = ∼140 μM) but shows in vivo toxicity.86 Compound 78 predominantly adopts a northern conformation in solution (58%), while it appears to be in the southern conformation in the crystal structure.87,88 In solution, nucleosides with anti-HIV activity typically adopt the southern conformation.89 Base modifications, as in 79a−d, yielded inactive or marginally active compounds.90,91

Isonucleosides with tricyclic bases inhibit HIV integrase, which is responsible for incorporating viral double-stranded DNA into the host chromosomal genome. Integrase recognizes the specific sequence (5′-ACTG···CAGT-3′) in viral DNA and removes the CA dinucleotide (underlined in the sequence, the 3′-processing step), before transferring the viral DNA into the host DNA by creating a specific incision into the host chromosomal DNA (strand-transfer step). Monophosphate 80 inhibits this strand-transfer step (IC50 = 68 μM), and 82 inhibits both the 3′-processing and the strand-transfer steps (IC50 = 75 and 53 μM, respectively).92 BVisoddU (83; BV = bromovinyl) inhibits HSV-1 replication at concentrations between 6 and 30.3 μM, which is almost 100fold higher than the concentration range at which 5-(E)(bromovinyl)deoxyuridine (BVDU) inhibits HSV-1 replication. Like BVDU, 83 is not active against HSV-2 strains VV, VZV, VSV, and CMV. Since BVDU and BVisoddU express binding strengths relatively similar to that of thymidine kinase, it is inferred that the difference in activity is probably related to different effects on the level of the HSV-1 DNA polymerase.93,94 Nair and co-workers described the synthesis of 84a−c,76 a homologue of the potent anti-HIV compound isoddA, and 85a,b,95 a 2′,3′-vinylic analogue, none of which significantly inhibit HIV (Figure 11). Likewise, the 3′-α-methylisonucleoside 86 lacks activity against HIV-1, HSV, VZV, and CMV.96 H

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Figure 10. Isonucleosides and deoxyisonucleosides.

Interesting results were obtained with the ring-expanded oxetanocin analogues 87a−d.97 The adenine congener 87a displays potent ganciclovir-like broad-spectrum antiviral activity against HCMV (EC50 = 4.4 μM), HSV-1 and HSV-2 (EC50 = 33.9 and 37.7 μM), and HBV (EC50 = 2.6 μM). It also exerts antiproliferative activity against several cancer cell lines (L1210, IC50 = 10.2 μM; KB cells, IC50 = 196 μM). The guanine analogue 87c selectively inhibits HSV-1 and HSV-2 replication (EC50 = 7.8−10 μM). Compounds 87b and 87d display activity against HSV-1, HSV-,2 and HCMV in the 56.8−117.3 μM range, while 87d also shows anti-HBV activity (EC50 = 23.5 μM). The corresponding (bromovinyl)uridine analogue 88 displays very potent activity against different strains of HSV and VZV (submicromolar to double-digit nanomolar concentrations) without a cytostatic effect, thereby being more effective than acyclovir against VZV. Because of its activity against simian varicella virus, extensive animal studies were carried out

in African green monkeys, but further development seems to have halted.98 Only the L-thymidine analogue 89b exhibits antiHIV activity (EC50 = 0.8 μM, IC50 > 200 μM, TI50 > 250).99 In general, exocyclic methylene compounds (90a−e, 91a−e, 100a−f) and spirocyclopropyl compound 92 lack anti-HIV activities possibly due to their increased rigidity. Compound 90 also lacks activity against other viruses.100,101 Their enantiomeric compounds 91a−e are inactive against HIV-1, HSV-1, and HSV-2, but show good activity against HCMV and, surprisingly, also against HBV. With an EC50 value of 47.3 μM, the uracil derivative 91b remains more than 10-fold less active against HCMV than ganciclovir in AD-169 cells, while the adenine derivative 91a expresses moderate activity against HCMV (EC50 = 134.7 μM) in addition to interesting anti-HBV activity (EC50 = 1.5 μM). The spironucleoside 92 lacks activity against HIV-1, HSV, VZV, and CMV.96 I

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Figure 11. Series of 2′- and 4′-substituted isonucleosides and their 4′-thio congeners.

The AZT mimic 93a102,103 fails to show promising anti-HIV activity. The synthesis of diastereomers 94 is reported without biological evaluation. 104 The locked nucleosides 95a,b, designed as S-type conformers of lamivudine, are inactive against HIV-1 and HSV-1.105 The enantiomeric compounds 96a−d and 97a−d fail to inhibit HIV significantly.106,107 The 4′-thio congeners 98a−f were found to be inactive against all tested viruses (HIV, HSV1, HSV-2, VZV, and HCMV) and demonstrated no cytotoxicity.108 Remarkably, unlike 74, the 4′-thio derivative 99 and other base analogues are inactive against HIV.109,110 The thiosugar with a 6-chloropurine base (100f) exhibits very weak anti-HCV activity.111,112 All the other nucleosides of the series are inactive against HIV-1, HBV, HCV, and HCMV.100,101,113,114 An epimeric mixture of the 4′-thio

counterparts of 87 (101a,b) was found to lack anti-HIV activity.115 The 3′-fluoro-4′-thioisonucleosides 102a−c do not exhibit anti-HIV, anti-HSV, or anti-EMCV activity. The cytidine analogue 102c combines activity against vesicular stomatitis virus (VSV; EC50 = 44.6 μM) with significant cytotoxicity in HeLa cells (CC50 = 68.8 μM). Unlike lamivudine from which it was structurally derived, it fails to inhibit HIV.116,117 For compound 103, an isostere of 3TC (2′,3′-dideoxy-3′-thiacytidine = lamivudine), no biological activity could be detected.118,119 The 3′-isonucleoside 104 is inactive against HCV,120 and no biological data have been released for the homologues 105a−c (Figure 12).121 The synthesis of the isonucleoside of adenine 106 and its enantiomer 107 is reported, but no activity data were included.122 The anti-HIV activity of nucleosides 108a−d J

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Figure 12. Series of hydroxymethyl-transposed isonucleosides and dinucleotides containing isonucleosides.

is not significant.123 Antiviral data of the base-transposed furanose and pyranose nucleosides 109a−c and 110a−c are awaited.124 Evaluation of a series of hydroxymethyl-transposed isonucleosides (111−114) indicates that they are marginally active or inactive against HIV-1, HSV, VZV, and CMV.125 Compound 115 was screened against HCMV and proved active (IC50 = 15.2 μM, CC50 = 507.1 μM).126 For the thio compounds 116a−d, no biological data have been reported.127 The dinucleotide 117, composed of an isonucleotide and a natural nucleotide (pidApdC), is reported to inhibit HIV integrase. It curtails both the 3′-processing and the strandtransfer steps (IC50 = 19 and 25 μM, respectively), although more weakly than the natural dinucleotide pdApdC (IC50 = 6 and 3 μM, respectively). Interestingly, pdApidA (118) reduces the efficiency of the integrase enzyme only in the strandtransfer step (IC50 = 41 μM).128−130

Figure 13. Isonucleoside phosphonates for which the biological activity has been reported.

2.5. Isonucleoside Phosphonates

The isodideoxyadenine phosphonate (isoddAP, 119; Figure 13) demonstrates relatively potent anti-HIV activity, though its EC50 (9.5 μM) is almost 2.5 times higher than that of tenofovir, which is attributed to less effective activation to the triphosphate derivative inside cells. This phosphonate possesses a rather attractive profile toward mutant strains. The activity

drops more than 10-fold in M184V RT (activity measured against the RT in MT-2 cell lines), and a factor of 3.2 and 2.2 against 6TAMs (thymidine analogue mutations) and K65R mutant strains, respectively. This is in sharp contrast to tenofovir but in line with the activity profile of abacavir. The extrusion of pyrimidine-based nucleosides by TAMs is well K

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documented, with adenine-based acyclic nucleosides being the most vulnerable to these mutations.131 Phosphonate 120 shows a much lower activity compared to the parent compound (S,S)-isoddA.132 Derivatives 121a−d do not show antiviral activity in vitro.133 Compounds 122a−c and 123a−c were found inactive against HCMV,126 but the carbocyclic guanine phosphonate 124 shows reasonable activity against HIV in CEM cells (IC50 = 20 μM).134 2.6. Homonucleosides and Homonucleoside Phosphonates

Idenix Pharma reported the synthesis of 2′-C-branched nucleosides 125a−d,135 which were found inactive against HCV, HIV, and other viruses and not cytotoxic (Figure 14).

Figure 15. C-nucleosides and nucleosides with non-natural bases.

carboxamide 137 are inactive or show very low potency against HIV.90,91

3. CHEMICAL SYNTHESIS OF NUCLEOBASE- OR 4′-HYDROXYMETHYL-TRANSPOSED NUCLEOSIDES AND NUCLEOTIDES

Figure 14. Homonucleosides and homonucleoside phosphonates.

3.1. Scope of This Section

The lack of activity may be explained by the entropy price that is paid due to the additional rotational flexibility between the sugar and the nucleobase or the change in spatial distance between the base and the primary alcohol in these isohomonucleosides. The locked AZT mimic 126a lacks antiHIV-1 activity as well as toxicity in MT-4 cells.136 The 1,2disubstituted carbocycle 127137,138 shows antitumor activity (IC50 = 24.3, 52.1, and 59.5 mM against cell lines Molt4/C8, L1210/0, and CEM/0, respectively), and its pyrimidine analogues exhibit marginal activity against HIV-1 and HIV-2 ( P5′ Phosphoramidates. Nucleic Acids Res. 1995, 23, 2661. (190) Gryaznov, S. M.; Chen, J. K. Oligodeoxyribonucleotide N3′– > P5′ Phosphoramidates - Synthesis and Hybridization Properties. J. Am. Chem. Soc. 1994, 116, 3143. (191) Matray, T. J.; Gryaznov, S. M. Synthesis and Properties of RNA Analogs-Oligoribonucleotide N3′– > P5′ Phosphoramidates. Nucleic Acids Res. 1999, 27, 3976. (192) Gryaznov, S. M.; Lloyd, D. H.; Chen, J. K.; Schultz, R. G.; DeDionisio, L. A.; Ratmeyer, L.; Wilson, W. D. Oligonucleotide N3′– > P5′ Phosphoramidates. Proc. Natl. Acad. Sci. U. S. A. 1995, 92, 5798. (193) Wu, X.; Delgado, G.; Krishnamurthy, R.; Eschenmoser, A. 2,6Diaminopurine in TNA: Effect on Duplex Stabilities and on the Efficiency of Template-Controlled Ligations. Org. Lett. 2002, 4, 1283. (194) Howard, F. B.; Frazier, J.; Miles, H. T. A New Polynucleotide Complex Stabilized by 3 Interbase Hydrogen Bonds, Poly-2-Aminoadenylic Acid + Polyuridylic Acid. J. Biol. Chem. 1966, 241, 4293. (195) Rackwitz, H. R.; Scheit, K. H. The Stereochemical Basis of Template Function. Eur. J. Biochem. 1977, 72, 191. (196) Cheong, C.; Tinoco, I., Jr.; Chollet, A. Thermodynamic Studies of Base Pairing Involving 2,6-Diaminopurine. Nucleic Acids Res. 1988, 16, 5115. (197) Seela, F.; Becher, G. Pyrazolo[3,4-d]Pyrimidine Nucleic Acids: Adjustment of dA-dT to dG-dC Base Pair Stability. Nucleic Acids Res. 2001, 29, 2069. (198) Matray, T.; Gamsey, S.; Pongracz, K.; Gryaznov, S. A Remarkable Stabilization of Complexes Formed by 2,6-Diaminopurine Oligonucleotide N3′– > P5′ Phophoramidates. Nucleosides, Nucleotides Nucleic Acids 2000, 19, 1553. (199) Boudou, V.; Kerremans, L.; De Bouvere, B.; Lescrinier, E.; Schepers, G.; Busson, R.; Van Aerschot, A.; Herdewijn, P. Base Pairing of Anhydrohexitol Nucleosides with 2,6-Diaminopurine, 5-Methylcytosine and Uracil as Base Moiety. Nucleic Acids Res. 1999, 27, 1450. (200) Krishnamurthy, R.; Pitsch, S.; Minton, M.; Miculka, C.; Windhab, N.; Eschenmoser, A. Pyranosyl-RNA: Base Pairing Between Homochiral Oligonucleotide Strands of Opposite Sense of Chirality. Angew. Chem., Int. Ed. Engl. 1996, 35, 1537. (201) Ebert, M.-O.; Jaun, B. Oligonucleotides with Sugars other than Ribo- and 2′ -Deoxyribofuranose in the Backbone: the Solution Structures Determined by NMR in the Context of the ’Etiology of AO

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Nucleic Acids’ Project of Albert Eschenmoser. Chem. Biodiversity 2010, 7, 2103. (202) Wilds, C. J.; Wawrzak, Z.; Krishnamurthy, R.; Eschenmoser, A.; Egli, M. Crystal Structure of a B-Form DNA Duplex Containing (L)alpha-Threofuranosyl (3′-2′) Nucleosides: A Four-Carbon Sugar Is Easily Accommodated into the Backbone of DNA. J. Am. Chem. Soc. 2002, 124, 13716. (203) Pallan, P. S.; Wilds, C. J.; Wawrzak, Z.; Krishnamurthy, R.; Eschenmoser, A.; Egli, M. Why Does TNA Cross-Pair More Strongly with RNA Than with DNA? An Answer From X-ray Analysis. Angew. Chem., Int. Ed. 2003, 42, 5893. (204) Reck, F.; Wippo, H.; Kudick, R.; Bolli, M.; Ceulemans, G.; Krishnamurthy, R.; Eschenmoser, A. L-alpha-Lyxopyranosyl (4′-3′) Oligonucleotides: A Base-Pairing System Containing a Shortened Backbone. Org. Lett. 1999, 1, 1531. (205) Ebert, M. O.; Mang, C.; Krishnamurthy, R.; Eschenmoser, A.; Jaun, B. The Structure of a TNA-TNA Complex in Solution: NMR Study of the Octamer Duplex Derived from Alpha-(L)-Threofuranosyl-(3′-2′)-CGAATTCG. J. Am. Chem. Soc. 2008, 130, 15105. (206) Heuberger, B. D.; Switzer, C. Nonenzymatic Oligomerization of RNA by TNA Templates. Org. Lett. 2006, 8, 5809. (207) Schmidt, J. G.; Nielsen, P. E.; Orgel, L. E. Enantiomeric CrossInhibition in the Synthesis of Oligonucleotides on a Nonchiral Template. J. Am. Chem. Soc. 1997, 119, 1494. (208) Bohler, C.; Nielsen, P. E.; Orgel, L. E. Template Switching between PNA and RNA Oligonucleotides. Nature 1995, 376, 578. (209) Chaput, J. C.; Ichida, J. K.; Szostak, J. W. DNA PolymeraseMediated DNA Synthesis on a TNA Template. J. Am. Chem. Soc. 2003, 125, 856. (210) Chaput, J. C.; Szostak, J. W. TNA Synthesis by DNA Polymerases. J. Am. Chem. Soc. 2003, 125, 9274. (211) Kempeneers, V.; Vastmans, K.; Rozenski, J.; Herdewijn, P. Recognition of Threosyl Nucleotides by DNA and RNA Polymerases. Nucleic Acids Res. 2003, 31, 6221. (212) Horhota, A.; Zou, K.; Ichida, J. K.; Yu, B.; McLaughlin, L. W.; Szostak, J. W.; Chaput, J. C. Kinetic Analysis of an Efficient DNADependent TNA Polymerase. J. Am. Chem. Soc. 2005, 127, 7427. (213) Ichida, J. K.; Zou, K.; Horhota, A.; Yu, B.; McLaughlin, L. W.; Szostak, J. W. An in Vitro Selection System for TNA. J. Am. Chem. Soc. 2005, 127, 2802. (214) Kurz, M.; Gu, K.; Al-Gawari, A.; Lohse, P. A. cDNA - Protein Fusions: Covalent Protein - Gene Conjugates for the in Vitro Selection of Peptides and Proteins. ChemBioChem 2001, 2, 666. (215) Zhang, S.; Chaput, J. C. Synthesis and Enzymatic Incorporation of Alpha-L-Threofuranosyl Adenine Triphosphate (tATP). Bioorg. Med. Chem. Lett. 2013, 23, 1447. (216) Yu, H.; Zhang, S.; Dunn, M. R.; Chaput, J. C. An Efficient and Faithful in Vitro Replication System for Threose Nucleic Acid. J. Am. Chem. Soc. 2013, 135, 3583. (217) Pinheiro, V. B.; Taylor, A. I.; Cozens, C.; Abramov, M.; Renders, M.; Zhang, S.; Chaput, J. C.; Wengel, J.; Peak-Chew, S. Y.; McLaughlin, S. H.; Herdewijn, P.; Holliger, P. Synthetic Genetic Polymers Capable of Heredity and Evolution. Science 2012, 336, 341. (218) Yu, H.; Zhang, S.; Chaput, J. C. Darwinian Evolution of an Alternative Genetic System Provides Support for TNA as an RNA Progenitor. Nat. Chem. 2012, 4, 183.

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