Recent advances in synthetic approaches for medicinal chemistry of C-nucleosides

C-nucleosides have intrigued biologists and medicinal chemists since their discovery in 1950's. In that regard, C-nucleosides and their synthetic analogues have resulted in promising leads in drug design. Concurrently, advances in chemical syntheses have contributed to structural diversity and drug discovery efforts. Convergent and modular approaches to synthesis have garnered much attention in this regard. Among them nucleophilic substitution at C1' has seen wide applications providing flexibility in synthesis, good yields, the ability to maneuver stereochemistry as well as to incorporate structural modifications. In this review, we describe recent reports on the modular synthesis of C-nucleosides with a focus on D-ribonolactone and sugar modifications that have resulted in potent lead molecules.

Because of their key role in many biological processes, modifications to the nucleoside structure have been widely employed in the design of drugs, most notably in the fields of virology and cancer research [13][14][15]. Variations in the nucleoside scaffold are typically accomplished by the insertion, deletion or transposition of functional groups or atoms [23][24][25][26][27][28][29]. The varied properties of such modified nucleosides arise from changes in hydro-   [31]. The extent of leaving group stabilization and approach of the nucleophile determine charge accumulation on the sugar ring. A concerted process leads to a transition state-like species shown in the figure, while a greater accumulation of positive charge leads to an oxocarbenium ion intermediate. gen bonding motifs, electronic effects, hydrophobic interactions, acid-base properties and chemical reactivity [25][26][27][28][29][30][31][32][33][34][35][36][37]. One such modification is the change in the nature of the glycosidic bond [29,37].
Although the glycosidic bond is stable under physiological conditions, cleavage of the bond is common and is highly dependent on the nature of the nucleobase and local pH. In addition, the rate of glycosidic bond cleavage is higher for purines than pyrimdines [38][39][40][41][42][43][44]. Moreover, the glycosidic bond in 2'-deoxy ribonucleosides has a higher susceptibility to cleavage than in the corresponding ribonucleosides [38][39][40][41]43]. The rate of glycosidic (C-N) bond cleavage is enhanced by decreasing pH and enzymes, which modify the localized acid-base environment [31,35,36]. The C-N bond cleavage proceeds either by activation of a nucleophile that attacks C1' or by stabilization of the leaving group, which could either be the nucleobase or an oxocarbenium ion [31,36]. As such, the oxocarbenium ion is a species formed during the glycosidic bond cleavage, which may be present as an intermediate or a transition state depending upon the accumulation of the positive charge on the sugar ring ( Figure 2). As a result, any change in the nucleobase-sugar connectivity (C-N) affects the formation of the oxocarbenium ion and thus influences the stability (or instability) of the nucleoside analogues.
Replacing the hemiaminal (O-C-N) connectivity of the canonical nucleosides with an O-C-C bond (Figure 3) results in a class of compounds called "C-nucleosides" [45][46][47][48][49][50][51]. Further modification to a C-C-C connectivity results in "carbocyclic C-nucleosides" (Figure 3) [52,53]. C-nucleosides feature (hetero)aryl aromatic groups such as 9-deazapurines, pyrimidines, pyridines and phenyl groups connected by a C-C bond to a sugar (or sugar mimic) as shown in Figure 4 [30,45- Figure 4: Examples of natural and synthetic C-nucleosides. Pseudouridine and formcycin are among several naturally occurring C-nucleosides that are being studied for their role in RNA biology and antibiotic properties respectively. In the recent past, synthetic C-nucleosides, such as immucillin-H and GS-5734, have shown potent activity against purine nucleoside phosphorylases (PNP) and broad spectrum antiviral activities. 47,50,[54][55][56][57]. The change in the nature of the glycosidic bond is accompanied by i) increased hydrolytic stability, ii) altered hydrogen bonding motifs, and iii) altered molecular recognition properties [25,29,37,58]. Because of these changes, C-nucleosides have been useful in the study of RNA and DNA processing enzymes, as well as drug design efforts and novel supramolecular structures [12,29,59].
Pseudouridine is a naturally occurring C-nucleoside that was first discovered in the 1950s [45][46][47]50]. Subsequently, many more C-nucleosides were discovered and their medicinal properties evaluated ( Figure 4) [18,29,37,[45][46][47][48][49][50][60][61][62]. Due to advances in synthetic methodologies over the years, the repertoire of C-nucleosides has since expanded and has enabled the discovery of clinically useful molecules. Some of the more prominent biologically active analogues that have advanced to clinical evaluations include the immucillins developed by Schramm et al, and Gilead's antiviral pyrrolo[2,1-f]triazine C-nucleosides (GS-5734 and GS-6620) [32,[63][64][65]. Thus, this review attempts to capture the progress in the synthesis efforts and subsequent drug discovery of the C-nucleosides over the past few years. In the first section, the structural and stereochemical underpinnings of nucleophilic substitutions to D-ribonolactone are discussed, a method that has seen wide applications. Next, we describe reports of different applications and structural variants that have expanded the diversity of the C-nucleosides. Finally, we discuss a modular synthetic approach to carbocyclic C-nucleosides that is also based on the nucleophilic substitution of ribonolactone.

Review
Nucleophilic addition to D-ribonolactone and its stereochemistry Two prominent methods of C-nucleoside syntheses involve either i) the linear construction of a (hetero)aryl moiety on a C1'-functionalized ribose or ii) coupling of a pre-synthesized (hetero)aryl with a ribosyl moiety ( Figure 5A) [48,49,62]. The C-C bond formation usually involves a functional group at C1' of the ribosyl moiety that is amenable to additional functio-nalization ( Figure 5B). Like other nucleoside coupling approaches (other than the well-known Vorbrüggen coupling reaction [66], the synthesis of C-nucleosides typically gives a mixture of stereoisomers (α and β) at the anomeric carbon [48,49,54,62,67,68]. Since the naturally occurring nucleosides (and most biologically active nucleosides) are β-anomers, achieving 100% stereospecificity in C-C bond formation is an important goal, but often difficult to attain [62].
Codée and coworkers elaborated on the mechanism and stereochemistry of this reaction by calculating the energies of different oxocarbenium conformers using a free energy surface (FES) mapping method [80,81]. These studies were based on the Woerpel's model comprising of two stable conformers, namely 3 E and E 3 , in equilibrium ( Figure 6B) [84,85]. The nucleophile approaches from the side presenting the least number of eclipsing interactions with the C2' substituent ( Figure 6B) [80]. Examining the energies of the various conformers of the permethylated furanosyl oxocarbenium intermediate revealed   that the E 3 conformer with the C5'-OMe oriented over the positively charged furanosyl ring ( Figure 6C) has a large stabilizing effect due to C5'-O5 dipole interactions. In addition, the C2' pseudoequatorial methoxy and C3' pseudoaxial methoxy groups further stabilize the intermediate in E 3 conformer, thereby favoring the E 3 confomer over the 3 E. In the case of an anomeric phenyl group (Ph, Figure 6C), stabilization of the positive charge (C=O + ) through conjugation, via parallel alignment, helps to overcome the unfavorable steric interactions between the C2'-OMe and the Ph group [81]. Because E 3 is the favored conformer, an inside attack of the nucleophile (H − ) results in an α orientation in the final product, which is evident from the the synthesis of 1 (OBn-substituted Pseudouridine, Figure 6D). Despite the greater stability of the E 3 conformer, it is the faster reacting conformer (E 3 or 3 E) that ultimately affects the ratio of diastereomers in the final product [80]. This difference in reactivity results in the differences in various α/β mixtures obtained during the synthesis of C-nucleosides using the D-ribonolactone approach.
imidazo [2,1-f] [1,2,4]triazine were active as nucleosides in HCV1b RNA replication assays, and as triphosphates they inhibit the NS5B polymerase as did the triphosphates of the guanosine analogues [71]. The library of adenosine analogues was further expanded by introducing functional groups at C7 (16)(17)(18)(19), which exhibit potent activity in RNA replication assays, with the carboxamide group in particular imparting high potency but also high cytotoxicity [72].
A further modification to the imidazo[2,1-f][1,2,4]triazine C-nucleoside scaffold was reported by Dang et al., wherein they synthesized a series of 2'-β-Me analogues possessing a 1',2' cyclopentyl ring (Figure 9) [73]. A representative synthesis (compound 23) is shown in Figure 9, which involves installing an allyl group at C1' (20) and converting the C2'-CN to an aldehyde (21) followed by a Wittig reaction to install a second allyl group at C2' (22). Second generation Grubb's catalyst was used for the ring formation, followed by hydrogenation to give the desired cyclopentane ring (23) [73]. The biological data of these compounds has yet to be reported. (24)(25)(26) that mimic the riboside of favipiravir in their effort to develop novel anti-influenza compounds ( Figure 10A) [74]. Protected D-ribonolactone 27 was treated with lithiated pyridine to obtain lactol 28 ( Figure 10B). Deoxygenation and reduction gave 29, wherein the isopropylidene group was also removed. Conversion of the cyano to an amide group, followed by removal of the silyl protecting group gave 24, which proved to be the most promising compound. The fluorine on 29 was replaced with a methoxy group after re-installing the isopropylidene protecting group. The cyano group was then converted to an amide and the methoxy converted to a hydroxy group. Removal of the protecting groups on the sugar gave 25, which exhibited potent activity against the H1N1 influenza strain (A/WSN/33) in cell based assays [74]. The pyrimidine compound 26 was synthesized using an identical approach and is not shown here. The activity of 24 and 25 as nucleosides was comparable to favipiravir and its riboside. Furthermore, they found that the triphosphate of 24 (24-TP) was incorporated opposite U and C of an RNA template by the influenza polymerase [74]. These experiments indicate that the H-bonding motifs of 24 allow it to mimic both A and G ( Figure 10A) [74]. Despite the mis-incorporation, an unmodified sugar moiety may not result in obligate chain termination. While 24-TP is incorporated opposite U and forms more of the full length product than terminated product, its incorporation opposite C results in greater truncated product. Thus, the putative mechanism of action of 24 is through mutagenesis of viral genomic RNA and inhibition of viral polymerase [74].

Carbocyclic C-nucleosides
In an attempt to synthesize carbocyclic C-nucleosides, Maier et al. found that reaction of aryl lithiums with pentanone 37 results in carbocyclic C-nucleosides with a C1'-hydroxy group (38 and 39, respectively, Figure 12A) [52,53]. They synthesized cyclopentanone 37 in 7 steps starting from norbornadiene (40, Figure 12B). Furthermore, silyl protection (TIPS) of the C2' and C3' was observed to be critical for the stability of 37 and to facilitate functional group interconversions as shown in Figure 12A [53].

Conclusion
With increasing reports of emerging and reemerging infectious diseases globally, there is a need to develop more effective and safer drugs. In that regard, C-nucleosides have recently shown great potential, which in turn, has resurrected interest in this class of molecules [29]. Several antiviral C-nucleosides have been discovered in the past five years and are now in advanced stages of clinical applications. The overarching features of these compounds with regards to changes in the nucleobase and sugars allow optimal interactions with enzymes resulting in potent and often times, selective, inhibitory activities [18,65,74,96]. As continuing efforts to design greater diversity in C-nucleosides, methods of their synthesis have become critical to more effective drug discovery. For example, the pyrrolo[2,1-f] [1,2,4]triazine scaffold has been key to the discovery of several highly active molecules [53,69,[71][72][73]86]. Modular and convergent synthetic routes have proved valuable in this regard both in terms of increasing diversity and reducing the time and length of the syntheses [70][71][72][73]76]. Efforts have been aided by advances in the synthesis of modified sugars and sugar mimics, particularly D-ribonolactone analogues [53,73,75,97]. Furthermore, chemical and theoretical studies have elucidated the mechanism and stereochemical preferences of reactions involving D-ribonolactone [80,81,84,85]. Therefore, the chemist has better control over the reactions with more predictable outcomes. In the coming years, new applications may be reported. Moreover, with the biological potential of C-nucleosides now being revisited, studies of naturally occurring C-nucleosides and their biosynthetic pathways have garnered renewed interest, as has the pursuit of new biosynthetic C-nucleosides [98][99][100][101][102][103][104]. Previously reported C-nucleosides are also being revisited and may be repurposed with increased knowledge of new biological targets [29,65,86,96]. In summary, these efforts, in concert with improved synthetic advances, provide strong impetus for the next wave of C-nucleoside design and the discovery of nucleoside therapeutics.