Design and Synthesis of Bicyclo[4.3.0]nonene Nucleoside Analogues

Nucleoside analogues are effective antiviral agents, and the continuous emergence of pathogenic viruses demands the development of novel and structurally diverse analogues. Here, we present the design and synthesis of novel nucleoside analogues with a carbobicyclic core, which mimics the conformation of natural ribonucleosides. Employing a divergent synthetic route featuring an intermolecular Diels–Alder reaction, we successfully synthesized carbobicyclic nucleoside analogues with high antiviral efficacy against respiratory syncytial virus.

N ucleoside analogues (NAs) are effective antiviral drugs and are used to treat many pathogenic viral infections. 1 The continuous emergence of new viruses requires the development of novel antivirals, and structural diversity is key to overcoming drug resistance and enhancing combination therapies.Herein, we report the design and synthesis of NAs with a bicyclo[4.3.0]nonenecarbobicyclic core [IV (Figure 1C)] as a novel template for antiviral NAs with activity against respiratory syncytial virus (RSV).While a traditional approach focuses on modifying the nucleobase, exemplified by the antivirals ribavirin [Ia (Figure 1A)] 2 and molnupiravir (Ib), 3 other strategies involve simplification, substitution, or modification of the ribose core (see Figure S1).Substitution of the ribose ring oxygen with a methylene group creates an important antiviral class, the carbocyclic nucleoside analogues (Figure 1B).However, these NAs are uncommon, as the absence of anomeric stabilization often leads to unnatural conformations and reduced biological activity. 4To address this, carbobicyclic analogues have been developed to lock the pseudosugar ring in its bioactive conformation.Notably, bicyclo[3.1.0]hexaneanalogue N-MCT [IIb (Figure 1B)] 5 has shown superior drug-like properties 6 and promising antiviral activities.Another strategy for generating a favorable conformation involves substituting the ribose ring oxygen 7 with an exocyclic alkene, a modification exemplified by entecavir (IIa). 8he synthesis of conformationally locked nucleosides presents notable challenges, primarily due to the necessity of designing them ab initio and their high density of stereochemical information. 9For instance, the synthesis of IIa can be achieved only through a complete total synthesis route in 8−15 synthetic steps. 8Similarly, N-MCT IIb, another locked carbocyclic analogue, is achieved after 11 steps from the same precursor III. 10 As a result, the exploration of structure− activity relationships (SARs) in carbocylic NAs is often hindered.
The design of NAs that can act as alternatives to ribonucleosides while retaining bioactive conformations is challenging.As an example, saturated bicyclo[4.3.0]nonaneNAs are candidates with the potential for functionalization and SAR studies.However, their application is compromised by their unfavorable conformation, lack of biological activity, and inefficient synthesis (16−19 steps as shown in Figure S2).9c We envisioned that the incorporation of an endocyclic double bond into the unsaturated bicyclo[4.3.0]nonenescaffold [IV (Figure 1C)] could align these analogues conformationally with natural ribonucleosides.This modification not only simplifies the synthesis process but also potentially enhances the biological activity, addressing the limitations of previous designs.Building upon our expertise in carbohydrate chemistry, 11 we have designed a concise and divergent route yielding a structurally distinct class of bicyclo[4.3.0]noneneNAs.
Initially, we launched the synthesis of carbobicyclic analogues of ribavirin.Ribavirin itself is a Food and Drug Administration-approved antiviral drug for treating infections caused by RSV.Our goal was to generate a stereo-and regiodiverse library of analogues, which facilitates the exploration of the SAR of our compounds.The stereodivergence is achieved by the Diels−Alder reaction between diene VI and dienophile V. Diene VI itself should be available from VII by the Mitsunobu reaction.The alternative reaction pathway in which the Diels−Alder reaction occurs before the Mitsunobu reaction was not successful.9c We synthesized Diels−Alder precursor 6 from commercially available enone 2 12 in four steps (see Scheme 1).First, αiodination of enone 2 under a Baylis−Hillman type pathway followed by Luche reduction gave alcohol 3 stereospecifically. 13The reduction product could be employed in the Mitsunobu coupling with triazole 4 yielding 5a and 5b in 78% total yield as separable regioisomers. 2,14Notably, with THF, iso-ribavirin type isomer 5b was afforded as the major product (5a:5b ratio of 1:4).While using DCM, both regioisomers were obtained in a nearly equimolar ratio, a phenomenon that had not previously been reported.Both isomers can be converted to the corresponding dienes 6a and 6b in excellent yield using a Stille cross-coupling. 15n the intermolecular Diels−Alder reaction, an activated dienophile was necessary, and thus, vinyl boronate was used to allow the efficient installation of a pseudo-C5′-OH functionality (Scheme 2) 16 through facile oxidative cleavage of the boronate with NaBO 3 .We envisioned that the dienophile approaches diene 6 from the upper side, avoiding the steric interactions with the isopropylidene acetal group.To our delight, the desired alcohol 7 was isolated as a single isomer.For the two other main isomers, separation was possible after esterification to 8 and 9 (for details, see Figure S3).Adducts 7−9 could be isolated in 67−70% total yield from 6.The synthesis of the designed ribavirin analogues was then completed in two or three steps (see Table 1).First, the methyl ester of the triazole nucleobase was transformed into the amide, 3,14a−c,17 and then nucleoside analogues 10−12 were achieved by deprotection.In detail, ribavirin analogue 10a and isomer 10b were obtained by treatment of 7 with methanolic ammonia and subsequent acidic treatment (TFA in H 2 O/ MeOH).An additional esterification step 18 yielded C5′-OH esters 10c and 10d.In the case of 8 and 9, amidation did not lead to cleavage of the iso-butyric acid esters (at pseudo-C6′), and analogues 11c, 11d, 12c, and 12d were isolated after acidic deprotection.Amidation with subsequent one-pot K 2 CO 3mediated ester cleavage, followed by acidic acetonide deprotection, gave 11a, 11b, 12a, and 12b in good yields.In short, a total of 12 ribavirin analogues were synthesized in seven or eight steps from commercially available enone 2.
The relative configurations of isomers 10a−12a among others (see the Supporting Information) were determined by two-dimensional NMR studies, and all other intermediates were assigned accordingly.This assessment is in accordance with the X-ray single-crystal analysis of 12b, which validated our initial hypothesis that the bicyclic core might have a riboselike conformation [P = +24.0°;C3′-endo (see Figure S4)].This conformation is also observed in natural nucleosides and therefore is promising for biological activity.4b Superposition of the three-dimensional X-ray structure of 12b with the carbasugar entecavir 19 revealed an identical conformation of the carbasugar core of both structures (Figure 2A).More importantly, the comparison with the authentic ribose-based ribavirin 20 revealed a nearly identical core conformation albeit slightly twisted.Thus, the nucleoside analogues adopted a favorable drug-like conformation.This is in stark contrast to the case for the previously reported saturated bicyclo[4.3.0]-nonanenucleoside analogues, which have a biologically inactive C1′-exo conformation.9c We then assessed their effectiveness in inhibiting RSV replication by using an established model (RSV-A, HEp-2 cells). 21First, we tested parental compounds 10a−12a and 10b−12b at concentrations of 2 and 40 μM, respectively, and observed promising antiviral activities (Figures S5 and S6).The ribavirin analogues (10a−12a) compared to the isoribavirin (10b−12b) exhibited similar levels of activity.Additionally, analysis of the prodrug variants [10c−12c (see Figure S8)] revealed no significant difference in antiviral potency compared to that of their active form (10a−12a).Although there is no difference in activity, these subtle structural differences impact the pharmacokinetic properties.The iso-butyrate analogues demonstrated more favorable predicted values for intestinal permeability (P app ), solubility (log S), lipophilicity (log D), and the partition coefficient (log P) compared to thoose of the non-ester analogues (see table S1).In addition, the predicted intestinal permeability of analogues 10−12 is higher than that of ribavirin, suggesting a potential for better oral bioavailability. 22e then followed up to determine the IC 50 against RSV and the cytotoxicity (CC 50 ).All three derivatives, 10a−12a showed excellent anti-RSV activity with IC 50 values in a low micromolar range [0.53−1.66μM (Figure 2B)], even lower than that of ribavirin (IC 50 = 8.83 μM).Analogue 11a was the most active compound, with activity that was 16 times higher than that of ribavirin.It is noteworthy that all three derivatives have a CC 50 value of >40 μM, indicating a high selectivity and thus a wide therapeutic window (see Figure S9).Immunostaining of RSV-infected cells validated the screening results (Figure 2C), where the treatment with 12a (2 μM) leads to a significant inhibition (see also Figure S6).
To further explore the potential of bicyclic nucleoside analogues fully, we were interested in whether the antiviral activity was limited to ribavirin type analogues.Therefore, we synthesized carbobicyclic uridine analogues 19−21 to rule out the possibility that the antiviral effect stemmed from the triazole base alone.Uridine analogues 19−21 were synthesized in analogy to the previous synthesis as shown in Scheme 3.

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benzoyl group was partially cleaved under the thermal conditions, leading to difficulties during the isolation of the Diels−Alder adduct.Thus, debenzoylation prior to the Diels− Alder reaction was essential.The conversion of uracil diene 15 was relatively retarded compared to that of its ribavirin analogue; the optimized molar ratio of the dienophile furnished diverse cycloadducts 16−18 in moderate yield with regio-and diastereoselectivity comparable to that of the ribavirin analogues.The final esterification and/or deprotection led to carbobicyclic uracil analogues 19−21 in seven or eight steps from 2.
During NMR analysis of the uracil analogues, we observed significant peak broadening, suggesting that the analogues exist as two structural conformers at room temperature.Variabletemperature NMR studies showed that both conformers merge at increased temperatures [315 K (see Figure S10)].The desired connectivity (N1 vs O2) was confirmed by NMR (characteristic 1 H and 13 C shift at C1′) 25 and NOESY.In addition to NOE, the relative configuration was further confirmed in analogy with ribavirin type analogues, as the identical core structure (e.g., 10 and 19) shares characteristic 1 H NMR chemical shifts and splitting patterns (see Figures S11−S13).
In the subsequent cellular assays, uridine analogues 19a and 21a showed good antiviral activity against RSV and almost no cytotoxicity whereas 20a at 40 μM showed only a nonsignificant reduction of viral load (see Figures S5 and S6).We demonstrate a dose-dependent inhibition for pseudo-C5′-OH analogue 19a with an IC 50 of 6.94 μM and low cytotoxicity [CC 50 > 40 μM (Figure S9)].Collectively, these data confirmed that the carbobicyclic core contributes to antiviral activity.Although our compounds are structural analogues of traditional nucleosides, their mechanisms of action may differ from those of the known nucleoside analogues.Further studies are essential to elucidate their antiviral mechanisms.
In summary, we report herein the design, synthesis, and biological activity of a new class of nucleoside analogues with a bicyclo[4.3.0]nonenecore.The synthesized ribavirin type carbobicyclic analogues exhibit a ribose-like conformation and demonstrate promising antiviral activity with minimal cytotoxicity.The versatility of our synthetic approach will allow for further diversification through the incorporation of various nucleobases such as adenosine, guanosine, and 5-fluorouracil.Thus, this new antiviral structural motif opens opportunities to combat emerging and future outbreaks of viral diseases and antiviral drug resistance.