Nucleotide Analogues Bearing a C2′ or C3′-Stereogenic All-Carbon Quaternary Center as SARS-CoV-2 RdRp Inhibitors

The design of novel nucleoside triphosphate (NTP) analogues bearing an all-carbon quaternary center at C2′ or C3′ is described. The construction of this all-carbon stereogenic center involves the use of an intramoleculer photoredox-catalyzed reaction. The nucleoside analogues (NA) hydroxyl functional group at C2′ was generated by diastereoselective epoxidation. In addition, highly enantioselective and diastereoselective Mukaiyama aldol reactions, diastereoselective N-glycosylations and regioselective triphosphorylation reactions were employed to synthesize the novel NTPs. Two of these compounds are inhibitors of the RNA-dependent RNA polymerase (RdRp) of SARS-CoV-2, the causal virus of COVID-19.


Introduction
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a positive-sense RNA virus and the causal agent of coronavirus (CoV) disease 2019 . CoVs employ a multi-subunit replication/transcription machinery. The virus enters the cell by endocytosis using the ACE 2 receptors and is then uncoated. ORF1a and ORF2 of the positive strand RNA are then translated to produce non-structural protein precursors (nsp), including proteins that further cleave the precursor to form mature functional helicase and RNA-dependent RNA polymerase [1] (RdRp, or alternatively nsp 12). The latter has been recognized as an optimal target for drug design, due to its crucial role in RNA synthesis, lack of host homologues and high structural conservation between coronaviruses.
in the Remdesivir ribose moiety and the serine 861 side chain in the nsp 12 subunit of RdRp [7]. The truncation of serine 861 to alanine or glycine renders the RdRp less sensitive or insensitive to inhibition by Remdesivir [8]. An alternative mechanism was recently proposed involving the RdRp-dependent RNA proofreading. The mechanism of NHC (Molnupiravir), presently evaluated in clinical trials and is attributed to the triple level incorporation of NHC-TP, resulting in increased mutations and, ultimately, in a process known as "lethal mutagenesis". From a drug design standpoint, the antiviral activity of nucleoside analogues (NAs) depends on their efficient transport into cells where a first phosphorylation takes place (e.g., by cytidine kinase). These two critical steps are rate-limiting in the entire cascade that leads, through subsequent phosphorylation by other kinases, to the active nucleoside triphosphate (NTP) analogues that compete with natural nucleoside triphosphates. To circumvent these limitations, phosphoramidates were developed as monophosphate lipophilic pro-drugs that facilitate intracellular transport. The pro-drug is cleaved intracellularly, releasing the NA monophosphate that is then transformed into the corresponding bioactive triphosphorylated analogue. The main objective of the present study was to evaluate a novel series of triphosphorylated nucleotide analogues to test their activity Remdesivir (RDV) was approved by the Food and Drug Administration (FDA) for the intravenous treatment of COVID-19 in hospitalized adult and pediatric patients [5]. RDV-TP (1), the active agent, is incorporated into RNA by the RdRp enzyme and then acts as a delayed chain termination of RNA synthesis at position i + 3 [6]. The Remdesivirinduced RdRp stalling is caused by a translocation barrier between the C1 -cyano group in the Remdesivir ribose moiety and the serine 861 side chain in the nsp 12 subunit of RdRp [7]. The truncation of serine 861 to alanine or glycine renders the RdRp less sensitive or insensitive to inhibition by Remdesivir [8]. An alternative mechanism was recently proposed involving the RdRp-dependent RNA proofreading. The mechanism of NHC (Molnupiravir), presently evaluated in clinical trials and is attributed to the triple level incorporation of NHC-TP, resulting in increased mutations and, ultimately, in a process known as "lethal mutagenesis".
From a drug design standpoint, the antiviral activity of nucleoside analogues (NAs) depends on their efficient transport into cells where a first phosphorylation takes place (e.g., by cytidine kinase). These two critical steps are rate-limiting in the entire cascade that leads, through subsequent phosphorylation by other kinases, to the active nucleoside triphosphate (NTP) analogues that compete with natural nucleoside triphosphates. To circumvent these limitations, phosphoramidates were developed as monophosphate lipophilic pro-drugs that facilitate intracellular transport. The pro-drug is cleaved intracellularly, releasing the NA monophosphate that is then transformed into the corresponding bioactive triphosphorylated analogue. The main objective of the present study was to evaluate a novel series of triphosphorylated nucleotide analogues to test their activity directly in vitro against RdRp [9]. If active, the corresponding phosphoramidate pro-drugs would then be installed on the parent NA for further evaluations. The nucleoside analogues reported in the literature thus far display additional substituents at C1 (cyano for Remdesivir Figure 1A) and different C2 or C3 substituents (methyl or fluorine, Figure 1B). This was suggestive that further modification of the C2 or C3 positions would be tolerated.
Our laboratory has a long-standing interest in the development of new methodologies to improve the synthesis of nucleoside analogues. In parallel, we have been studying carbon-centered free radicals on acyclic molecules and their reactivity in atom transfer reactions, leading to the generation of all carbon stereogenic quaternary centers. Together, these findings [10][11][12][13][14][15][16] led to the conceptualization and synthesis of novel nucleoside analogues bearing a quaternary all-carbon stereogenic center at C3 or C2 [16][17][18].
The presence of these all-carbon quaternary centers also provides structural properties that may influence the recognition of these nucleosides or nucleotides by given targets (enzymes or a receptors). For instance, nucleosides and nucleotides are flexible molecules adopting conformations ranging between North (C3 endo, Figure 1C) or South (C2 endo, Figure 1C). Increasing the populations of nucleosides in their bio-active conformation may translate into a greater binding affinity to the target. We hypothesized that the presence of the quaternary center could induce a conformational bias when located at C2 or C3 , the former would favor the North conformation (RNA-like) and the latter, the South (DNA-like). These conformational changes would be induced to minimize the steric effects (gauche effects) of the quaternary centers with their proximal substituents ( Figure 1C). The X-ray analyses of some of our analogues having stereogenic quaternary centers at C2 or C3 supports these conformational biases. In solution, these molecules will, however, still possess some plasticity, allowing for conformational realignment during binding, contrary to locked nucleosides analogues [19]. The presence of the hydroxyl on this center could also potentially act as an extended pharmacophore, providing different proximal binding. On the other hand, binding to enzymes susceptible to steric hindrance at these positions could lead to inactivity. We thus embarked on the syntheses of a small library of nucleotides having stereogenic centers at C2 or C3 bearing adenine or cytosine nucleobases ( Figure 1D) to investigate their inhibitory profile against RdRp.

Results and Discussion
The synthesis of the all-carbon quaternary stereogenic centers was based on our findings that a carbon-centered free radical flanked by an ester and a secondary carbon bearing an electronegative substituent (hydroxyl) could stereoselectively participate in kinetically controlled atom transfer reactions. Hydrogen and allylation transfer reactions have previously been studied both experimentally and theoretically by our group [20,21]. We recently prepared radical precursor 2 using an enantioselective Mukaiyama aldol reaction in good yield and with high diastereoselectivity in favor of the 3R isomer ( Figure 2) [14]. The secondary alcohol was then transformed into dimethylallysilyl ether 3. Acyclic intermediate 6, bearing the quaternary stereogenic center, was synthesized by a sequence involving an intramolecular atom transfer cyclization, an elimination reaction under photoredox catalysis and subsequent ester reduction with protection of alcohol generated.
Allylic oxidation of 6 using SeO 2 led to a modest 50% yield of the corresponding ketone 7 (Figure 2) [14]. The 2,4-syn diol 8 was then obtained from ketone 7 by reduction using catecholborane in the presence of cesium chloride. The key intermediate 8 was transformed to furanosides 9 and 10. The corresponding β-cytosine nucleoside analogue 11 was obtained from 9 by taking advantage of anchimeric participation by the acetate at C2 . On the other hand, α-cytosine NA 12 was accessed from 10 using Me 2 BBr [22].
Biological evaluation of the corresponding novel nucleotides necessitated an improvement in the overall synthesis of these C3 quaternary substituted nucleosides. As reported herein (Figure 2), this was accomplished by introducing the hydroxyl at C2 through a stereoselective epoxidation of glycal 13 [23,24] to the epoxide 14, followed by hydrolysis and stereoselective N-glycosylation to generate novel nucleoside analogues 16.
Molecules 2022, 27, x FOR PEER REVIEW 4 of through a stereoselective epoxidation of glycal 13 [23,24] to the epoxide 14, followed hydrolysis and stereoselective N-glycosylation to generate novel nucleoside analogues Different pathways to prepare key intermediate 21 were explored. Cyclization of dehyde 19 in the presence of PTSA in THF/H2O led to lactols 20a,b with a 1.4:1 anome ratio (Scheme 2). Mesylation and elimination with Et3N generated glycal 21 in 51% yie Alternatively, methylfuranoside 22a,b was derived from aldehyde 19 in the presence PTSA in anhydrous methanol. Methylfuranoside 22a,b were then treated with TMSO and 2,6-lutidine to give glycal 21 in an excellent yield. through a stereoselective epoxidation of glycal 13 [23,24] to the epoxide 14, followed by hydrolysis and stereoselective N-glycosylation to generate novel nucleoside analogues 16.  Dondoni's dimethyldioxirane (DMDO) [25], generated in situ with a catalytic amount of acetone and a stoichiometric amount of potassium peroxymonosulfate (oxone), was chosen as the oxidant. When glycal 21 was subjected to DMDO oxidation, the ribolike epoxide 23a was obtained as the major epoxide in a 7:1 ratio relative to the arabinolike epoxide 23b (Scheme 3). The stereoselectivity is rationalized by spiro-like transition states TS A and TS B (Scheme 3), where DMDO approaches the olefin from the inside face of the glycals' envelope-like conformations [26][27][28]. Attack from the bottom face in TS A avoids significant steric clash with the C5′ substituent in TS B (Scheme 3). Hydrolysis of the crude epoxides 23a and 23b in THF/H2O gave the ribo-like lactols 24a,b as the major compounds (63%, isolated by flash column chromatography). These lactols were then protected with acetyl groups to afford acetate ribofuranosides 25a,b in 85% yield. Scheme 3. Synthesis of diacetate ribofuranosides 25a,b. ‡ represents the "Transition State" With ribo-like diacetate furanosides 25a,b in hand, stereoselective N-glycosylations of 25a,b were performed in the presence of a silylated base (adenine or cytidine) and TMSOTf (Scheme 4). The 1′,2′-trans ribo-like nucleosides 26-28 were obtained in excellent diastereoselectivity (>20:1 dr) and yield (75-85%) in accordance with anchimeric assistance of the C2′ acetyl group. NMR spectroscopic analysis experiments confirmed the 1,2′trans stereochemistries (2D NOESY) and, in the case of adenine coupling, N 9 isomers (HMBC). Subsequent cleavage of the C5′-OTBDPS protecting groups with 3HF‧NEt3 provided corresponding nucleoside analogues 29-31 (86-87%), which were the key precursors for the formation of the nucleoside C5′O-triphosphates. Further, cleavage of the C2′and C3′ acetyl-protecting groups with NaOMe provided 1′,2′-trans ribo-like nucleoside analogues 32-34 (80-82%). Dondoni's dimethyldioxirane (DMDO) [25], generated in situ with a catalytic amount of acetone and a stoichiometric amount of potassium peroxymonosulfate (oxone), was chosen as the oxidant. When glycal 21 was subjected to DMDO oxidation, the ribo-like epoxide 23a was obtained as the major epoxide in a 7:1 ratio relative to the arabino-like epoxide 23b (Scheme 3). The stereoselectivity is rationalized by spiro-like transition states TS A and TS B (Scheme 3), where DMDO approaches the olefin from the inside face of the glycals' envelope-like conformations [26][27][28]. Attack from the bottom face in TS A avoids significant steric clash with the C5 substituent in TS B (Scheme 3). Hydrolysis of the crude epoxides 23a and 23b in THF/H 2 O gave the ribo-like lactols 24a,b as the major compounds (63%, isolated by flash column chromatography). These lactols were then protected with acetyl groups to afford acetate ribofuranosides 25a,b in 85% yield. Dondoni's dimethyldioxirane (DMDO) [25], generated in situ with a catalytic amount of acetone and a stoichiometric amount of potassium peroxymonosulfate (oxone), was chosen as the oxidant. When glycal 21 was subjected to DMDO oxidation, the ribolike epoxide 23a was obtained as the major epoxide in a 7:1 ratio relative to the arabinolike epoxide 23b (Scheme 3). The stereoselectivity is rationalized by spiro-like transition states TS A and TS B (Scheme 3), where DMDO approaches the olefin from the inside face of the glycals' envelope-like conformations [26][27][28]. Attack from the bottom face in TS A avoids significant steric clash with the C5′ substituent in TS B (Scheme 3). Hydrolysis of the crude epoxides 23a and 23b in THF/H2O gave the ribo-like lactols 24a,b as the major compounds (63%, isolated by flash column chromatography). These lactols were then protected with acetyl groups to afford acetate ribofuranosides 25a,b in 85% yield. With ribo-like diacetate furanosides 25a,b in hand, stereoselective N-glycosylations of 25a,b were performed in the presence of a silylated base (adenine or cytidine) and TMSOTf (Scheme 4). The 1′,2′-trans ribo-like nucleosides 26-28 were obtained in excellent diastereoselectivity (>20:1 dr) and yield (75-85%) in accordance with anchimeric assistance of the C2′ acetyl group. NMR spectroscopic analysis experiments confirmed the 1,2′trans stereochemistries (2D NOESY) and, in the case of adenine coupling, N 9 isomers (HMBC). Subsequent cleavage of the C5′-OTBDPS protecting groups with 3HF‧NEt3 provided corresponding nucleoside analogues 29-31 (86-87%), which were the key precursors for the formation of the nucleoside C5′O-triphosphates. Further, cleavage of the C2′and C3′ acetyl-protecting groups with NaOMe provided 1′,2′-trans ribo-like nucleoside analogues 32-34 (80-82%). With ribo-like diacetate furanosides 25a,b in hand, stereoselective N-glycosylations of 25a,b were performed in the presence of a silylated base (adenine or cytidine) and TMSOTf (Scheme 4). The 1 ,2 -trans ribo-like nucleosides 26-28 were obtained in excellent diastereoselectivity (>20:1 dr) and yield (75-85%) in accordance with anchimeric assistance of the C2 acetyl group. NMR spectroscopic analysis experiments confirmed the 1,2 -trans stereochemistries (2D NOESY) and, in the case of adenine coupling, N 9 isomers (HMBC). Subsequent cleavage of the C5 -OTBDPS protecting groups with 3HF·NEt 3 provided corresponding nucleoside analogues 29-31 (86-87%), which were the key precursors for the formation of the nucleoside C5 O-triphosphates. Further, cleavage of the C2 and C3 acetyl-protecting groups with NaOMe provided 1 ,2 -trans ribo-like nucleoside analogues 32-34 (80-82%). The synthesis of the C2′ quaternary series was then explored. The five-membered ring lactone 40, bearing the quaternary center at C2′, was prepared by a route previously reported by our group [13] (Scheme 5). The Mukaiyama aldol reaction of aldehyde 35 with tetrasubstituted silylated-enolether 36 provided 37a,b. The α-bromomethylesters 37a,b were subjected to lactonization, followed by installation of a TBS at the C5 primary alcohol. Installation of vinyldimethylsilane at the C3′ secondary alcohol provided lactones 39a,b, which were subjected to an atom transfer cyclization/elimination reaction using photoredox catalysis to give lactone 40. An alternative synthetic sequence was then optimized to reduce the number of steps to reach 43a,b [13]. A TBS-protecting group was introduced on the secondary hydroxyl of 40, followed by ozonolysis resulting in aldehyde 41 in excellent yield. After the simultaneous reduction of both the lactone and aldehyde using Red-Al, benzoylation provided furanosides 43a,b. With benzoylated furanosides 43a,b in hand, stereoselective N-glycosidation of 43a,b with 2,6 dichloropurine in the presence of TMSOTf at −10 C led to the regioselective formation of N 9 nucleoside analogue 44 with high diastereoselectivity (β:α, 10:1) and good yield (73%, Scheme 6). The selective formation of the β-anomer is attributed to an anchimeric assistance from C2′ benzoate. The 1′,2′-trans stereochemistry was confirmed by 2D NOESY experiments, while the N 9 regioselectivity was verified by the key indicative three bond correlation between the H1′ of sugar and C4 of purine in 1 H/ 13 C 2D HMBC NMR The synthesis of the C2 quaternary series was then explored. The five-membered ring lactone 40, bearing the quaternary center at C2 , was prepared by a route previously reported by our group [13] (Scheme 5). The Mukaiyama aldol reaction of aldehyde 35 with tetrasubstituted silylated-enolether 36 provided 37a,b. The α-bromomethylesters 37a,b were subjected to lactonization, followed by installation of a TBS at the C5 primary alcohol. Installation of vinyldimethylsilane at the C3 secondary alcohol provided lactones 39a,b, which were subjected to an atom transfer cyclization/elimination reaction using photoredox catalysis to give lactone 40. An alternative synthetic sequence was then optimized to reduce the number of steps to reach 43a,b [13]. A TBS-protecting group was introduced on the secondary hydroxyl of 40, followed by ozonolysis resulting in aldehyde 41 in excellent yield. After the simultaneous reduction of both the lactone and aldehyde using Red-Al, benzoylation provided furanosides 43a,b. 27 The synthesis of the C2′ quaternary series was then explored. The five-membered ring lactone 40, bearing the quaternary center at C2′, was prepared by a route previously reported by our group [13] (Scheme 5). The Mukaiyama aldol reaction of aldehyde 35 with tetrasubstituted silylated-enolether 36 provided 37a,b. The α-bromomethylesters 37a,b were subjected to lactonization, followed by installation of a TBS at the C5 primary alcohol. Installation of vinyldimethylsilane at the C3′ secondary alcohol provided lactones 39a,b, which were subjected to an atom transfer cyclization/elimination reaction using photoredox catalysis to give lactone 40. An alternative synthetic sequence was then optimized to reduce the number of steps to reach 43a,b [13]. A TBS-protecting group was introduced on the secondary hydroxyl of 40, followed by ozonolysis resulting in aldehyde 41 in excellent yield. After the simultaneous reduction of both the lactone and aldehyde using Red-Al, benzoylation provided furanosides 43a,b. With benzoylated furanosides 43a,b in hand, stereoselective N-glycosidation of 43a,b with 2,6 dichloropurine in the presence of TMSOTf at −10 C led to the regioselective formation of N 9 nucleoside analogue 44 with high diastereoselectivity (β:α, 10:1) and good yield (73%, Scheme 6). The selective formation of the β-anomer is attributed to an anchimeric assistance from C2′ benzoate. The 1′,2′-trans stereochemistry was confirmed by 2D NOESY experiments, while the N 9 regioselectivity was verified by the key indicative three bond correlation between the H1′ of sugar and C4 of purine in 1 H/ 13 C 2D HMBC NMR experiments. Nucleoside 45 was formed from treatment with ammonia in methanol to give the 2-chloroadenosine and deprotection of the C2′ benzoate, followed by desilylation With benzoylated furanosides 43a,b in hand, stereoselective N-glycosidation of 43a,b with 2,6 dichloropurine in the presence of TMSOTf at −10 • C led to the regioselective formation of N 9 nucleoside analogue 44 with high diastereoselectivity (β:α, 10:1) and good yield (73%, Scheme 6). The selective formation of the β-anomer is attributed to an anchimeric assistance from C2 benzoate. The 1 ,2 -trans stereochemistry was confirmed by 2D NOESY experiments, while the N 9 regioselectivity was verified by the key indicative three bond correlation between the H1 of sugar and C4 of purine in 1 H/ 13 C 2D HMBC NMR experiments. Nucleoside 45 was formed from treatment with ammonia in methanol to give the 2-chloroadenosine and deprotection of the C2 benzoate, followed by desilylation in the presence of 3HF·NEt 3 . The 2-chloroadenosine analogue 45 was then hydrogenated to the adenosine derivative 46 in 78% yield. The cytidine analogue 47 was also prepared from the benzoylated furanoside, as reported by our group [13]. to the adenosine derivative 46 in 78% yield. The cytidine analogue 47 was also prepared from the benzoylated furanoside, as reported by our group [13]. Scheme 6. Synthesis of 1′,2′-trans ribo-like adenine nucleoside analogues.

Synthesis of Nucleoside Triphosphates (NTPs)
Having established synthetic routes to access nucleoside analogues bearing an all carbon quaternary stereocenter at C2′ or C3′, we next investigated the synthesis of their C5′ triphosphate derivatives. The synthesis of NTPs represents a challenging task [29] Initial triphosphorylation attempts using Taylor's method, using trimetaphosphate and mesitylenesulfonyl chloride, were unsuccessful [30]. The one-pot synthesis approach o Huang and co-workers [31] was more appropriate for our substrates. The phosphoryla tion is accomplished under mild conditions using tributylammonium pyrophosphate in the presence of salicyl phosphorochloridite (SalPCl).

Synthesis of Nucleoside Triphosphates (NTPs)
Having established synthetic routes to access nucleoside analogues bearing an allcarbon quaternary stereocenter at C2 or C3 , we next investigated the synthesis of their C5 triphosphate derivatives. The synthesis of NTPs represents a challenging task [29]. Initial triphosphorylation attempts using Taylor's method, using trimetaphosphate and mesitylenesulfonyl chloride, were unsuccessful [30]. The one-pot synthesis approach of Huang and co-workers [31] was more appropriate for our substrates. The phosphorylation is accomplished under mild conditions using tributylammonium pyrophosphate in the presence of salicyl phosphorochloridite (SalPCl).
The phosphorylation of nucleoside analogues bearing a C2 quaternary stereocenter was then conducted using the same strategy. Triphosphorylation of unprotected NAs 46-47 furnished the corresponding NTPs 5-7 in low, but acceptable yields, for this challenging transformation (5-13%, Scheme 8).

General Information
All the anhydrous solvents were purchased from Sigma Aldrich, USA. All the glassware was purchased from Pyrex USA. Reactions requiring anhydrous conditions were performed under an atmosphere of nitrogen in flame-dried glassware using standard syringe techniques. Molecular sieves (Sigma Aldrich, USA) were used to prepare the anhydrous solvents. The sieves (4 Å , 1-2 mm beads) were activated by heating at 180 °C for 48 h under vacuum prior to their addition into new bottles of solvent purged with argon. All commercially available reagents were used as received: TBSCl was purchased from TCI America, USA and TBSOTf was obtained from Oakwood Chemicals, USA. Remaining all reagents were purchased from Sigma Aldrich, USA. Flash chromatography was done on silica gel 60 (0.040-0.063 mm, Silicycle, QC, Canada) using an automated purification system. Thin-layer chromatography (TLC) was done on pre-coated (0.25 mm) Merck F-254 silica gel aluminum plates. Visualization was performed with short UV wavelengths and/or revealed with potassium permanganate solutions. See the 1 H, 13 C, and 2D NMR

General Information
All the anhydrous solvents were purchased from Sigma Aldrich, Saint Louis, MO, USA. All the glassware was purchased from Pyrex USA. Reactions requiring anhydrous conditions were performed under an atmosphere of nitrogen in flame-dried glassware using standard syringe techniques. Molecular sieves (Sigma Aldrich, USA) were used to prepare the anhydrous solvents. The sieves (4 Å, 1-2 mm beads) were activated by heating at 180 • C for 48 h under vacuum prior to their addition into new bottles of solvent purged with argon. All commercially available reagents were used as received: TBSCl was purchased from TCI America, USA and TBSOTf was obtained from Oakwood Chemicals, USA. Remaining all reagents were purchased from Sigma Aldrich, USA. Flash chromatography was done on silica gel 60 (0.040-0.063 mm, Silicycle, QC, Canada) using an automated purification system. Thin-layer chromatography (TLC) was done on pre-coated (0.25 mm) Merck F-254 silica gel aluminum plates. Visualization was performed with short UV wave-lengths and/or revealed with potassium permanganate solutions. See the 1 H, 13 C, and 2D NMR spectra data in the Supplementary Materials. The 1 H NMR spectra were recorded at room temperature on a 700 MHz, 500 MHz and 400 MHz NMR spectrometer and the data is reported as follows: chemical shift in ppm referenced to residual solvent (CDCl 3 δ 7.26, CD 3 OD δ 3.31 and D 2 O δ 4.79 ppm), multiplicity (s = singlet, d = doublet, dd = doublet of doublets, ddd = doublet of doublets of doublets, t = triplet, td = triplet of doublets, m = multiplet, app = apparent), coupling constants (Hz), and integration. The 13 C NMR spectra were recorded at room temperature using 176 MHz and 126 MHz with the data reported as follows: chemical shift in ppm referenced to residual solvent (CDCl 3 δ 77. 16 and CD 3 OD δ 49.00 ppm). The 31 P NMR spectra were recorded at room temperature using 162 MHz. Infrared spectra were recorded on a Fourier transform infrared spectrophotometer from a thin film of purified product or with single reflection diamond attenuated total reflection module, and signals are reported in cm −1 . Mass spectra were recorded through electrospray ionization with positive ion mode. A Hybrid Quadrupole Orbitrap mass analyzer was used for high-resolution mass spectrometry (HRMS) measurements. Optical rotations were measured at room temperature from the sodium D line (589 nm) using the formula: [α] D = (100)α obs //( ·(c)), where c = (g of substrate/100 mL of solvent) and = 1 dm. The sequence from 35 to 40 in the Scheme 5, and compounds 5 and 47 [13] were prepared as reported in our previous publications [13,14]. (17). To a solution of secondary alcohol 5 [14] (3.90 g, 9.14 mmol, 1.00 equiv.) in anhydrous DCM (45 mL, 0.20 M), imidazole (1.56 g, 22.9 mmol, 2.50 equiv.) was added, immediately followed by triethylchlorosilane (2.30 mL, 13.7 mmol, 1.50 equiv.). The resulting mixture was stirred at room temperature for 16 h. The mixture was diluted with DCM (25 mL) and saturated NH 4  (+)-((2S,3R)-2-(((tert-Butyldiphenylsilyl)oxy)methyl)-3-methyl-2,3-dihydrofuran-3-yl)methyl acetate (21). To a solution of methoxy lactols 22a,b (0.12 g, 0.25 mmol, 1.0 equiv.) in anhydrous DCM (1.3 mL, 0.2 M), 2,6-lutidine (0.12 mL, 1.0 mmol, 4.0 equiv.) and TMSOTf (0.09 mL, 0.5 mmol, 2 equiv.) were added at 0 • C [32].The resulting solution was stirred for 30 min at room temperature. The mixture was then diluted in DCM (5 mL) followed by washing with water (5 mL), and the aqueous layer was extracted with DCM (3 × 5 mL). The combined organic layers were dried over MgSO 4 , filtered and concentrated under reduced pressure. Purification by silica gel flash chromatography (Hexanes/EtOAc, 90:10) provided glycal 21 (96 mg, 90%) as a colorless oil, which was confirmed to be identical to the compound formed from 20a,b (see above).
General Procedure D: If the nucleside analogues hydroxyl or amine functional groups are protected with acetyl (Ac) or benzoyl (Bz) follow the Gerenaral Procedure C until centrifiguation. Residue (solid) inside the centrifuge tube was dissolved in NH 4 OH (0.02 M) and was stirred for overnight. The mixture was concentrated under reduced pressure. The residue was purified by reverse phase C18 flash chromatography (MeCN in 20 mM triethylammonium acetate buffer, pH = 7) to provide corresponding nucleoside triphosphate triethylammonium salt, which was then lyophilized to provide pure solid nucleoside triphosphate as a white powder.

Conclusions
The syntheses of novel NTP analogues bearing a quaternary all-carbon stereogenic center at C3 and C2 have been achieved. The stereogenic quaternary center at C2 or C3 were generated by photocatalyzed cyclization/elimination free-radical-based reactions through five-exo-trig cyclization in the C2 quaternary series and seven-endo trig cyclization in the C3 quaternary series. The installation of the hydroxyl at C2 was significantly improved through a stereoselective epoxidation, providing access to NA-containing quaternary carbon at C3 . A modified approach was also presented for the synthesis of NAs of C2 quaternary center series. The synthesis and purification of the corresponding nucleoside triphosphates are reported for the first time. Optimization for NTPs bearing C2 quaternary stereogenic center is under development.
Finding novel molecules that could act as antiviral agents against SARS-CoV-2, or other emerging viruses, is an important venue for the present and future treatment of these infections. We have reported herein two inhibitors, 6 (LCB-2344) and 7 (LCB-2279), against SARS-CoV-2 RdRp, which are lead molecules for further optimization. The stereogenic quaternary center at C2 will be further modified to improve the potency of the novel series of molecules. Currently the study is underway to explore the monophosphorylated pro-drugs of these molecules and their antiviral efficacity.